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Structure and function of cytochromes P450:

a comparative analysis of three crystal structures


Charles A Hasemann', Ravi G Kurumbail', Sekhar S Boddupalli2 ,
Julian A Peterson 2 and Johann Deisenhofer 1, 2*
1Howard Hughes Medical Institute and 2Department of Biochemistry, University of Texas Southwestern Medical Center,
5323 Harry Hines Boulevard, Dallas, TX 75235-9050, USA

Background: Cytochromes P450 catalyze the oxidation distribution in the three structures is similarly asymmetric
of a variety of hydrophobic substrates. Sequence identities and defines a molecular dipole.
between P450 families are generally low (10-30%), and
consequently, the structure-function correlations among Conclusions: Based on this comparison we believe that
P450s are not clear. The crystal structures of P450,,rp and all P450s will be found to possess the same tertiary struc-
the hemoprotein domain of P4 5 0 BM-3 were recently ture. The ability to precisely predict other P450 substrate-
determined, and are compared here with the previously contact residues is limited by the extreme structural
available structure of P450cam. heterogeneity in the substrate-recognition regions. The
Results: The topology of all three enzymes is quite simi- central I-helix structures of P4 50te,,p and P4 5 0 BM-3 sug-
lar. The heme-binding core structure is well conserved, gest a role for helix-associated solvent molecules as a
except for local differences in the I helices. The greatest source of catalytic protons, distinct from the mechanism
variation is observed in the substrate-binding regions. The for P450ca,,,,,. We suggest that the P450 molecular dipole
structural superposition of the proteins permits an im- might aid in both redox-partner docking and proton
proved sequence alignment of other P450s. The charge recruitment for catalysis.

Structure 15 January 1995, 2:41-62


Key words: crystal structure, cytochrome P450, electrostatics, hemoprotein, monooxygenase

Introduction hydroxylation of the bicyclic monoterpene camphor


Cytochromes P450 catalyze the monooxygenation of a (Cl(H 160). The biochemistry of this enzyme has been
variety of hydrophobic substrates [1,2]. P450s have been extensively characterized, and those results correlated
identified in bacteria, yeast, fungi, worms, plants, insects, with the high-resolution structures of the isolated
fish and mammals [3]. A partial inventory of P450- enzyme [12] and the enzyme in complex with substrate
dependent processes would include steroid metabolism [13], carbon monoxide [14], inhibitors [15,16] and sub-
[4], drug deactivation [5], procarcinogen activation [6], strate analogs [17-19], as well as a mutant enzyme [20].
fatty acid metabolism [7,8], xenobiotic detoxification [9] The vast majority of our understanding of P450 struc-
and catabolism of exogenous compounds as a source of ture-function is based on the P 4 50can, model [21].
energy [10]. As the list of biologically significant oxida-
tion reactions catalyzed by P450s has grown, so has inter- Extension of the P4 50ca,ll structural paradigm to other
est in the relationship between P450 structure and the P450s has proved difficult, primarily because of the lim-
mechanisms of substrate recognition and catalysis. In the ited sequence identity among P450s (generally 10-30%).
case of the human enzymes, a better understanding of This is especially true when trying to model the eukary-
P450 substrate binding might lead to the rational design otic enzymes, which are membrane bound, unlike the
of inhibitors and/or precursors that would block or take prokaryotic enzymes which are found free in the cyto-
advantage of the activity of these enzymes in vivo. A great plasm. The dichotomy between the prokaryotic and
deal of interest also surrounds the possibility of harness- eukaryotic enzymes goes further than just their mem-
ing the oxidative power of these enzymes for industrial brane status. P450s can be broadly divided into two
purposes, such as the catalysis of stereospecific oxidations classes based on sequence homology and the identity of
in vitro. Finally, 'designer' P450s might offer environmen- their redox-partner protein. The class I P450s (bacter-
tal applications; for example, bacterial hosts might be ial/mitochondrial) receive their NADH-derived (bacter-
used to clean up the toxic organic compounds that pol- ial) or NADPH-derived (mitochondrial) electrons from a
lute the world. In all cases, a better understanding of the two-protein redox chain, (FAD reductase-iron-sulfur
relationship between the amino acid sequences of P450s protein-P450); the class II enzymes (microsomal)
and their three-dimensional structures is crucial. receive NADPH-derived electrons directly from an
FAD/FMN-containing reductase.
The crystal structure of the extensively studied P4 50 ca,,,
(CYP101, Pseudomonas ptida) has been available for These factors combine to generate a superfamily of
some time [11]. P4 50a,,, catalyzes the stereospecific enzymes whose differences seem to out-number their

*Corresponding author.

I Current Biology Ltd ISSN 0969-2126 41


42 Structure 1995, Vol 3 No 1

similarities. One approach to understanding the struc-


ture-function relationships in such a complex family of
proteins is to compare the crystal structures of several
family members. The premise of such a study is that
regions of functional significance will be conserved
among the structures, despite the divergence of their
amino acid sequences. Towards this end, we have
recently determined the three-dimensional structure of
an additional class I enzyme (P4 5 0terp [22]), as well as the
first structure of a class II enzyme (the hemoprotein
domain of P4 50BM-3 [23]).

P450terp is a bacterial enzyme (CYP108, Pseudomonas sp.)


which catalyzes the oxidation of the monoterpene
hydrocarbon a-terpineol (Co1 H l 8 0) in the catabolic
assimilation of this compound as a sole source of carbon
and energy [24]. Like P450cam, P450terp is grouped with
the class I P450s based on sequence homology and its
requirement for an iron-sulfur protein as an electron
donor. P45 0 BM_3 (CYP102, Bacillus megaterium) catalyzes
the oxidation of a variety of fatty acids, and although
P450BM-3 is also a soluble bacterial product, it is
grouped with the class II P450s based on both sequence
homology and its requirement for an FAD/FMN-con-
taining reductase domain as a source of electrons.
P4 50 BM-3 is unique among P450s in that the holo-
enzyme contains both an FAD/FMN-containing reduc-
tase domain and a heme-containing P450 domain [25].
The catalytic activity of the holoenzyme could be recon-
stituted by combining the two individual domains [26].

We report here a comparison of the three P450 crystal


structures (P4 50terp, P 4 50cam and the hemoprotein
4 50
domain of P BM_3) . The enzymes have similar tertiary
structures, with a conserved core of secondary-structure
elements. The preservation of several key structural fea-
tures among the enzymes supports many structure-func-
tion relationships proposed after study of the P45 0 ca,,,
structure alone. Several previously unappreciated features
of P450 structure are also revealed by comparison of the
three structures.

Results and discussion


The P450 fold
The crystal structures of P450ca, (1.63 A resolution,
Rcryst=0. 19), P4 50terp (2.3 A resolution, RCr t=0.19)
and the hemoprotein domain ofP 4 50BM-3 (2.0 resolu-
tion, Rcryst=0.17, hereafter simply referred to as
P45 0 BM-3) were reported elsewhere [11,22,23]. The
coordinates for all three structures are available from the
Brookhaven Protein Data Bank [27] as entries 2CPP, Fig. 1. Similarity of the overall fold and secondary structure
1CPT and 2HPD respectively. The three structures have content of the three P450s. The molecules are viewed from
13 ao-helices in common (A, B, B', and C-L) and five the substrate access (distal) face. -Helices are presented
P-sheets (31-135). We have taken a departure from the as purple coils, 310 -helices and r-helices as green coils,
structural nomenclature of Poulos et al. [13] in several [3-strands as red arrows, and loops as yellow tubes. The
heme molecules are shown as ball-and-stick models. The
instances. First, we have completely renamed the visible secondary structures of each molecule are labeled (-
13-sheets in a format that is more compatible with the helices A, B', D-G, I and L, and 3-sheets 131-135), as are the
PDB convention and that reflects the common motifs of amino (N) and carboxyl (C) termini. (Figure prepared using
the three structures [now a five-stranded sheet (31), a the program RIBBONS [91].)
Structure and function of cytochromes P450 Hasemann et al. 43

Table 1. Secondary-structure positions.

P4 4 50 4 50
P450cam P 50am
BM 3 P
P450a m P450erp
P450ter P
P450BM-3

ct-helix p-sheet
A' - P8-L17 - 1-1 D52-T56 P39-A43 E38-A44
A G37-A43 D23-Q38 K24-G37 1-2 H62-T66 P50-T55 R47-S53
B R67-D77 K56-Q66 S54-C62 1-3 G315 L320 G335-L340 G350-V355
B' P89-Y96 D81-S92 S72-C83 1 4 D297-T302 F317-L322 P329-K336
C E107-G120 F108-L118 E93-1102 1-5 H80-S82 L69-S71 R66-N70
C' M121-K126 Q122-K128 - 2-1 Y305-F307 T325 V327 T339-L341
D L127-R143 L129-D146 Y115 R132 2-2 V310-L312 Q330-1332 E344-L347
E P157-C168 Y160-G171 P142-N159 3-1 G146-N149 G149-D152 1139-V141
F 1174-R186 E177-L181 H171-R190 3-2 L402-W406 V421-A428 F444-K451
G T192-K214 A208-C233 A197-S226 3-3 S382-A384 K397-S404 H420-E424
H D218-N225 D237-N244 D232-N239 4-1 Q390-K392 R408 V410 D432-T436
T234-K266 D253-R285 D250-K282 4-2 G398-Q400 G417 N420 T438-E442
S267 R277 N286-D296 N283-L298 5-1 G226-V228 5245-L247 G240-D242
J' - - S304-Q310 5-2 R231-1233 N250(1252 G246-L249
K R280-F292 L299-A312 L311-W325 6-1 L294-A296 P313-S316
K' S341-N346 L356-H361 6-2 1395-5397 F414-G416 -
K" - - D363-G368
L G359-1378 G379-L395 G402-K419

310-helix p-bulge
a P19-V23 P18-A22 N16-N21 F350-G353 F370-G373 F393-G396
b N33-A36 S101-M105 L103-S108 L356-C357 M376-C377 A399-C400
c V44-E47 P173-D176 Q109-G114
d P170-D173 - N163 R167
e P321-D328 - R375-E380
f D407-T411 - N381-1385
i-helix
E' F150-E156 F153-L159 -
F - K182-F188 -

Positions of the secondary-structure elements for each protein are presented, with the start and end amino acids noted by their single-letter code, and
their sequence position. The absence of an element from a protein is indicated by a dash.

three-stranded sheet (3) and three two-stranded sheets P4 5 0 BM-3molecules in the asymmetric unit [23]. No
(P2, 34, and 135)]. We have also divided the C and E analogous rotations were apparent among the ot and
helices (C and C*, E and E*) to reflect the significant domains of the three molecules, and so we elected to
deviation from ideal helical structure observed in these treat the molecules as intact units [choosing the more
regions. Two additional a-helices are observed in similar of the P4 5 0 BM-3 models (molecule 2) for compar-
P 4 50terp, one near the amino terminus (A'), the other ison]. Table 2 shows the root mean square (rms) devia-
between the K and L helices (K'). P4 50 BM-3 has three tions for the three possible pairwise superpositions, and
additional ot-helices, J' between the J and K helices, and the pairwise percentage sequence identity. When the rms
both K' and K" between the K and L helices. We have deviations of these molecules are plotted as a function of
also identified a short -structure (6) which is common their percentage sequence identity, it is clear that these
to P450ter and P450ca m . Finally, we refer to the region structures have diverged in a fashion consistent with a
between te Cys-pocket (the 3-bulge region which con- variety of proteins related by evolution (Fig. 2) [28]. The
tains the thiolate heme ligand) and the K' helix as the roughly 2.0 A rms deviation among the cores of these
'meander', based on its extended wandering topology. A structures is consistent with their -18% sequence iden-
summary of the sequence positions of the secondary tity. This deviation is quite large, and was responsible for
structures in all three molecules is presented in Table 1. the failure of a molecular-replacement solution for
P450terp based on the structure of P450a,,, [22].
Fig. 1 shows the overall fold and secondary-structure
content of the three structures as ribbon diagrams. The We have aligned the sequences of the three P450s based
three structures clearly possess the same set of secondary- on the structural superposition. Fig. 3 shows this
structure elements in very similar folds. To quantify this sequence alignment, with the positions of helices,
similarity, we began our analysis of the structures by 3-strands, and the highly conserved 3-bulge or 'Cys-
determining an optimal superposition of the three mol- pocket' indicated. As in Fig. 1, it is clear that the same set
ecules. In our analysis of P4 5 0 BM-3 we observed a con- of secondary structures is maintained in all three mol-
certed rotation between the oa and 13domains of the two ecules. It is also evident that both P 4 50 terp and P45 0 BM-3
44 Structure 1995, Vol 3 No 1

