MCDB 110

Prof. Rothman

http://www.chemistry.wustl.edu/~courses/genchem/LabTutorials/Cytochromes/images/Proteins.jpg

Lecture 15 Oxidative
Phosphorylation

Electron Transport and ATP Production

ATP synthesis requires transport of electrons
through the chain and vice-versa
Electron transport and ATP production are coupled:

If no ADP present or ATP synthesis blocked no

electron transport, no reduction of O2

Respiration rate and ATP requirements are tightly

regulated: respiratory control

Stoichiometry of Coupling
P/O ratio: number of ATP/2 e- through
the ET chain (i.e., per O reduced)
Determined experimentally by measuring
O2 consumption and ATP production
Depends on substrate:
Substrate
P/O
NADH
2.5
Succinate (FADH2)
1.5

The Chemiosmotic Hypothesis

(Peter Mitchell, 1961; 1978 Nobel Prize)

An electrochemical gradient of H+ generated

during ET provides the energy for ATP synthesis

Redox energy from ET

transport of H+
from matrix to IMS

Electrochemical potential (pH & charge gradient)

is harvested to make ATP

Coupling of ET to ATP Synthesis Explained

Ac-S-CoA

H+ gradient built up until

G of H+ transport
= G of ET

Matrix

TCA

ET then stops unless

gradient is dissipated by
H+ passing through ATP
synthase

NAD+

NADH
FADH2

FAD

H+

TCA, ET, and ATP

synthesis are all coupled
and require O2

ETS

H+

H+

ATP

ADP
+ Pi

H 2O
O2

ATP
IMS
+ synthase

H
IMS

Proton-Motive Force
Energy to drive ATP synthesis is combination of
pH and charge difference across membrane:

pmf = membrane potential + pH gradient

pmf =

2.3RT
F

pH

pH = pH (outside) pH (inside) = -1.4 pH

= + 0.14 V.
pmf = 0.14 V. + 0.084 V = 0.224 V
Charge potential is predominant force

Energy in pH Gradient
Converting pmf to Go (pmf = E):
Go = -nFEo = -22 kJ / mol
Compare: ADP + Pi ATP Go = +31 kJ / mol

2 H+ per ATP minimum

Evidence for Chemiosmotic Theory

1. IM impermeable to H+s and other ions
2. ET causes H+s to be transported
Start: mitochondria,
excess ADP, succinate,
no O2

3. Elimination of pH gradient by uncouplers

blocks ATP synthesis

Uncoupling of ET and phosphorylation

Uncouplers cause ET in absence of ATP synthesis:
IM

DNP
Outside
(IMS)

Inside
(matrix)

oligomycin inhibits ATP synthase: no ET

uncouplers (hydrophobic weak acids, such as
DNP): shuttle H+s across IM

Evidence for Chemiosmotic Theory (cont.)

4. Artificial electrochem. gradient ATP synthesis

Load up mitochondria with K+ at high pH

Lower pH (pH gradient), no K+ (charge gradient)
ATP synthesized without ET!

ATP Synthase
Proton flux through ATP synthase
drives ATP synthesis
ATP synthase:
Large; lollipops in electron
microscope
Two separable components:
F1: faces matrix;
synthesizes ATP
F0: spans IM; H+ channel

Can run in reverse

IMS

Matrix

Mechanism of ATP Synthase

Pi reacts directly with ADP in one step
Bound ATP made from ADP + Pi w/o pmf!
Binding and conformational change makes ATP
synthesis favorable
pmf produces release of bound ATP
Rotational catalysis: worlds smallest motor

Mitochondrial Transport and Shuttle Systems

Compartmentation requires transport
across IM
Reducing equivalents (e-) from glycolysis
(NADH) move from cytosol to matrix

NADH cannot be transported across

IM
Strategy shuttling systems for electrons

Malate shuttle (e.g., liver, heart)

NAD+

Net: NADH (cyto) NADH (mito)

(no ATP cost)

Glycerol-3-phosphate shuttle (e.g., brain)

Net Cost 1 ATP: NADH (cytosol) FADH2 (mito)

(explains 30-32 ATP for glucose CO2)

Transport of ATP and Phosphate

ATP/ADP shuttle:
Antiporter: ADPcytosolATPmatrix
driven by ; ATP4- and ADP3- exchanged
Phosphate shuttle:
Symporter of phosphate and H+
driven by pH

Natural Uncoupler in Heat Generation

Newborn and hibernating animals

Brown adipose tissue: rich in mitochondria;

mitochondria contain thermogenin
Allows H+ to flow across IM into matrix
without ATP synthesis
Leakage produces heat
Skunk cabbage: heat releases odor

MCDB 110

Prof. Rothman

http://upload.wikimedia.org/wikipedia/commons/a/a9/Sunlight_Th
rough_Leaves.jpg

Lecture 16 Photosynthesis

Photosynthesis sustains life on earth

Energy biosphere
Fixation of CO2
Source of O2

Carbon Cycle of the Ecosystem

Respiration

ATP

Photosynthesis

6 H2O
+ 6 CO2

Mitochondria

hv
Chloroplasts

C6H12O6
+ 6 O2

Photosynthesis: two sets of reactions

Light Reactions
light energy + H2O
chemical energy (NADPH + ATP) + O2

Dark Reactions (also occur only in the light!)

redox energy + ATP + CO2 hexose sugars

Photosynthesis Occurs in Chloroplasts (Euks.)

Similar to mitochondria
Triple membrane system:
Thylakoid: light reactions
Stroma: dark reactions

Pigments Collect Light Energy

Chlorophyll is the primary light collector
Hydrophobic side chain anchors in thylakoid
membrane
Absorbs some of the wavelengths of sunlight

Accessory pigments absorb complementary

wavelengths of light

Chlorophylls

Chlorophyll

Bacteriochlorophyll

Accessory Pigments: the Glory of Autumn

Absorption Spectra of Major

Photosynthetic Pigments