Oxidative Phosphorylation

Pratt and Cornely, Chapter 15

Goal: ATP Synthesis

Overview• Redox reactions• Electron transport chain• Proton gradient

• ATP synthesis• Shuttles

Analogy: How does burning coal put flour in the grocery store?

Standard Reduction Potential

Half Reactions

• Reduction potential written in terms of a reduction half reaction

• Aox Ared

• Example:NADH + FMN NAD+ + FMNH2

Redox reactions: electricity

• 2 e- transfer• Calculate DG by

reduction potential

• NADH: Eo’ = -.32• FMN: Eo’=-.30• DGo’ = -nFDEo’ = -2(96485)(0.02)= -3.9 kJ/mol

N

R

H2N

O H H

NR

N

N

NH

R

R

O

O

H+

NADH FMN

N

R

H2N

O

NR

HN

NH

NH

R

R

O

O

NAD+ FMNH2

Numerous Redox Substrates• O2: high “reduction potential”• Substrates– Organic cofactors– Metals (iron/sulfur clusters)– cytochromes

Coenzyme Q: Mobile Carrier

• FADH2 is a one e- donator

• Many reactions, including metals

• Ubiquinone is a key intermediate

• Can diffuse through nonpolar regions easily

Oxygen: the final electron acceptor

• Water is produced—has very low reactivity, very stable

• Superoxide, peroxide as toxic intermediates

• Overall reactionNADH + H+ + ½ O2 NAD+ + H2O

Flow Through Complexes

Compartmentalization

Protonmotive Force

• NADH + H+ + ½ O2 NAD+ + H2O + 10 H+ pumped

• succinate + ½ O2 fumarate + H2O + 6 H+ pumped

Complex I

• NADH Q through– FMN– Iron-sulfur clusters

• “Q pool”• 4 protons pumped– Proton wire

Complex II

• Non-NADH sources– Complex II (citric acid

cycle)– Fatty acid oxidation– Glycerol-3-phosphate

shuttle (glycolysis)

• Bypasses Complex I – Loss of 4 protons

pumped

Complex III

• QH2 cytochromes• 4 protons pumped• Through Q cycle• Problem 10: An iron-sulfur

protein in Complex III donates an electron to cytochrome c. Use the half reactions below to calculate the standard free energy change. How can you account for the fact that this process is spontaneous in the cell?

FeS (ox) + e- FeS (red) Eo’ = 0.280 VCyt c (Fe3+

) + e- cyt c (Fe2+) Eo’ = 0.215 V

Complex IV• Cytochromes O2

• Stoichiometry of half of an oxygen atom

NADH into Matrix

• NADH of glycolysis must get “into” matrix

• Not direct• Needs either

– malate-aspartate shuttle (liver)

– Glycerol-3-phosphate shuttle (muscle)• Costs 1 ATP worth of

proton gradients, but allows for transport against NADH gradient

Glycerol-3-phosphate Shuttle

• Glycerol phosphate shuttle (1.5 ATP/NADH)

• Produces QH2

• Operational in some tissues/circumstances

Overall

• Chemiosmosis• 10 protons shuttled

from matrix to intermembrane space

• Makes pH gradient and ion gradient

Problem 39

• How did these key experiments support the chemiosmotic theory of Peter Mitchell?– The pH of the intermembrane space is lower than

the pH of the mitochondrial matrix.– Oxidative phosphorylation does not occur in

mitochondrial preparations to which detergents have been added.

– Lipid-soluble compounds inhibit oxidative phosphorylation while allowing electron transport to continue.

Proton Gradient

• Gradient driven by concentration difference + charge difference– Assume DpH 0.5 and 170mV membrane potential

• Free energy of ATP hydrolysis = -48 kJ/mole• How many protons needed to fuel ATP

formation? Minimum of 3

Protonmotive Force• Flow of electrons is

useless if not coupled to a useful process– Battery connected

to wire• Proton gradient

across mitochondrial membrane

Using the Gradient

• Coupled to ATP synthesis• Uncouplers used to show

link of oxygen uptake and ATP synthesis

Complex V: ATP Synthase

• Molecular motor• Rotor: c, g, e– Proton channel

Proton Channel• Protons enters channel

between rotor and stator

• Rotor rotates to release strain by allowing proton to enter matrix

• 8- 10 protons = full rotation– Species dependent

• “Stalk” (g) moves inside the“knob”—hexameric ATP synthase

• Knob held stationary by “b”

Hexameric Knob

Binding-Change Mechanism• Stalk causes ATP synthase to

have three different conformations: open, loose, tight

• In “tight” conformation, energy has been used to cause an energy conformation that favors ATP formation

Remember Analogy

• Fuelelectricitywater pumped uphillflows down to grind flour

• But we don’t have bread until flour is transported to where it needs to go!

• Compartmentalization: ATP is in matrix, but must get to the rest of the cell

Active Transport of ATP

• ATP must go out, ADP and Pi must go in• Together, use about 1 proton of protonmotive

force

Energy Accounting• ATP costs 2.7 protons– 8 protons produces 3 ATP

• NADH pumps 10 protons when 2 e- reduce ½ O2

– 4 protons in Complex I, 4 protons in Complex III, and 2 protons in Complex IV

• P/O ratio--# of phosphorylation per oxygen atom– 10H+/NADH (1 ATP/2.7 H+) = 3.7 ATP/NADH– 6H+/QH2 (1 ATP/2.7 H+) = 2.3 ATP/QH2

• In vivo, P/O ratio closer to 2.5 and 1.5 due to other proton “leaking”– i.e. importing phosphate

Uncouplers

• “Uncouple” protonmotive force from ATP synthase– DNP pKa / solubility

perfectly suitable

• Other respiration poisons– Cyanide—binds Complex

IV in place of oxygen

Net ATP Harvest from Glucose

• Glycolysis = 2 ATP– Plus 3 or 5 ATP from

NADH– What leads to

difference in this case?

• Pyruvate DH = 5 ATP• Citric Acid Cycle = 20

ATP• Total: 30-32

ATP/glucose

Problem 47

• A culture of yeast grown under anaerobic conditions is exposed to oxygen, resulting in dramatic decrease in glucose consumption. This is called the Pasteur effect. Explain.

• The [NADH]/[NAD+] and [ATP]/A[ADP] ratios also change when an anaerobic culture is exposed to oxygen. Explain how the ratios change and what effect this has on glycolysis and the citric acid cycle in yeast.