terminus for the F helix, and a shift of four amino acids


Table 2. Pairwise structural similarity and sequence identity.
towards the amino terminus of the G helix. Because the
F and G helices are antiparallel, these motions occur in
Cam Terp BM-3
the same direction. The shift in alignment, which corre-
Cam 23.0%a 12.9%a sponds to one turn of helix, is consistent with the amphi-
pathic nature of these helices, and the tendency of the
a
Terp 0.5 8 8 b 15. 9 % hydrophobic side chains to interdigitate into the adjacent
(1.91 A x 100/325) hydrophobic environment. This observation illustrates a
general problem in relating primary sequence informa-
BM-3 0 .8 02 b 0.63 1b
tion to structure, namely that regions with reasonable
(1.98 A x 100/247) (1.90 A x 100/301)
sequence conservation, such as the F and G helices, will
Structures were superimposed as described in the Materials and meth-
not always superimpose in space.
ods section. aSequence identity calculated as no. of identities/(no. of
aligned + no. of gapped) as aligned in Fig. 3. b Structural alignment score To describe the distribution of similarity among the three
calculated as (rms deviation in core x 100)/(no. of positions included). structures, we have determined regional rms deviations
of CO positions. These are presented as both a total
deviation among the three structures, and as the devia-
tion of each from an 'average structure'. The results of
this analysis are presented in Table 3. Statistics on the dis-
tribution of these regional three-way rms deviations can
a) be used to partition the structures into four categories
0 of structural conservation (see Materials and methods).
rZ First, there is a very well conserved core structure in the
C:
neighborhood of the heme and extending into the heli-
cal domain (color-coded dark blue in Table 3) which
includes the central and carboxy-terminal parts of the I
-C helix, strand 1 of 1-sheet 6 (6-1), the Cys-pocket, the L
helix and the J helix. A second tier of conservation (cyan
20 40 60 80 in Table 3) includes four strands of the large sheet (131-1,
Identity (%) 131-2, pl31-3, 11-4), the C, E and K helices, 132-2, 6-2
and surprisingly, the meander region. These first two
groups include all the 'interior' structures. The first tier
Fig. 2. Plot of the relationship between structural similarity and of less well conserved structure (green in Table 3)
sequence identity. The test-set data () were tabulated by
Chothia and Lesk [28] in a study which concluded that the rms includes the B, C*, D and K' helices, the amino-terminal
difference in the core regions of homologous molecules was pre- portion of the I helix, -sheets 133 and 4 and -strands
dictably correlated to their sequence identity. This relationship is 131-5 and 132-1. This group is dominated by surface fea-
shown to hold true for the P450s in this study (A). tures of the molecules. The last and most divergent group
(red in Table 3) includes the most flexible regions of the
contain several large insertions compared with P45 0 ca,,. structures, as assessed by atomic B-factors, including the
It is noteworthy that, with the exception of the amino amino termini, the A, B', F, G and H helices, and 5. It
terminus, P4 5 0ca,,, features no significant insertions rela- is of considerable interest that this last group includes the
tive to either P4 5 0 terp or P450BM_3. In a comparison of surface helices thought to be involved in substrate re-
these three structures with a large number of P450s, cruitment and binding (helices A, B', F and G). Fig. 4
P450ca,,,,, seems to represent the minimal P450 structure. shows the distribution of structural similarity. This repre-
As discussed below, the location of the insertions in sentation gives a feeling for the distribution of similarity
P 4 50terp and P4 50 BM-3 is significant in relation to the among the structures, with the conserved regions sur-
function of P450s. rounding the heme, and extending into the core of the ot
domain. Equally important, the figure shows the two
The structure-based alignment is not the same as that major foci of variability, the substrate-docking region
which would be produced from sequence conservation (helices A, B', F and G), and the proposed reductase
alone, particularly in the regions of very low sequence interaction face of the molecules (primarily comprising
identity. The structure-based alignments yield identities the J/J' and meander insertions in P450BM3 )
which are 3-4% lower than the corresponding identities
derived from the sequence alignments (e.g., P 4 50a ,,m ver- Extension of the sequence and structural alignments to
sus P45 0BM-3 16.6% compared with 12.9%, as in Table 2). other P450s
One major goal of this study was to extend our structural
A dramatic example involves the sequence alignment results to the many remaining P450s for which no crystal
shifting the F and G helices of P450ca,,, by one helical structures exist. The insight gained from the structural
turn relative to the structural alignment. This results in a superposition of these three enzymes has allowed us to
sequence shift of three amino acids towards the carboxyl attempt a general alignment of the bacterial, microsomal
Structure and function of cytochromes P450 Hasemann et al. 45

Fig. 3. Sequence alignment of P4 5 0 cam, P4 5 0terp and P4 5 0BM-3 based on their structural superposition. Gaps in the alignments are indi-
cated by dots, and the position number of the amino acid at the far right of each line is shown. Names of the a-helices and 3-strands
are indicated above the sequences. The extent of the secondary-structure elements of each sequence are boxed and color-coded
(a-helices magenta, 310-helices green, rr-helices orange, 1-strands red, and the Cys-pocket in blue). Note that this amino acid align-
ment is based on the actual spatial superposition of the structures, and not optimization of sequence conservation.

and mitochondrial P450s. Most influential in this align- comparisons. These errors were primarily in the amino-
ment was an understanding of the structure of P45 0BM-3. terminal half of the P450s, where the sequence identity is
Because of this enzyme's unique status as a bacterial extremely low (10-12%). To align the identifiable pat-
enzyme that is highly related to the microsomal P450s, terns that represent the conserved secondary-structure
P45 0BM-3 offers an opportunity to predict the structure elements, a large amount of 'hand-fitting' of the
of the eukaryotic P450s with a level of confidence not sequences was done. As discussed in the paragraphs that
previously attainable. The combination of all three struc- follow, this manual alignment was guided by several
tures enhances the power of prediction by allowing us landmarks that we believe point the way to a correct
to develop a consensus for those elements that are well alignment. This alignment cannot be absolutely correct
conserved, as well as indicating the regions that are in the absence of structural information for any of the
structurally variable or disposable. eukaryotic P450s.

The multiple sequence alignment shown in Fig. 5 The importance of structural information in this
includes some of the best characterized representatives sequence alignment is substantiated by the fact that the
of each of the major P450 families, together with the 'correct' sequence alignment for P450can,, P450terp and
sequences of P450cam, P450terp and P 4 50 BM-3. We P 45 0BM-3 produced by the alignment programs is actu-
attempted several automated procedures to align these ally a very poor representation of what we know to be
sequences but in each case found the alignments to the correct alignment based on structure. This is espe-
be incorrect, based on our insights from the structural cially true regarding the loop regions between secondary
46 Structure 1995, Vol 3 No 1

structures and in the exceedingly variable regions (amino Several observations from this alignment merit discus-
termini, A helix, B' helix and F-G region). As we saw in sion. First, the amino-terminal half of the sequences
the alignment of P450can,, P450terp and P 4 50BM-3, it is (between the amino terminus and P5) is the most poorly
quite possible for the optimal sequence alignment of conserved. For the amino termini of the eukaryotic
helices to be shifted by units of helical turn relative to the enzymes (before helix A) we have simply used the best
structural alignment. Because of this, the helices with no sequence alignment, as the structures impart no know-
well-conserved sequence motifs, and a prevalence for ledge about the presumed membrane anchors found
insertion and/or deletion at their termini, might easily be there. Whereas the amino-terminal border of the A
incorrectly aligned by ±3-4 amino acids. Such an align- helices is rather uncertain, the pattern of hydrophobicity
ment will obey the conserved pattern of hydrophobicity in the region indicates that the other enzymes do contain
for the helix, but be structurally incorrect. With these an A helix. The first structural landmark is at the car-
caveats in mind, we have a reasonably high confidence in boxyl terminus of the A helix, where all of the eukary-
the correctness of this alignment. otic sequences and P4 50 BM-3 share the sequence XG
Structure and function of cytochromes P450 Hasemann et al. 47

Fig. 4. C, trace of P4 5 0BM-


. 3 Stereo pairs
of the model are shown, as viewed from
both the distal (a) and proximal (b)
sides. The tube is colored according to
the rms deviation of the C, at each posi-
tion in the three structures as aligned in
Fig. 3. The transition from cool to warm
colors (dark blue to light blue to green
to yellow to orange and red) represents
the progression of structural conserva-
tion from most similar to most variable.
The backbone of P4 50 BM-3 was chosen
for this figure because it contains the
largest number of insertions relative to
either P4 5 0cam or P45 0 terp. Regions of
P4 5 0 BM-3 which are insertions were
assigned the highest score, and thus
appear red. The amino (N) and carboxyl
(C)termini are labeled.

(Gly96, Fig. 5), where X is a large hydrophobic residue. and arginine interact with a propionate side chain of the
This hydrophobic amino acid anchors the carboxy-ter- heme (see below).
minal end of the P 4 5 0BM-3 A helix, while the glycine
initiates the turn towards 31-1. We believe that the The region between the F and G helices is considerably
structures of all P450s will include an A helix, in an ori- variable, both in length and sequence content, making
entation similar to that observed in these structures. The prediction of the borders between the helices and the
region amino-terminal of the A helix could serve as a loop between them difficult. This region is also the loca-
flexible linker to the membrane-spanning region in the tion of the shift between the structural and sequence
membrane-bound enzymes. This region could possibly alignments for P450Can, relative to P450te r and
contain a helix analogous to the A' helix of P450terp, as 4 50
P BM-3. The distance between the G and I he ices is
it is evaluated to be helical by structure-prediction quite variable among the sequences. This region includes
algorithms, and others have suggested this as the location the H helix, which has a fairly consistent sequence motif,
of the A helix [29,30]. surrounded by G-H loop insertions and/or variable
length insertions in the 35 region. The region between
The B' helices are the most variably positioned regular the F helix and 135 is the most consistently poor region of
secondary-structure elements among the three structures superposition among the three structures.
(8.99 A rms), with very different lengths and orienta-
tions as well as very low sequence homology. The align- The region between the I and L helices is the most
ment of the B'-helix sequences is consequently the most highly conserved among P450s, and consequently is the
tentative region in Fig. 5. The phenylalanine residue in region where the multiple alignment is most meaningful.
31-5 serves to anchor this strand to the core of all three Like P450BM_3, the eukaryotic sequences appear to have
structures, and thus is a good marker for the alignment an extended J helix and a J' helix insertion relative to the
of the sequences at the amino-terminal flank of B'. The bacterial sequences. The alignment of the K helices is
B'-C turn is structurally similar in the three crystal substantiated by the conserved EXXR motif that is
structures, and reasonable homology exists among the invariant in all the sequences that we have examined.
eukaryotic sequences and P4 50 BM-3 in this region, The presence of a variable-length 36-1 segment in the
which serves to anchor the carboxy-terminal flank of B'. three structures is recapitulated in the eukaryotic
The amino terminus of the C helix is well determined, sequences; the exact location of the gaps being uncer-
as this is the location of the conserved WXXXR tain, but the segment is anchored at one end by the
sequence (Trpl75, Arg179, Fig. 5) where the tryptophan K-helix EXXR motif and at the other end by the
48 Structure 1995, Vol 3 No 1

P450&... ................... ...................... ' TETI QSNANLAPLP PiVPEHLVPDFQMYNFSNLS A.GVEAWA V LQESV.


PSOtrp ........................................................ MAR ATIPEhART VILPQGY ..... A
DDEVIYFAFKILRDFQF
11IATlro.Tvin ............ . .... STATPRPYSEIPSPGDDGWL NLYNFWRKSQR Q...................
FRHE NFQKYG. F
27_R.bbit YAALGCMLR WALLCPRVAG CGLcpGAA 'AAIPALPA OAAQAPGAG PCDRRRRRSL EELPRLGQLR FFYQAFVQGY LL.LKLQVL IRYG. P
21aJHm.n .. ....... .......... MLGLLLLP LLAGARLLWN WWKLRS.LHL PPLAP . ..LHLLQF....DLPIYLLG LTQKFC...F
l7._Ra . t .......... ..MWELVGLL LLILAYFFWV KSKTPG.AKL PRSLPSLPLV GSLPFLFFPRRG ..PHIFVFFK LQEKY.. P
l2_rat ................... M..HAFSQYISLA PELLLATAIFCLVFWVLRGT RTQVPKCLKS PPGPWGLPFIGILTLGK . ..NFhLSLTKLSQQYG D
2clRt . .YLVLVLTLSSLLLLWLRQSFGRGKLFPPGPTPLPIGNTLQIYK. ..DIGQSIKKFSKVYG
............................. .P
24_Rbb ........... .................. Y PVAGLVL GLCCLLLSLWQSRGL PPGPTPFPII GNILQIDV.. DISKSLTK FSERYG..p
2e4_Rabbit . ........ LL........
A VLIITVA ....... LLFISV KQMSWNL PPGPFPLPII GNLLQLDLK. ..DIFKSFGRLAERFG...P
2ibl at .......... M.................
R PTILLL. LA LLVGFLLLLV RGHPKRGNF PPGPRPLPLL GNLLQLDRG ... GLLNSFMQ LREKYG
...D
2&4JoOUS ........................... MLTSGLLLVAAVA FLSVLVLMSV WKQRKLSGKL FPPGPTFLPV GPLQLNTE ... QMYNSLMK ISQRYG..P
3a,4_ ....................... .ALIPDL
M.. AETWLLLAVSLVLLYLYGT HSHGLFKKLG IPGPTPLPFLGNILSYHK ...GFCMFDME CHKKYG..K
44A A..
........
t MVALSTR FTGSISGFLQ VASVLGLLLL LVKAVQPYLQ RQWLLKAFPQQ FPSPPFPWPFGAK.QFQGD..KELQQIMTCVENFF.. S
P50b3 i........................... ...........
TIKE MPQPKTPGEL KNLPLLNTD. KPVQALMK IADELG..E
........
9 ..............
nm M LPL FAYITSI V7EMpAAT M LLTGLPL%VNVYEGT$S I.IPGYFGI GPLISHGRFL GIGSACNY YNRVYG...E
cnsen. ..... .............. 1.11. 1111sv .G.p.rgklppgptptpfignqld- . ih.lk sekyg.. .p
....

M1- -A Bl 3, CC. 20
0
PiS0caw LVWRTCNG.. GAWIATRGQLIREAYIDY.. HFSSE..CP FIP7RAGAY...........DFIPTSMD P.PEQRQFRALANGWGMFVVDK...
P45ECrp LAMAHIEGYD PMWIATADVMQIGQp.. GLFSAMGSA ILYDMNEA MARSGSGCPHVIDSL.TSMDP.PT"TAYRGLTLNWFQPSIRK.
11&MAY0IYRK4LG..L ESVYAIIHP VAH/LFKMP..
27_Rabbt MNSYLG.PQ LFVNLASAPL
GIPER. YD IPLAYHRY YQKPI..GVLKKS...GTWKDRV VLNTEVAE AIKN.
VETNQA.. GY7V. .MDMQLWMRDH ADLAY . GVFTTAG.. ,H4YQLR ALNQLLKP AAL.
F Fig. 5. Multiple sequence alignment of
21a2-Huan IALALOL
"M4AMAKM. DVARLNSMT ADFAGM
7_a7.t.IYSLk1A.TTTTVI GYQL ARELIKK . KESR.
.... PLTYKLVSKNY
1..
GMVFTQSL
LSDG.
.....PDLS.GYS . LLWKAHK LTSAL.LUGlES.
GVAPADAG. SAWALARKLVFSTFSLFK
.. DGQK.
various P450s based primarily on
142-Fat VLQIAI.ST PVMLSGLT IKQALVKOG.DDKGR
2C11_Rat 17TLyLG.MO PFAVVIGYEA
VALVLG. EEFSG .
. ATNAIF.
"LSPTL
GSPVSER VNKGL .
SMTPFNPD$G.FASARRFLAGR
VAPAMA...
ALKSFSiASDPTSVSS
MQEIFF FSIMTLRTFG MGR .....
sequence conservation. Using the
ICLOSIAb VPIY.ALA.MPTAVLMVTWA GROALV
21_..abbi= VLVGL.SR RVVVLHOYKARMLN4K. NEFSGR
A ... ..
.
GHPIAEK VNKGL.......GIVPTA. .WMRR
GEIPAPMe KDK .......
G F..
IIFG.
KDTRR
A.
FSL RNFG MKRS ...
FSLTTLRDYGMCKQC.. correctly aligned sequences of P450te r ,
2h1.4.
3GatJ~ou..
4
VVIRML.P "FYRDMLTT IKMVGQA. ED? .
FRVFTIy.C.MIVVLCQM%VILVDQA. E .
GTIAVI FIKE.........GVIPANG...
GEQATMFL
.. .....
K
ERKALFR FLATMRDF MK .....
YGAAPSA. .:.P'RAKQLRASIATLRDFG VGMG.. P450cam and P4 50 BM-3, other P45%s
a-RUi4n lVWFYD.GQ PVLA4TDH IKTVLVXF SEFTNR AISIA . ,EWKRLR
¢&1_4* AFPR NEVIL
K AYLIVYfDPDY . GR4074
C...
..RPPPVF
...
....
.L
X... . LLSPTFTG LKE..
LAPIG..YGLLLLNG. ..QMPHR MTPAFYDI LKP.... were aligned in order to maximize
P450b.3 IFKFEAG.M VTYL5RL KMACM.. S DKN . ..LGALE YAA..DL7TAT N ILLSFSQQAMKG......
IMKKAEXM
19., n PFVWSGEZTLII 2 PID S SRFGSK....... LGCIGM HE ... . G.Pl ..ELWKTTRP FPMKAUSGP LVR ...... sequence alignment, without violating
Fonl.n... vftvyl-.F FYVl.. vkalvd... .itM .... .. . .
vkk ...... .gilftng. .. ..iFlfO
lrk.1-g gk......
conserved motifs observed in the crystal
structures. The approximate locations of
PF0.... . LACSLESLR .... Al TDYAEPFPI
P450rp , .'LEESIM AASVQRLL F. ..054CMFMTDCALYYPL ALV.
..RFLLGL
.. L.... ...... .........
EDD EPUMLALTOD
F...
.TF
FFGVMEFDEQAV... APR QSADEAARR
the secondary structures, the Cys-pocket
1a1.1-vin IPLLNPVSQDMVSLLH4KRIK
27_R.abbitTDALVIAS FVVRLDQLA
QQGSGAF/0 IKEDLFHFAFESITNFGE LM..LEST VNPEAQKFIDAVYNFHTSVpLLNV..PPELYRFRTKW
ESASQDQEDNADLLYHPAL AICYLPEK RC. .LEASIPKDTEFIR SVGMPIQNSV YV7....FLP KTR IPFY
and the meander regions in the three
21IJ2M- ..I4EFVEOLTCM7CERKAQ. PTFAI EE.EFSLLTCSIICYTFGD XI.. MDDL MPAYYCIQE VLKTWSMS IVDVIPFLR FFPNFPGLRRL
"._ Mat . .LALICQE AKSLCEOLAMD..E$IDL ST.PI FM NICAICFNISYE..NDPK LTAIKTFTEG1VDATIDRNL VDI-.PWLT IFPNOLEVI known crystal structures are shown
1&2_rt CYLESME ANMLISKG IEGHP
2.11_.._at . IMMRES AQCLVEELME .7.$APFDP
EIM.QVEEVA NCACPGK NP.M.RSAM MLNLVKSSG
TV.ILCAF NVICSIIFQ4DP .FYKDPT
FVENVTSGNA
FLNLMMFNE NFALFSSPWL
VAP.. FPVLRYPNFPALKRF
QVCNTFPAIIDYFPGEN.QV above the sequences. Groups f 10
EL-hbit
2.1_Rabbit.
. .ARVQE ARCLELK . .THALPCDP TV. CAPCNVICAIYLHRFD.. TDEE PLKLMRLM NIRILASPWL
.NDRIAKE AMILLELRK. .TIQPDP TF.VICTP? NVIAILFNDRPD..YDKQ ALRLMSLFNE NPYLLSTPWL
OVFNNFPALL DYPPGIXTL
QYNSNYL QYMPCSRKV amino acids are separated by spaces,
2bl.at . VS1Q5 E AQCLVERMK . .MOAPLDPTP.LFQCITANICSIVPGE RD..7GDRQFLMLLELFYR TFSLLSSFSSQOVEFPSGFL KYFPGAHRoI
2a4_MOU4e . IPM REEAGVLIDSFRK. .TNC"IDPTP.YLSTVS NVISSIVGDRFD..YEDKEFL.LLRNN1LG SLQFTATSMGQVYEMSSV KHLPGPQQQA and the alignment position at the end of
.W.MVPIIAQY GDVLVRNLRRAMT0MPVTL KD.VPGAYSM DVITSTSPGV
ND...GLNN PODPFVENTKLLRFDFLDFFFLSITVFPFLIFILEVLNI
3i.4NbM
4m1_4Ra ..YVK/DS IRLMLEMMEQ LAAQDSSIRI PQ.HISILTL DffMKCAPSH
NGSVQVDGNFMSYIQAIGNLNOLFHSRVRNIQ.. DTI YFS.NGHLF each row is shown. The single letter
P450b.3 . .YM lE AVOLVQKERM
L.NADRIEV PE.DNTRLTLDTIGLCGFNY . .ANPDDF..AYDENKRQF
RFNSPYRDP HPFITSHVRALDENMANMLQR
19_H.un . .MATVCAES LKTHLDLE V.TROSGYVE VL.TLLRRVMLDTSNTLFLRIF.. LDESA IVVYIGYFD AQAILIKF IF'.....KI LYKKYEKS amino acid code is used, with gaps in
Fonsensus .. l44riMeeaq.lv~..lFr.... ~.P.dpSf..1gcfPlN-iF-ilfd rfd..ykdeefl.l.iON. n.lNSpl GD.... 1. ky.Fa rf
-.
individual sequences indicated by a
dot. The consensus sequence is pre-
P m AEAA LIFEQR IF ...................... G I I .LGQV
...... M..-IT.DEAK -.
AS.
GLL
MID54IN AYVAIATAG
G LDVVNFLS1 MEFAKSPE
HDTTASSGO AIAGLMNp
sented as follows: upper-case letter,
P4trp ETIATTY FNG VRS C'............. KD4 SLIAN..SL ......
1al AviN DMHVAANDI
27_Rbbit MRYL
VN4EK4Y0TM
IFIGT
S
FYQD.. LRR K .TtRNYP GILY.CL
KLQErVAOAL
DOKNLX
ILEKMLLEDVK
.KS ......
AGSDGVQVS GYM. .ALL T .........
ANITIAGG VNTTSMTLQ.
GQLSPREALGALPEULLAG
-YEMARSLN
VTTSTLTN ALYRLSNPE
100% conservation; lower-case letter,
21&2_M.M KQAIEKXDI VELRQHKE SLVAQ .........RDMM DTI.GVA QPSMEGS. GQLLEGA MAAVDLIGC
17.Mat MGYAKVRNEV LTGIPEKCRE
KPDS . A.SALTDILIQAkKM S ANN $CMGD
TETTANTLAWAVVFLLRAFE
PODPSDEIL ATVGDIFGA IETTTVLK ILAFLVENPE
most frequently occurring amino acid in
1&2_rst KNF0DNU LRMTV
A
lE at
E:YQ DFN](NS
LKNPFYI]YVLEMMYMKM SLKD...
. ODIT GALFK.HSEN
....PRDPI CFLN.SMSO
I.. KNG .GLIPOEKV N-NAIFGAG FETVTTAIFSILLLVTEK
E..LRIPO..SEFLES V ATVTANPGAG TE=TSTTLRYGLLLLLKV
>25% of the sequences; dot, no indi-
2C4abbit LRNkAYT!SIM EEQK LDVN....
2e.1_Rbbitl VS RXEYTLARVXIH ELAPSC ....
PDI I.KHEK ... N N ..LEFTLGSLV IVFELFOAG TETTSTTLRYSLLLLLKHFE
.PAI DSALIEEM A.. SEATS PLYTLEWIA VTVAOMPFAOTETTSTTLYGLLILL.PR
vidual amino acid in 25% of the
2bA MaE SNLOEALDY
la4Mouse PKRLIGLEDF
IGHIVEK~RATLDPSA.
1TK$EHNSGTUAPNS .
P::PI TL.ME
..... ... KS H.TEPHHENLMISLLSLPFAG TETSSTTLRY
PRDFI DILI.OM.E M.R..K-N.TEFY
GFLLMLKYPH
LV LTLNLFFA TETVSTTLRYGFLbLAKYPD
sequences, or two or more amino
4.,uma CVpREVITOLRKV
4a1at NRACQLAAH
SRLEDT
TDGVIKKD QLQKAGELEK
......QKHRVDPLQLMID..N
VKKKRRLDFLDILLA
S.. KETESH.KALSALELVAQSIIFIFAG
A.
YETSGVLSFINYELATEPD
I SKDL AEVDTFMESHDTTAGVSW IFYAL.TFK acids at an equal frequency in >25%
P450b.3 QEDIKV4L VMIADRKA SGEQ......
19_u- VKDSLKAIEVLIAMCIS T LM ::ISD
TEN.H M P......
A......DDLL T GELDDENIR YQITFLIAG HETSGLLSFALYFLVSNFP
TELIL..AEK M..........DLTRENVNQCILEALIAA PDTMSVSLFFELFLIAKHPN
11 11
of the sequences. The trivial name,
1.kivkeh. aId.
onensukknl..i.dy .... Pdfi dilR..k ...... .. ltdenla1VdIffa teT-s. . .khpe
standard nomenclature, and source
organism for the sequences presented
I1. K 81-4Lo -21 fl22 -AL- L K' K Mfl d& 00
O450 HQELIEP .......... RIPAACEL LRRFSLV.A.DGRILTDYE FHG.VQLKKG DQILLPQMLS GEFENAC. FpNFVPSRQ are: P450Cam (CYP101, P. putida),
P4t50fp QLALAISP. .LILVDEA
P4 5 0 te, (CYP108, Pseudomonas sp.),
VIUTAMS. PMMYGLADTM VEIQNIKRO DAMYPS NREEFSN. PDEFITRFF
1141 MAviE VQEMLREEVLA.45A05.. .0 GDISXNLQMLPLLRASIKET LRLHPISVT.LQRYFEDLV LQD.YLIPAKTLVQVAIYAM REFAFA. FPTML
27R.abbilt IQAALRKEV GVVAAG...0VPQhKDFAN PLLKAVLMET LOLYPVIPA.NSRIVKMI EVGGFLFPNTOFVFCFYV;T SFDPSTFSM.FATFpYRWL
21&2 Human
1&a.
IQQRLQELAMLOPGASS$ RVPYMDARL PLLNATIAMV
VK.KKQEIDQRG...FSR TPTVNDRSHLLNLEATIREV
LRLRVVPLA LPHRTTRP$
LRIRPVAPML
SIS.YDIPEG
IPHAMNV$S IGE.PYVpMD
VIIPNLQG LDETVMER. PIEFPDMFL
ThF;'NLWALARDENEDQ.PDQFPERFL
11 A _Bovin (P450sc c, CYPI 1A1,
1a2.at
2RlRt
VQRKIHEELD
VTAMVQEIII
TIGE. ..0 OPRLSDRPQL
RVIGRN...4 $P
PYLVAFILEI YRYTSFVFY IPHSYTRDTS
DRSGMpYTDAVVEI0EYIDLVPTN LPHLVTRDIK
LNG.PEIPKERCIFINQWQVGASDKQK. PFVFRPERFL
FRN.YFIPK TWFIVSLSSILEDDKEFP. PEKFDPGFL
bovine), 27_Rabbit (P45026 oh , CYP27,
24..abbi A RVIGM...M SPCViDRSIM
VAARVQEEO
2eM1RUbb£i AMKLMEID RVIGPS...R MPSVRDRVQ.
PYTDAVIHIQRPIDLLPTNLPHAVTRDVKRN.YFIPK TDIITSLTSVLEDEMAPN.pKVFDPGFL
PYMDAVHEI QOIDLVPSN LPHEATRDTT TWIFTLDSL
TQIYVIPR
G LYDKQEFFD.FEFPEAFL rabbit), 21a2_human 'P450c 21 B,
2 2blRaft VAEKVQEIDQVIGSM.. .MRLLDDRSM PYTAVIHEI ORFSDLVPIG
&4¢M-ou. IMAKVHEEIRVIGRN...0QPKYEDRMM PYTYAVIHEI
VPHRVTKTM FRG.YLLPRN
QRFADLIPMGLAMRVTKMTKFRD.PLLPMC
TEVYFISASA pDSFNFPEFL
UHAFQYFDH.
TEVFPLGV LKDPKFFN. FKDFNPKHFL CYP21A2, human), 17a_Rat (P450 7 f,,
3a4_- -- VQRKLQEEIAVLPN.. .A PPTYTLQM EYLOP.YNET
4.1-a- EQQR-CREVAAVUGDG.,
LRLFPAR ING.MFIPK VVSMIFSYAHFDPSYTE. PMKFLPMRFS
MLERVTKKDSE
. SITWD.LDOIPYTMCIKA LRLYPV-O IVREUTRVT FPDGSLPK IQVTLIGL RfROPKVYpN. PSFAPSFF. CYP17, rat), a2_Rat (P450 d, CYP1A2,
P4503 VLQKAAEMA RVIVp ...
19 VEEAAMEAG
VPSYEGQYIQL
E TV0GM....DIKIDDIQKA
IM
YVCVLNEA LALMPTAP.FSLYAMEDTV LOEYPULKG )ELMLIPQ1 RDIW.D VAPFERF.
U KVAEFIYES MRYQPVDL. .MRAL.EDVIDG.YPVRKGTNIILNIOR R.LEFFPK.PNEFTLFA- rat), 2c 1_Rat (P450M4, CYP2C11, rat),
2c4_Rabbit (P4 50 PBc4, CYP2C4, rabbit),
-... n..s -.
klqeeid ,id-i.h.i..
sptGkdrsm pFldviEi 1RlipI-s.. 1pr-rdL. fr.11iPkA-i .lyh.d.kn. p.fd f

-3 - R-2 4-2 .3' ... 2e1l_rabbit (P4503 a, CYP2E1, rabbit),


P450caF VS
P450-P . ............. GO
NTF. H-GQ..............LARREIIVL KELTRIP..S
ACLGQ LAKLEMKIFF ELLPKL- .
Q1 RE. GD SOV Q-LF ADPAT
VELGPP RLVATN..FVGOPKn/FIR TKA ......
2bl_Rat (P4 50Ob, CYP2B1, rat),
111M_B
7 n SKKDLIHFR.....NL
2 abbit RKGQPETSKTQHPFGSVPFG
AGVRQCVGRRIAELEMTLFLIHILENFR .E..MQRIGDV MIFN .LI LTPDKPIFLVFRPFNQAFGA....
YCVRACLGORIAELEMQLLLARLIQRYEL. MLAPETOEVQSVA..RIVLVP.NKKVDLFLPTQR......
2a4_Mouse (P4 5 0 15,oh-1, CYP2A4,
21a2_Hun EPN
17..at
R.......ALAFG CGAPVCLGEP
PTSOL. .I TPTSYLPFGAGPOSCIGEA
LRLELFFL TRLLQAFTLL
LA0GMLFVFT
PSG.DALSL QPLPDCS.VILRQ.PFQVR LQPRGMGAS GQNQ.
ALLORFDL. ..DSDDKQILPRLEGPKV FLID..FFV ITVRIAMAG AEVGT
murine), 3a4_human (P4 50n 25,
la2 at TNTAIDMY LEM MFG A.KECIGEI PAKEVTFLPA
2cllRat DERGNKK.....DYFXPFAGKRICAGEA LARTELFLFF
AILLQLEF. ..TVpPGVMV
TTILONFNL. LVAYKDI
DLTPSY.GLTMKPRTCENVQ APFSK..
DTTPAISGFG-LPP.FYEACPIPVORADSL
....
SSL..
CYP3A4, human), 4alrat (P450LA,,
2c4 Rabbit DEGNFI ... .YMFS
241 Rabbit NEGKFKY ... SDYFKPF
AGK CVGEGLARMP.PLFL TSILONFKL..QSLVEPKD DITAVVNGFVSVPP.SYQL FIFI............
AGRVCVGEG LARELFLLL SAIQ.FNL. .KPLVDPEDIALRNYVGFGRVP.RYKLCVIPR- ........ CYP4A1, rat), P4 5 0 BM-3 (CYP102, B.
2blRat DANGALKK.
24.Mos
.. .$AFMPS TPKEICLEG IGRMMLFF TTILQNFSV..SSHLAPK DLTPSGIG KIPP.TYQITFSA ......
DDGQFKK ... .DAFVPP$AGKRYCFGEGLARELFLFLTNIMQNFHF..STGAFQAIAVGFRLVGPV T IP.TYTMS FLR.. megaterium), 19 Human (P4 50 Arom,
344 H-
4il at
KKRNID. ... PYIYTPFGSPRNCIGTMFALMKLAL IRVLQNFF. .F CKETQIPKLSLGLL QPFEK.pWLK
.GPAAFMR.. SPPFA OARNIGRO P-SRKVIV ALYLURPL. .LDPTKVFA P.... LV LKSKNIYLDLKKLY
VESRDGTVD A....
......... CYP19, human). The source of the
. .QGAFKPGNGAACIGQQFALIEATLYLGM4LKHFDF...
P450503
19_H.-
AENPAIP..
KNVPYR. YFQPF GPRAGKY IGAMAVWAIL VTLLRRFHVK
RDTRML IET...LT LSFF.GFVVK
TLQCVESI QKIR.
1
AKSKIFLG IPFFST
LS LFDETYML EIFTFNS RDLEH. amino acid sequences, and the method
consensus de.Asf. FI.*pDF9akr.C.Geo IAr.lflfl . .1l1f.l. .s..pkdi F...v
t k y ....i ...
of alignment are provided in the
Materials and methods section.

conserved arginine/histidine propionate ligand in 31-4 region in all three crystal structures (see below). In all
(Arg/His453, Fig. 5). P450s the Cys-pocket includes four nearly invariant amino
acids. A high degree of sequence conservation extends
Surprisingly good homology is observed in the K'-mean- from the Cys-pocket through most of the L helix, which
der region, which is anchored by several conserved pro- exhibits the lowest rms deviation among the three struc-
lines and aromatic residues. Also notable in the meander is tures. The variable lengths of the remaining [3 structures in
a conserved arginine/histidine/asparagine, which is part of the three crystal structures, as well as poor sequence con-
the ERR-triad that stabilizes the structure of the meander servation, make the alignment in the 33-[4 region
Structure and function of cytochromes P450 Hasemann et al. 49

difficult. This region of the structure was aligned in a man- true both in terms of rms deviation and sequence iden-
ner that preserved the interaction of the conserved hydro- tity. The structures close to the heme include the B'-C
phobic residues with the hydrophobic core of the mol- turn, the central I helix, 36-1/31-4, the Cys-pocket,
ecule. In addition, the location of prolines and glycines at and the amino-terminal L helix. The rms deviations
the turns between -strands is expected to be conserved. between these regions are among the lowest for the three
molecules (mean 1.54 A), i.e. well below the rms for the
We see a wide array of differences in comparing our entire structures. Seven of the eight invariant residues and
results to previous eukaryotic P450 sequence alignments 12 of the 23 strictly conserved residues presented in Fig.
based on the structure of P 4 50 ca,,, alone. The alignment 5 are involved in heme binding. Generally, there is a con-
reported by Gotoh [29] is the closest to our results, servation of non-polar residues that establish a suitable
except for the placement of the insertion in the meander hydrophobic environment for the heme. The more
region, which misaligns the arginine/histidine/asparagine strictly conserved residues are involved in propionate
residue in the ERR-triad (position 498, Fig. 5). The coordination or in the formation of structural motifs.
study by Gotoh included the identification of substrate-
recognition sites (SRSs) based on the position of The Cys-pocket
camphor in the binding site of P 4 50 ,,a,. Our alignment The heme of P450s is covalently bound to the invariant
is slightly different in SRS-2 and SRS-6, so we would cysteine found in a -bulge region called the Cys-pocket
adjust these boundaries for the eukaryotic P450s. (Cys5 2 6, Fig. 5). The -bulge appears to serve the role
of enveloping the cysteine in a hydrophobic environ-
Our alignment bears several significant differences from ment. The Cys-pocket region of P450terp is shown in
the widely quoted works of Nelson and Strobel [30,31]. Fig. 6. Three residues besides the cysteine are very
Most notable is the completely different placement of the strictly conserved [only one substitution at each position
E and F helices, which places a large insertion between in a compilation of 192 sequences [3] (D Nelson, per-
helices D and E, rather than between F and G. Our sonal communication)]. Two of these are glycines, one of
structure comparisons indicate that this variability is which is in a position that allows the formation of the
almost certain to lie in the F-G loop region. Several 3-hairpin turn (Gly522, Fig. 5). The second glycine
other efforts that have relied entirely on automated pro- (Gly528) serves two roles, allowing for a sharp turn from
cedures [32-34], or a combination of automated pro- the Cys-pocket into the L helix and for proximity to the
cedures and manual fitting [35], as the basis for alignment heme. The third conserved residue is a phenylalanine
are quite different from ours in multiple respects. Because that is quite close to the sulfur-iron bond in all three
the combination of the three structures provides a far structures. A recent report of mutations at this position
superior scaffold on which to build a multiple alignment, indicates that it affects the efficiency of electron transfer,
it is not unexpected that predictions based on the struc- but does not play a critical role [36]. This side chain
ture of P4 5 0ca,,, alone would be different from ours. completes the hydrophobic enclosure of the proximal
heme, in combination with other side-chain and main-
Functional implications of the structures chain atoms of the Cys-pocket. It has been demonstrated
Heme binding that the redox potential of the heme iron in model
The most highly conserved regions among the three systems is sensitive to the hydrophobicity of the heme
structures are those involved in heme binding. This is environment [37]. It is possible that the hydrophobic

Fig. 6. Stereoview of the conserved


amino acids involved in heme binding,
shown for P450,e rp. The -bulge known
as the Cys-pocket is shown as a pink
tube. The invariant cysteine ligand to
the heme iron, and the invariant phenyl-
alanine which completes the hydro-
phobic shielding of the cysteine-iron
bond, are shown. Amino acids that are
involved in propionate coordination are
also shown. Two of these are found in
the C helix (pink coil)': His 10 is a con-
served hydrogen-bond donor (position
175, Fig. 5), and Argl 14 (position 179,
Fig. 5) is a conserved basic residue
which forms a salt bridge to the D-ring
propionate. The Cys-pocket amino acid
at position 375 (His375, position 524,
Fig. 5), and the main-chain nitrogen at
position 372 (position 521, Fig. 5) inter-
act with the D-ring and A-ring propi-
onates in all three structures. The
conserved arginine (Arg319, position 453, Fig. 5) in -strand 1-4 (pink arrow) forms a salt bridge with the A-ring propionate, and a
water (red sphere) hydrogen bonds with that propionate and the amino acid at position 72 of P450terp (Asn72, position 135, Fig. 5).
50 Structure 1995, Vol 3 No 1

Fig. 7. Stereoviews of the meander


region and the conserved ERR-triad.
(a) C trace showing the superposition
of the meander regions of P450,terp
(green), P4 50 cam (red) and P450 BM-3
(purple). For the purpose of this Figure,
the structures were superimposed by
optimizing the fit of Cs in the region
between the K' helix and the Cys-pocket
(positions 482-518, Fig. 5) as well as
the Cas of the conserved glutamate and
arginine side chains in the K helix (posi-
tions 439 and 442, Fig. 5). Side-chain
atoms of the conserved glutamate and
arginine in the K helix, and the con-
served arginine in the meander (position
498, Fig. 5) are also shown. (b) Detail of
the interactions that stabilize the recur-
ring structure of the meander regions,
shown for P450 terp. The Ca trace of the
meander (magenta tube) and a portion
of the K helix (magenta coil) are shown.
Salt-bridge interactions between the glu-
tamate (Glu306) and arginine (Arg309
and Arg362) side chains, and hydrogen
bonds between the arginine side chains
and meander carbonyl oxygens are
indicated by narrow lines. The C,s of
positions 353 and 357 are numbered.

envelopment of the heme is important in establishing the charge to neutralize the A-ring propionate. The corre-
redox potential of the heme iron. Another possibility is sponding position in the other two structures [an
that this residue serves a function similar to the invariant asparagine in P450terp, (Asn72 in Fig. 6) and a serine in
tryptophan adjacent to the disulfide bonds of immuno- P 4 50can] forms a hydrogen bond with the propionate
globulins, shielding the sulfur-iron bond (or sulfur-sulfur through a water molecule. In total, both heme propi-
bond in immunoglobulins) from solvent-borne reducing onate groups of each structure are coordinated by one
agents [38]. positively charged residue (arginine/lysine) and at least
two other conserved hydrogen-bond interactions, either
Propionate coordination directly or through water molecules.
The heme propionate side chains require polar and/or
charged residues to accommodate their polarity and neg- The meander and the ERR-triad
ative charge in the hydrophobic interior of the molecule. A conserved Glu-Arg-Arg triad is associated with the
Further, propionate coordination has been suggested to meander region of each of the three structures. The glu-
have an influence on the redox potential of the heme tamate and arginine from the K helix (Glu439 and
iron [39,40]. This is accomplished by four residues that Arg442, Fig. 5) are invariant in all P450 sequences,
are well conserved throughout the P450s (illustrated in whereas the arginine from the meander (Arg498, fig 5)
Fig. 6 for P450te r ). Two are at the amino-terminal end appears as either arginine, histidine or asparagine in all
of the C helix (islO, Argll4 in Fig. 6, positions 175 P450s. The name 'meander' arose from our initial im-
and 179 in Fig. 5), one in 31-4 (Arg319 in Fig. 6, posi- pressions of this region as a structureless wandering of
tion 453 in Fig. 5), and one in the Cys-pocket (His375 -20 amino acids between the K' helix and the Cys-
in Fig. 6, position 524 in Fig. 5). A hydrogen bond is also pocket. It is apparent in Fig. 7a, however, that the term
found consistently (based on distance and geometry con- meander is a misnomer. In each structure, the ERR-triad
siderations between hydrogen-bond donors and recipi- forms a set of salt-bridge interactions, positioning the
ents, here and throughout the manuscript) between the terminal arginine nitrogens (N) to form hydrogen bonds
main-chain nitrogen corresponding to position 372 of with a series of carbonyl oxygens in the meander
P450terp and the A-ring propionate (position 521 in Fig. (Fig. 7b). This set of salt-bridge interactions and hydro-
5). Interestingly, P4 50 BM-3 does not have an arginine or gen bonds results in a highly conserved three-dimen-
histidine at position 453 in 1-4. Instead, a lysine from sional structure in the three proteins, despite the lack of
the 1-5 region (Lys135, Fig. 5) provides the positive any typical secondary structures in the region.
Structure and function of cytochromes P450 Hasemann et al. 51

There is considerable evidence that the ERR-triad is in


some way linked to heme binding by the protein.
Mutation of the K-helix glutamate [41] or arginine
[42-44] or the meander arginine [45] in four separate
enzymes has led to the production of completely inactive
protein. In the two cases where it was tested, this inactiv-
ity was demonstrated to result from the production of
apoprotein [41,45]. The meander sequence is located just
amino-terminal to the Cys-pocket motif, and conse-
quently the conserved ERR/meander structure is closely
associated with the Cys-pocket. We propose that the
ERR-triad acts to establish a folding motif in the meander
that locks the Cys-pocket in position, assuring the stable
association of heme with the protein. As a result, disrup-
tion of the ERR-triad leads to the loss of heme binding,
and all P450 enzymatic activity. Further, the meander
region is located near the heme on the proximal face of
the enzyme. This region is the likely site of redox-partner
interaction, implying that the structure of the meander
might play some role in this protein-protein interaction.

Substrate binding
The substrate footprints for the three P450 structures are
shown in Fig. 8. The footprint is based on the crystallo-
graphically determined position of camphor in the sub-
strate-bound form of P 4 50can, [13], on preliminary
crystallographic data for substrate-bound forms of
P4 50 BM-3 and on the docking of a model of ot-terpineol
into the substrate-binding pocket of P450terp [22] as well
as preliminary results with a substrate-bound form
of P4 50terp. Because the substrates for P4 5 0BM-3 are
C 12 -C2 0 fatty acids [25,46], the footprint is larger than
for the smaller ring structures of camphor (C1,H 1 6 0)
and -terpineol (C1 0H 1 8 0).

In an analysis based solely on the structure of P4 50ca,,


Gotoh [29] proposed that the eukaryotic family-2
enzymes (e.g. CYP2B1) would adopt a tertiary structure
similar to P450an,. On the basis of the position of sub-
strate binding in P 4 50 cam, he also proposed that there
would be six locations in the primary sequence of all
P450s that would be potential substrate-recognition sites
(SRSs) [29]. He also demonstrated that among the
family-2 enzymes these SRS sequences are hypervariable
with respect to the remainder of the proteins. Our find-
ing that the overall folds of P450ter and P4 5 0 BM 3 are
similar to that of P4 50can, validates Gotoh's premise that
such SRSs can be predicted.

We can extend the premise to say that, in addition to the


sequence hypervariability of the SRSs, there is spatial
hypervariability in SRS-1 (B' helix), SRS-2 (carboxy- Fig. 8. Substrate 'footprints' for the three P450s. The Cs trace
terminal region of the F helix), and SRS-3 (amino-ter- of each molecule is shown in blue, with the atoms of sub-
minal region of the G helix). In contrast, SRS-4 (central strate-contact residues highlighted. The heme molecules are
I helix), SRS-5 (6-1/31-4), and SRS-6 (34-hairpin) also shown (in pink). The identity of contact residues is based
show only limited spatial variability. on the camphor-bound form of P450cam [13], on docking
studies [22] and preliminary analysis of an ac-terpineol-bound
form of P4 50 terp (CA Hasemann and J Deisenhofer, unpub-
The superposition of the B' helices (SRS-1) of all three lished data), and on preliminary analysis of arachidonate [23]
structures, including the extended chain regions at either and laurate (SS Boddupalli, RG Kurumbail and J Deisenhofer,
end of the helices, is shown in Fig. 9a. P 4 50terp is the unpublished data) bound forms of P4 5 0 BM3.
52 Structure 1995, Vol 3 No 1

Fig. 9. Comparison of the variable


regions of the three P450s which might
be involved in substrate recruitment and
binding. P450terp (green), P4 50 cam (red),
and P4 5 0 BM-3 (magenta) were superim-
posed as described for Table 3 and Fig.
4. A stereo pair of the C, trace is shown
for each molecule as superimposed. The
heme of P450terp is shown in order to
demonstrate the proximity of these
structures to the site of catalysis.
(a) B' helices and flanking polypeptide
segments. The size and spatial disposi-
tion of each helix is quite different, as
are the lengths of the polypeptide seg-
ments that surround them. (b) F and G
helices, and the F-G loop. The orienta-
tion of this view is the same as for part
(a). The G helices of P450terp and
4 50
P BM-3 clearly follow a different tra-
jectory from that of the G helix of
P450cam. This leaves them further from
the heme, out of the region which might
play a direct role in substrate binding.
Similarly, the shorter F helix and F-G
loop of P4 5 0 cam is much closer to the
heme than observed for P4 5 0 BM-3, form-
ing a tighter 'lid' over the substrate-
access channel. Note that the loop
between the F and G helices of P4 50 terp
was disordered in the crystal, and thus
was not modeled.

most different, as a result of the amino acid insertions at regions among all P450s, whereas both P450terp and
both ends of the B' helix relative to the other two P4 50 BM-3 typify the F-G regions of most of the eukary-
sequences. This has resulted in the P450 B' helix otic P450s (Fig. 5). As discussed above, the position of
moving away from the heme to the extent that no helical the F-G region of P450ca,,, is shifted relative to its
side chains are predicted to be involved in substrate con- sequence alignment with P450terp and P4 50 BM-3. This
tact. In contrast, in P4 50 BM-3 and P4 50 ca,1 helical results in the observed ability of the F-G loop of P450can
residues are involved in substrate binding. Although sub- to pack tightly against the underlying B' helix, with the F
strate-contact residues have been mapped to the B' helix and G helices at roughly equal heights above the heme.
itself [47,48], the highest concentration of contact In contrast, the large F-G loop of P4 50 BM-3 has caused
residues is found in the extended chain at either end of the G helix to adopt a conformation above the F helix
the helices (31-5 to B', 137-144, and the B'-C turn, with respect to the position of the heme. For the regions
162-167; Fig. 5). Several naturally occurring or designed of P 4 50terp that are crystallographically ordered, the F-G
mutations that alter substrate specificity can be mapped region resembles that of P4 50 BM_3, and we predict that
to the B'-C turn [49-54]. In several cases, these changes the eukaryotic enzymes will also adopt this topology.
have erroneously been mapped to the B' helix itself, a The F-G region has the highest thermal parameters for
subtle distinction, but one that merits clarification for the each of the three structures, with particularly extreme
design of future experiments. values for P450terp, in which the F-G loop is crystallo-
graphically disordered. The high mobility of this region
The F and G helices of all three structures are shown most probably allows it to act as a flexible lid over the
superimposed in Fig. 9b. Again, one observes a high substrate-binding pocket.
degree of spatial variability, attributable primarily to the
different lengths of the F-G loops of the three sequences. Several amino acids in the F helix have been identified as
P450Ca,, is at one extreme, with one of the shortest F-G substrate-contact residues in other enzymes [50,55,56].
Structure and function of cytochromes P450 Hasemann et al. 53

Efforts to relate their precise location to a position in the To summarize, several substrate-contact regions are the
crystal structures are clouded by the structural variability most variable substructures in the three P450s. Variability
in this region, as cited above. The location of the G is most pronounced for the B' and F-G regions of the
helix, above the F helix, removes any potential for G- structure, which together form the majority of the sub-
helix amino acids to interact with substrate in the active strate-access channel. The extended structure of the B'
sites of either P4 50 BM-3 or P4 50 terp. For the enzymes helix of P4 50terp and the F-G loop of both P450terp and
with large F-G regions (most P450s), the inclusion of the P4 5 0 BM-3 provide for a much more open or accessible
F-G loop and/or the amino-terminal G helix as an SRS binding pocket than that seen in P450can,. The extremely
is probably not valid. Thus, only P450s with short F-G low degree of similarity in the binding sites of the three
regions (several bacterial enzymes, including P 4 50 ca,,,,) are enzymes precludes the ab initio prediction of binding-site
expected to possess SRS-3. structures for other P450s. For those with an interest in
redesigning P450 specificities, therefore, two primary
The remaining three SRSs show more limited structural options remain. First, one can work with one of the
variability. In two of the cases this is probably related to three enzymes whose structures are now available, select-
their location in the core of the P450 fold, and reflects ing the enzyme with the binding site most similar to that
the necessity to limit their variability for structural rea- desired in terms of size and accessibility. As a second
sons. SRS-4 is located in the central I helix, and SRS-5 option, one can work with an as yet structurally unchar-
is located in the region containing 6-1 and 131-4. As acterized enzyme. The improved sequence alignment
discussed below, the central I helix has been proposed to and general understanding of the substrate-binding struc-
play a role in proton delivery for catalysis. The I helix is tures that arises from the combination of the three P450
also a member of the four-helix bundle that forms the structures presented here does allow for a reasonable
core of the o-domain. Because of these central roles in hypothesis as to the substrate-contact residues in another
P450 structure and function no insertions or deletions enzyme. Experiments that target these regions could
are observed in the I helices of any of the P450s. then be designed to manipulate the substrate specificity
Similarly, the central and carboxy-terminal I helices are of almost any P450.
among the most conserved regions of the three structures
(Table 3). Sequence variation in the I helix has, however, Catalysis
been shown, both by mutation of the conserved aspar- The hydroxylation of substrates by P450s can be regarded
tate/threonine pair [57,58] and other residues [45,59-61] as a two-stage process (reviewed in [64]). The first stage
to play a role in substrate specificity and/or reaction involves the activation (splitting) of molecular oxygen,
kinetics. The region containing 6-1 and 131-4 is also at with the liberated oxygen atom eventually joining two
the core of the P450 fold, and contains several residues solvent-derived protons to produce water. The second
shown to exert an influence on substrate specificity stage entails hydrogen abstraction from substrate by the
[50,53,54,62,63]. This region contains limited insertions iron-bound activated oxygen, producing a substrate radi-
or deletions (1-2 amino acids) as aligned in Fig. 5, and cal intermediate [65]. The substrate radical and iron-
P450,,,,,a is one amino acid shorter than either P450terp or bound hydroxy intermediates then rearrange to produce
P450BM-3 in 6-1. Despite this variability in insertions, the hydroxylated product, regenerating the Fe2 + heme
SRS-5 is among the most spatially conserved regions iron in the process. The only influence of protein struc-
(Table 3), probably reflecting its role in heme binding. ture at this stage is in stabilizing the substrate in a given
Finally, SRS-6 is located at the turn in 34, and includes orientation, assuring the correct position and stereo-
part of 4-1, 6-2, and part of 34-2. As aligned in chemistry of product formation [47].
Fig. 5, this 3-turn region exhibits modest variation in
length and composition. The superposition of the three The splitting of molecular oxygen is generally dependent
structures in the 34 region is also somewhat worse than on P450 architecture in two ways. First, the interaction
average (Table 3). with the redox-partner protein for the delivery of

Fig. 10. The central region of the I


helices of P4 5 0terp and P4 5 0BM-3 are
more similar to one another than to that
of P4 50,,cam. C traces of the central I
helices of P4 5 0,te (green), P4 5 0cam
450
(red) and P BM-3 (magenta) are shown
in stereo with the heme of P4 5 0 terp.
While all three helices have a disruption
in the normal helical hydrogen-bonding
pattern which results in a slightly
'untwisted' helix in this region, the
location of this distortion is different
in P450cam compared with P4 50terp and
P4 50 BM-3 For this figure the three
molecules were superimposed to minimize the deviation between C. positions in the central I helices only (positions 373-393, Fig. 5).
This was done to assure that no differences in distant regions of the molecule would affect the superposition of this region.
54 Structure 1995, Vol 3 No 1

Fig. 11. Features of the central I helices


of P45 0cam and P450terp which may be
involved in the delivery of catalytic pro-
tons for oxygen scission. Stereo pairs of
the I and F helices are shown as
magenta coils, with important side-
chain and main-chain atoms included.
The hemes are presented as ball-and-
stick models, with the heme iron repre-
sented by a green sphere, and the axial
ligand to the heme (either water or
hydroxide) shown as a red sphere.
Several other relevant waters are also
represented as red spheres. Hydrogen-
bond and salt-bridge interactions are
indicated with solid white lines.
(a) Atomic coordinates for the substrate-
free form of P450 cam were used for this
figure 12], so that the disposition of the
distal axial ligand (hydroxide or water)
could be shown. The disruption of nor-
mal helical hydrogen bonds is at the
'back' of the helix as viewed here. The
hydrogen bond between the invariant
threonine (Thr252) and the carbonyl
oxygen of Gly248 is shown, as is the
hydrogen bond from the carbonyl oxy-
gen of Gly249 to the water which has
intercalated into the 'back' of the helix.
The conserved acid side chain (Asp251)
is also shown, forming salt bridges to
Arg186 and Lys178, and hydrogen
bonding to Thr185. The water mol-
ecules adjacent to Arg186 and Lys178
are at the exterior of the molecule, and
represent the closest approach for bulk
solvent-derived protons to the central I
helix. (b) The I helix of P450terp, viewed
from the same perspective as for
P450cam in (a). In contrast to P450cam, the disruption of helical hydrogen bonds is at the 'front' of the helix. The hydrogen bond
between the invariant threonine (Thr271) and the carbonyl oxygen of Ala267 is shown, as are the hydrogen bonds from the carbonyl
oxygen of Thr266 and the main-chain nitrogen of Asp270 to the waters which have intercalated into the 'front' of the helix. Note also
that there is a hydrogen bond between the carbonyl oxygen of Ala267 and the axial ligand (water or hydroxide) to the heme iron. The
conserved acidic side chain (Asp270) is also shown, forming a salt bridge with Lys419, and hydrogen bonding to GIn1 85. As shown for
P4 50cam, the water molecules adjacent to Lys419 are at the exterior of the molecule, and again represent the nearest source of bulk
solvent protons for catalysis.

electrons is in some way modulated by a shift in the the futile expenditure of reducing equivalents into an
redox potential of the heme iron, linked to the presence unoccupied P450, and resulting in the production of
of substrate in the binding site. The series of P 4 50cam hydrogen peroxide and water, rather than oxygenated
structures has led to the theory that the coupling of spin- product. The structures of P450terp and P4 5 0 BM-3 are
state, potential, and substrate binding is achieved by the consistent with this P 4 50cam-,,,based theory. In their sub-
change in solvation of the substrate-binding pocket [21]. strate-free structures, both enzymes have well-hydrated
binding sites, low-spin irons, and presumably are at a low
Upon substrate binding, solvent is excluded from the redox potential. On the basis of docking studies with
binding site, and the distal axial ligand may be displaced. P 4 50terp and a model of oa-terpineol, the substrate-bound
In those cases where the ligand is not displaced, it is pre- enzyme is expected to experience complete desolvation
sumed to shift from a strong-field hydroxide ion to a of the active site [22]. The findings of preliminary model
weak-field water molecule. In both cases, the heme iron building based on data from arachidonate-bound
shifts from a low-spin to a high-spin configuration P4 5 0 BM3 crystals is also consistent with this correlation
because of the change in the nature (or presence) of the between solvation of the binding site and iron spin-state
sixth axial ligand. The redox potential of the iron is also (RG Kurumbail, SS Boddupalli and J Deisenhofer,
linked to the solvation state of the binding site, increasing unpublished data).
upon substrate binding (from -300 mV to -170 mV in
the case of P 4 50cam), presumably because the dehydrated The second influence of P450 structure on catalysis is in
porphyrin environment favors the Fe 2+ state. The coup- establishing an appropriate environment for the timely
ling of redox potential and solvation state of the distal delivery of reactants (electrons, oxygen and protons).
heme is in evolutionary terms advantageous, preventing The route which electrons take from the redox-partner
Structure and function of cytochromes P450 Hasemann et al. 55

protein to the P450 heme iron will remain unknown in structures, although the exact locations of the atoms in
the absence of an atomic structure of a complex between the motif have changed considerably.
these proteins. Because of its non-polar character, mol-
ecular oxygen can diffuse freely to the distal face of Fig. 11 shows the organization of the central I helices for
the heme molecule as it is needed. One reactant in the P4 50 can and P4 50terp (because of the high degree of
activation of oxygen remains, namely protons. similarity between P4 50terp and P4 5 0 BM-3 in this region,
only P450terp is presented). For P450cam , (Fig. 1 la) it has
Two solvent-derived protons join one atom of oxygen been proposed that the role of the I-helix disruption is to
to form water in the balanced P450 reaction. As men- form a binding pocket for dioxygen binding to the heme
tioned above, solvent must be excluded from the P450 iron [11,13]. Because of the shift in local conformation,
active site to avoid the futile production of hydrogen this pocket clearly does not exist in P4 5 0terp (or
peroxide from molecular oxygen. This means that pro- P4 50 BM_3) . The pocket is instead occluded by the car-
tons must pass through a protein intermediary to gain bonyl oxygen which is hydrogen bonded to both the
access to the iron-bound oxygen. Again, on the basis of water axial ligand and the conserved threonine (Fig.
the structure of P 4 50can,, models have been proposed lib). Thus the role of the conserved threonine is clearly
which implicate the conserved acidic amino acid and not to form a binding pocket for dioxygen in either
the adjacent threonine at the center of the I helix (posi- P4 5 0terp or P45 0 BM 3- Evidence even exists to suggest
tions 382 and 383, Fig. 5) as the amino acids that supply that the threonine is not critical to the formation of the
catalytic protons [20,66]. It has also been speculated that I-helix distortion of P4 50can,, in that the crystal structure
the role of the threonine in the center of the I helix is of the Thr-Ala mutant at this position [20] retains the
to cause a deformation of the helix which then acts as distortion in the helix.
a pocket for binding molecular oxygen [13]. The struc-
tures of P450terp and P4 5 0 BM_3 in this region call for A model for proton delivery in P 4 50Cam has evolved to
a re-examination of the general applicability of these include the delivery of a proton to iron-bound molecular
theories to all P450s. oxygen via the hydroxyl oxygen of the conserved threo-
nine [20,66,67]. The threonine side chain is proposed to
As can be seen in Fig. 10 (and Table 3), CR positions in adopt a new torsion angle, bringing the hydroxyl hydro-
the central region of the I helices of P450terp and gen into a position where it might interact with the
P4 5 0 BM_3 are quite superimposable, whereas the corre- reduced oxygen intermediate. The situation in P 4 50terp
sponding residues of P4 50 can, are not. In the first descrip- and P4 5 0 BM-3 is different, however, as shown in Fig. 1lb
tions of P 4 50anm by Poulos et al. [11], the existence of an for P4 50terp. In these cases, the atom closest to the axial
irregularity in the hydrogen-bonding pattern in the I ligand is the carbonyl oxygen that is hydrogen bonded
helix was noted immediately adjacent to the heme. The both to the conserved threonine and to the water axial
disruption of normal helical hydrogen bonding is also ligand (at a distance of 3.0 A for P450terp and 2.7 A for
evident in P450terp and P4 5 0 BM 3, though the position of P4 5 0 BM-3) . At no torsion angle is the threonine hydroxyl
the irregularity has changed. oxygen closer than 4.1 A to the axial ligand in either
P 4 50terp or P45 0 BM-3. The proximity of this carbonyl
For all three structures, the loss of two helical hydrogen oxygen to the axial ligand implies that it may play a role
bonds results in the formation of a 'groove' in the helix. in the diffusion of a proton to the activated oxygen. The
Solvent molecules have intercalated into the groove of all source of this proton in the case of P4 50terp or P 4 5 0 BM-3
three I helices, occupying some of the potential hydro- could be either the conserved threonine (as in P4 50can,)
gen-bond interactions lost upon disruption of the helix. or the ordered solvent found in the I-helix groove.
For P 450 can,, the groove is at the back of the helix, as
depicted in Figs 10 and 1la, with the maximum width of The function of the conserved acidic residue (glutamate
the groove in the helix occurring between C, positions or aspartate) adjacent to the conserved threonine in
380 and 384 (numbering as in Fig. 5). For both P450terp nearly all P450s remains elusive, despite the addition of
and P 4 5 0 BM_3 the groove is at the top and front of the the structures of P 4 50terp and P4 5 0 BM-3. Consistent with
helix, as depicted in Figs 10 and lb, and is widest the simple observation that it is highly conserved among
between C positions 378 and 382, one-half turn of P450s, several mutagenesis studies have demonstrated
helix before the groove in P450can,m. The different dispo- diminution in activity when the acid is replaced
sition of the grooves between P 4 50can, on the one hand [59,61,66,68]. Gerber and Sligar [66] have proposed a
and P 4 5 0terp and P4 5 0 BM-3 on the other causes a change distal-charge relay system, in which the aspartate of
in the relationship between the heme iron and the con- P4 50 cam, adopts two alternative rotamer conformations:
served acidic residue/threonine pair. It is noteworthy that its crystallographically observed position exposed to sol-
in all three structures a hydrogen bond is formed vent; and a position in which it might donate a proton
between the conserved threonine and the carbonyl oxy- directly to the conserved threonine, acting as a catalytic
gen of the amino acid four positions amino-terminal to base. The need for such a model for P 4 50can, arises from
the threonine. This motif of a distorted helix with water the lack of another donor that could supply solvent-
and the conserved threonine satisfying some of the lost derived protons to the threonine. In both P 4 50terp and
helical hydrogen bonds is conserved among the three P4 5 0 BM-3 this need is obviated by a series of hydrogen
56 Structure 1995, Vol 3 No 1

bonds through the water(s) found in the groove in the


helix, which might act as a conduit for catalytic protons
[22,23]. Although some solvent molecules are also asso-
ciated with the P450a,,, I helix, they do not have the
same access to bulk solvent as in P450terp and P4 5 0 BM 3,
because of the different locations of the helical distortion.
Furthermore, in all three structures, the acidic side chain
is involved in interactions that would be disrupted in
each cycle of proton delivery (hydrogen bonds and/or
salt bridges, Fig. 11). The energy consumed in such a
maneuver seems to preclude this as an attractive model
for proton delivery, especially in the cases of P4 50terp and
P 45 0 BM_3, where no such motion need be invoked.
Although we cannot rule out the distal-charge relay
mechanism (particularly for P4 50 a,,, where there is a
need for a proton conduit), we propose that the acidic
side chain primarily plays a structural role via the inter-
actions noted above. It may also play a role in the
electrostatics of the enzyme, as discussed below.

In total, these findings offer an interpretation of a basic


paradox in the P450 literature. Mutations of the con-
served threonine in several enzymes have produced a
variety of results. Replacement with serine generally
results in the most subtle change [43,58,59,67,69]. The
effect of other amino acid substitutions ranges from the
virtually complete uncoupling of some enzymes to the
production of enzymes with improved catalysis
[43,58,67,69]. In addition, some naturally occurring
P450s activate oxygen and catalyze hydroxylation reac-
tions despite the absence of the conserved threonine or a
serine [CYP3A1 (pcnl), CYP56 (Dit2) and CYP7
(Choll7a) [3]]. On the basis of the P450canm paradigm,
the conserved side-chain hydroxyl plays a critical role in
efficient catalysis, presumably because of its role in pro-
ton delivery. Should the arrangement found in P4 50terp
and P4 50 BM-3 prove to be generally applicable, however,
the threonine would be less necessary than previously
thought. Thus enzymes with no threonine or serine
(either mutant or natural) might function quite well by
substituting protons derived from the ordered solvent
molecules in the I-helix distortion. The proximity of the
carbonyl oxygen noted above might assist in the diffusion
of this proton from the groove in the helix to the acti-
vated oxygen. Thus a spectrum of possibilities for proton
delivery might exist. At one end of the spectrum, the
threonine side chain of P4 50 a,,m plays an obligatory role
in delivering protons, possibly via the distal-charge relay

Fig. 12. Electrostatic potential at the molecular surfaces of (a)


P4 50terp, (b) P4 50am and (c) P4 5 0 BM-.3 The consistent positively
charged (blue) patch near the center of the face of each molecule
is located directly above the heme, and is a good candidate for
the docking site between a P450 and its electron-donating redox
partner. Molecular surfaces were color-coded by an interpolated
value of the electrostatic potential at a point as calculated in the
program GRASP [81]. Surface points are colored in a spectrum
from blue to red corresponding to positive and negative potential
respectively, with neutral points colored white. The deepest
shades of blue and red correspond to potentials of >+6.0 kcal
and >-6.0 kcal respectively
Structure and function of cytochromes P450 Hasemann et al. 57

involving the conserved acidic residue. In the center of


this spectrum are P450terp and P4 50BM_3, for which the
role of the threonine is more nebulous. Solvent-derived
protons can be directly withdrawn from the hydrogen-
bond network in the I-helix groove, and the shortest
route of diffusion to the heme iron passes by the carbonyl
oxygen, and not the threonine side chain. At the other
end of the spectrum are those enzymes with no threo-
nine at all, where a proton is delivered in the complete
absence of a hydroxy side chain. Perhaps a unifying
theory would state that the conserved carbonyl oxygen/
threonine hydrogen bond acts as a gate for the regulated
transfer (or diffusion) of a proton to the axial ligand. The
proton might pass through this gate via the carbonyl or
hydroxy oxygen, whichever is closer to the axial ligand.
Rules for predicting which conformation the I helix
of a given P450 will adopt, or whether only two such
conformations exist, remain to be determined.

Redox-partner interactions
A considerable amount of experimental evidence sug- Fig. 13. Cartoon representation of the 'molecular dipole' for
gests that P450s interact with their electron donor at the P4 50BM-3 A C, trace is shown, with a plane cutting through the
proximal face of the molecule. Manipulation of ionic mid-region of the molecule. The plane is colored by an interpo-
lated value for the electrostatic field on a grid at the surface of
strength shows that a complementary charge interaction the plane. The charge separation that leads to the molecular
is involved in redox-partner docking [70-72]. Mutation dipole (yellow arrow) is evident. The proximal (redox-partner
studies indicate that the P450 provides positively charged docking) and distal (substrate access) faces of the protein are
residues [44,73-76], whereas the electron donor has con- indicated.
served negative charges [77-80] which are critical for
proper interaction of the partners leading to electron positions in space are found in the J' insertion (Lys425,
transfer. On the basis of these findings, we analyzed the Lys428 and Lys431).
electrostatic properties of the three models [81]. Fig. 12
shows the electrostatic potential at the proximal surface Differences in charge distribution and topology of the
of each of the P450s. It is clear that a common region proximal face associated with the J' and meander
of positive charge is centered over the Cys-pocket of sequence insertions of P4 5 0 BM-3 might be invoked to
each molecule (the closest approach to the heme iron explain the difference in redox-partner association
in each case). The distribution of the amino acids that between the class I and class II enzymes in this study. It
contribute to this positive charge differs between the two would be even more satisfying if these insertions were
class I enzymes (P450terp and P450can) and the class II found in all P450s which accept electrons from an
enzyme P45 0BM-3. FAD/FMN reductase, and would then explain the
redox-partner association for all the P450s. Unfortu-
In the class I enzymes, part of this positive field is attri- nately, the existence of a J' insertion is not solely charac-
butable to positively charged amino acids that interact teristic of class II enzymes, but rather is common to all
with the negative charge of the heme propionates (one or the eukaryotic enzymes, both microsomal and mitochon-
more of positions 175, 179 and 524; Fig. 5). Both drial (Fig. 5 and [3]). The mitochondrial enzymes accept
enzymes have a positive charge at the same position in electrons from the iron-sulfur protein adrenodoxin,
the L helix (Arg/Lys533). The remaining charges occupy which is similar in amino acid sequence to the bacterial
similar regions in space, but not the same positions in iron-sulfur redoxins, although these redoxins will not
the sequence. These include a B-helix residue (Argl22 substitute for one another in electron transfer. The
in P450cam, Lys126 in P450 rp) and a meander residue microsomal enzymes interact with an FAD/FMN-con-
(Lys500 in P450cam, Arg502 in P450terp). Although the taining reductase which is unrelated to the iron-sulfur
distribution of charge observed in Fig. 12 looks similar proteins. Thus, the changes in charge distribution and
for P4 5 0 BM-3 and the two class I enzymes, several amino topology associated with the J' and meander insertions
acids are in different positions. Analogous positive cannot be the common characteristic of enzymes that
charges coordinate the propionates (positions His179 and interact with an FAD/FMN reductase.
Arg524), and a meander residue lies in a position analo-
gous to the class I meander charge (Lys517). In contrast While analyzing the charge character of the proximal
to the class I enzymes, however, the positive charges faces of the enzymes, we discovered a common asymmet-
in the P4 50 BM-3 B helix face away from this region, ric distribution of charge in all three enzymes. This
and the amino-terminal L helix contains no positive charge distribution leads to the formation of a 'molecular
charges. Instead, the charges that occupy the analogous dipole' which has probable consequences for the function
58 Structure 1995, Vol 3 No 1

of P450s. Fig. 13 displays this dipole for P4 5 0 BM-3' structural identity with -2.0 A rms deviation in their
though the qualitative features of the P4 5 0 BM-3 charge cores. There should also be some entirely unpredictable
distribution are the same in all three enzymes. We pro- variety in their substrate-binding regions and amino ter-
pose that this dipole might act in two ways to promote mini, as well as in their various sequence insertions. The
P450 catalysis. First, this charge distribution would accel- membrane-bound enzymes presumably have an addi-
erate the diffusion-limited interaction of the P450 and its tional membrane anchor at their amino terminus, but we
redox partner. The.electrostatic field would steer the neg- believe that the remainder of the P450 fold shown here
atively charged region of the electron donor away from will be conserved, beginning with the A helix. Several
the negatively charged distal face, and towards the posi- membrane-bound P450s have been altered at their amino
tively charged proximal face of the P450. Because of the termini and have been found to remain membrane-asso-
solvent shielding effect, the dipole is probably not observ- ciated despite expression of protein lacking the amino-
able to the incoming protein at a great distance, but once terminal anchor [84,85]. The sequence alignment based
in close proximity the dipole would clearly assist in the on our structural comparison suggests that there are no
correct alignment of the two molecules. Once oriented additional structural motifs found in the membrane-
in this way, the negatively charged region of the redox bound enzymes that might act as an additional conven-
partner would be drawn into close contact by the electro- tional membrane anchor. We suggest that an as-yet
static field of the P450 to achieve the correct fine undetermined region of the membrane-bound enzymes
positioning for electron transfer. must contain a hydrophobic patch, superimposed on
the common P450 fold, which might aid in stabilizing
The dipole might assist P450 function at a second level as membrane association.
the orientation of the dipole is consistent with the elec-
trochemical flow of the reactants involved in the activa- Our results imply that the region that is of greatest inter-
tion of oxygen. Thus, both the proximal-to-distal flow of est for the structure-function relationships of the P450s,
electrons from the redox-partner protein and the distal- namely the substrate-binding region, is extremely struc-
to-proximal flow of protons from solvent will be posi- turally diverse. We believe that because of this diversity,
tively influenced by the electrostatic field of the P450. sequence alignments alone will not be reliable predictors
Electrostatic guidance of substrates has been proposed to of substrate-contact residues. This structural diversity
be extremely important for both acetylcholinesterase [82] makes teleological sense from the point of view of gener-
and superoxide dismutase [83]. The hypothesis that these ating a variety of binding specificities. It is however
findings might be extended to the smaller charged parti- inconvenient for those who would like to redesign P450
cles (electrons and protons) being recruited to the P450 specificity for the reasons speculated on at the beginning
active site is testable. Experiments to alter the surface of this article. The concept of SRS regions remains valid,
charge distribution of the distal face of the enzyme with- however, and experiments targeted to these regions
out affecting the folding and stability of the enzyme might still prove valuable.
should be possible. A limited form of such an experiment
has already been performed. The position and charge of Finally, we have discovered several unexpected features of
the conserved acidic residue at the center of the I helix P450 structure. These include the ERR-triad, which
are consistent with the dipole moment of the enzyme. encodes a motif important for the structure of the mean-
Several enzymes have been mutated to remove this acidic der region and the 'folding in' of the heme, or in stabiliz-
residue, resulting in decreased activity of the enzymes ing the folded holoenzyme structure. In addition, the
(see above). It will be instructive to re-evaluate those demonstration of a common molecular dipole provides a
results with an eye towards the effect on the molecular new target for P450 structure and function studies.
dipole of the enzymes studied. Despite the availability of three crystal structures, the
exact role of the conserved acidic residue/threonine pair
Summary of the overall impact of the structures in the I helix, and the associated irregularity in helical
One of the major implications of this study is quite structure, remains a question yet to be answered.
simple. Eukaryotic P450s undoubtedly look a lot like the
bacterial enzymes. This can be stated with confidence
because of the similarity of the sequence of P4 5 0 BM-3 to Biological implications
the microsomal sequences, and the concomitant simi- Cytochromes P450 catalyze monooxygenation
larity of the structure of P4 5 0 BM-3 to the structures of reactions for a variety of medically important
P 4 50erp and P450,,,a. The structure of P450ca,,. might substrates including steroid hormones, prosta-
be thought of as the 'minimal' P450, in that its sequence glandins, procarcinogens, and a variety of phar-
is among the shortest, and the structure consequently has macological compounds. P450s comprise two
very few extra loop regions relative to the other P450s. classes: the bacterial (soluble)/mitochondrial
The remaining P450s will fold the same way as the three (membrane-bound) enzymes (class I), and the
enzymes presented here, with variations on the theme membrane-bound eukaryotic microsomal enzymes
in loop regions. This is not to say that we believe that (class II). Because the majority of the medically
the atomic structures of new P450s are predictable. Quite relevant enzymes are membrane-bound class I or
the contrary, they are expected to have a similarly poor class II enzymes, efforts directed towards the
Structure and function of cytochromes P450 Hasemann et al. 59

purification and crystallization of these molecules between the resulting rms deviations was only on the order of
have not yet been successful. +0.1 A, suggesting that this choice was, in any case, not parti-
cularly critical. The sets of Ca pairs used to align the structures
and calculate the rms deviations presented in Table 2 are:
P4 5 0 BM-3
from Bacillus megaterium is however an
P450tcr/P450ca,, (1-5/14-18, 12-16/24-28, 30-33/44-47,
exception, in that it is a soluble class II enzyme. 37-44/50-57, 49-73/60-84, 76-80/85-89, 100-118/98-116,
Our comparison of the crystal structures of 126-145/124-143, 148-172/145-169, 176-185/170-179,
P450cam, P450terp (isolated from separate Pseudo- 186-190/179-183, 216-222/201-207, 238-247/219-228,
monas species, having catalytic activity for cam- 250-313/231-294, 314-404/294-384, 405-410/387-392,
phor and ac-terpineol respectively), and P4 5 0 BM-3 411-426/392-407); P4 5 0BM-3/P 4 50 c,,, (35-43/49-57,
reveals that their overall folds are quite similar 47-64/60-77, 65-69/79-83, 99-104/111-116, 111-131/
despite their low sequence identity. The fact that 123-143, 138-143/147-152, 144-158/154-168, 173-181/
P4 5 0 BM-3 is a class II enzyme whereas P450,a m and 173-181, 245-249/229-233, 260-293/244-277, 313-332/
P450terp are class I enzymes allows for a direct 280-299, 333-342/299-308, 343-368/308-333, 370-378/
comparison of the structure-function relationships 334-342, 390-425/347-382, 432-435/390-393, 438-447/
396-405, 450-453/411-414); P450,ep/P 45 0 BM-_3 (29-33/
for the two P450 classes.
31-35, 38-46/37-45, 49-57/47-55, 69-72/66-69, 99-105/
83-89, 110-147/96-133, 150-155/138-143, 157-174/
Based on knowledge from this structural compari- 144-161, 184-189/177-182, 215-231/208-224, 232-245/
son, we have made a more confident alignment of 227-240, 252-297/249-294, 298-329/312-343, 330-353/
the remaining class I and class II P450 sequences 345-368, 354-363/370-379, 367-402/390-425, 408-412/
than was previously possible. This improved align- 432-436, 414-428/437-451).
ment should assist in the interpretation of struc-
ture-function relationships of the other biologi- Given the superposition of the three models, the structure-
cally important P450s. The conserved regions of based sequence alignment of Fig. 3 was prepared. The results
the enzymes are found in the core of the protein, of Isq_improve include a pairwise listing of the positions used
in the calculation of the transformation, and these positions
probably representing the conservation of regions
were aligned in Fig. 3. A visual inspection was made for
important to correct protein folding and binding regions that were not included in the transformation (the
of the heme cofactor. Unfortunately, the substrate- loops), and structurally analogous (but spatially separated)
recognition regions of the proteins are the most regions were aligned. Finally, for the two regions of extreme
structurally divergent, involving large-scale shifts structural variability (amino terminus to the start of the A helix,
of the B', F and G helices. This casts significant B' helix), a sequence alignment that was consistent with the
doubt on our ability to precisely predict the set of structures was performed.
amino acids that determines substrate specificity
for other P450s whose three-dimensional structures Rms-deviation calculations
have not been determined. However, it should now Given the three-way superposition generated above, and the
structure-based alignment of the sequences, a deviation
be easier to achieve the goals of designing specific
between aligned positions could be calculated. Briefly, a pro-
inhibitors or agonists of medically relevant P450s, gram was written which calculated deviation statistics (defined
or redesigning the specificity of potentially useful below) for each position in the structural alignment that had
enzymes for industrial oxygenation chemistry. three atoms to compare (i.e. no calculations were done at posi-
tions that include a gap). The overall number reported in Table
3 includes all such positions, whereas the regional statistics
were calculated for subsets of the alignment.
Materials and methods
Structure superpositions and structural alignment The three-way rms reported in Table 3 was calculated as:
The structures were superimposed using the lsqexplicit and
Isqimprove routines in the program O [86]. The Isqexplicit E(AM[AAB+ABC+AAC]/3N)
function simply superimposes two structures based on the least- where M is the set of N positions (A, B and C) compared, and
squares fit of two atom lists. Typically, a stretch of analogous Aab=[(xa-xb) 2 +(y,-yb) 2 +(za-zb)2 ]. Individual terms of this
Ca positions in the I helix was supplied. The Isq_improve rou- summation were used to color code the Ce, positions of Fig. 4.
tine improves a starting transformation by iteratively seeking to
maximize the number of atom pairs included in the least- The individual rms values reported in Table 3 (i.e. Cam rms,
squares calculation (below a threshold deviation), while mini- Terp rms and BM-3 rms) each represent deviation of the indi-
mizing the rms deviation between those atoms. The exact vidual structures from a mean structure, and are calculated as:
algorithm has not been published. We found that the result was
quite insensitive to the seed transformation, given that it was [I,M(AAI1)/N
reasonably close to correct. Because the procedure only works where M is the set of N positions (x, y and z), and
in a pairwise manner, the potential exists for bias in the result AAml=[(X-Xm) 2 +(y-y )2 +(Z-zm) 2 ], with (x,),y,,z) being the
based on the choice of structure pairs. We tried all pairwise mean coordinate for that position in the three structures.
superpositions, and always found that the structure of P450ter,
was the 'middle' structure. Therefore, the superposition pre- The regional rms deviations were partitioned into four groups
sented in Table 3 used the CO, positions of P4 50tep as the target based on their statistical distribution. The mean () and
structure, with the C, positions of both P4 5 0,,,,, and P4 5 0 BM3 standard deviation (). were calculated for the distribution of
superimposed onto it. In any combination, the variance individual regional rms scores for the regions which are
60 Structure 1995, Vol 3 No 1

represented in all three structures (i.e. not A' and K') and charged whereas solvent-exposed histidines would be neutral.
excluding the extreme outliers (A, B' and G). Thus 31 terms This ApK, was used as a guide, with only histidine nitrogens at
contributed to a mean rms = 2 . 2 1 with a standard deviation the positive extreme of the ApK a range being charged.
r=0.69. The four basic groups reported in the text and color
coded in Table 3 are then: rms<1.52 (-r) [dark blue], 1.52 Because the net charge on each molecule is not zero, the 'mol-
(jI--c)<rms<2.21(.) [cyan], 2.21 ()<rms<2.9 (p+cr) [green], ecular dipole' is not a pure dipole. The actual procedure is to
rms>2.9 (+cr) [red]. place a monopole equal to the excess charge at the charge-
weighted average position of that excess charge. A dipole is
Multiple sequence alignment then calculated for the magnitude of the balanced charge mul-
Several efforts were made to accomplish an automated multiple tiplied by the distance between the charge-weighted average
alignment. The program PILEUP in the GCG package [57] position of the positive and negative charges. This is then
was used, but, invariably, obvious problems could be identi- referred to as the 'molecular dipole'. The dipole vector shown
fied, despite the judicious application of various combinations here has its tail midway between the charge-weighted average
of input parameters. We also attempted multiple alignments positions of the positive and negative charges, and parallel to
based on the sequence profile and three-dimensional profile the vector between them.
methods of Eisenberg and colleagues [87], as well as the multi-
variate combination of profiles [88]. The profile methods also Acknowledgemtents: We thank Drs Robert Diamond and Mike
failed to provide reasonable alignments in the amino-terminal Hartshorn for discussions on the superposition of multiple struc-
half of the protein. We finally resorted to 'hand-fitting' the tures. This work was supported by the Howard Hughes Medical
sequences in the program LINEUP, also from the GCG Institute D, CAH and RGK) and by a grant from the National
package. The starting set for the multiple alignment included Institutes of Health (GM43479; SSB andJAP).
the sequences presented in Fig. 5, as well as the following;
P4 50 pinF1 (CYPI103, Agrobacterium tunmefaciens), P450 c (CYPIAl,
rat), P450 a (CYP2AI, rat), P4 5 0 LM2 (CYP2B4, rabbit), References
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