ELECTRON TRANSPORT

Thermodynamics

· NADH oxidation is highly exergonic.

NAD⁺ + H⁺ + 2e ⇌ NADH E₁°’ = -0.315 V (1)

0.5 O₂ + 2H⁺ + 2e ⇌ H₂O E₂°’ = 0.815 V (2)

(2) – (1)

0.5 O₂ + NADH + H⁺ ⇌ H₂O + NAD⁺

DE°’ = E₂°’ - E₁°’ = 1.13 V

DG°’ = -nFDE°’ = -2 ´ 96494 J V^-¹ mol^-¹ ´ 1.13 V = -218 kJ mol^-¹

· In Comparison:

ATP ® ADP + Pi DG°’ = -30.5 kJ mol^-¹

ATP ® AMP + PPi DG°’ = -32.2 kJ mol^-¹

Efficiency of ATP Synthesis

· (3 ´ 30.5/218) ´ 100% = 42% under biological standard conditions

· ~70% under physiological conditions in active mitochondria

Major Electron Transport Carriers

· NADH; FMN; Q; FeS; Cyt (b_L + b_H); Cyt c₁; Cyt c; Cyt (a + a₃); Cu_A; Cu_B; O₂

· Hydrogen carriers: FMN, NAD⁺, Q

FMN + H⁺ + e ⇌ FMNH· FMNH· + H⁺ + e ⇌ FMNH₂

or FMN + 2H⁺ + 2e ⇌ FMNH₂

NAD⁺ + 2H⁺ + 2e ⇌ NADH+ H⁺

Q + H⁺ + e ⇌ QH· QH· + H⁺ + e ⇌ QH₂

or Q + 2H⁺ + 2e ⇌ QH₂

· H⁺ + e = H· (hydrogen atom) H⁺ + 2e = H^- (hydride)

· E carriers: FeS, cytochromes (e-transport heme proteins), copper proteins

Fe³⁺ + e ⇌ Fe²⁺

Cu²⁺ + e ⇌ Cu⁺

Sequence of Electron Transport and

Four Complexes In The E-Transport Chain

See Fig. 17-8 for Complexes I, III, and IV.

Complex I (NADH-Coenzyme Q Oxidoreductase)

4 H⁺

↑

· NADH ® FMN ® 2 FeS ® Q

· Contains 1 FMN and 6 to 7 iron-sulfur clusters (FeS).

· Passes 2e from NADH to Q.

· Pumps 4H⁺ from mitochondrion matrix to cytosol.

· Mechanism not well understood. One model: differential H⁺ binding and release resulting from protein conformational change.

· H-bonded groups in proteins and water are like a proton wire.

Strong acids with lower pK_as. AH ⇌ A^- + H⁺ with high tendency for à.

Strong bases with higher pK_as. B + H⁺ ⇌ BH⁺ with high tendency for à.

Place AH next to B: AH + B à A^- + BH⁺

Proton is transferred from AH to B.

Bacteriorhodopsin as a model for proton pump through a proton wire (Fig. 17-12)

Complex II (Succinate-Coenzyme Q Oxidoreductase)

· Contains succinate dehydrogenase covalently bound FAD (Succinate to Fumarate), 3 Fe-S clusters and Cyt b₅₆₀.

2 H⁺

↑

· Succinate ® FAD ® 3 FeS clusters® Cyt b₅₆₀ ® Q

(succinate DH)

· I and II do not operate in series.

Complex III (Coenzyme Q-Cytochrome c Oxidoreductase or cytochrome bc₁)

· Contains 2 Cyt b (b_L and b_H for low and high potentials, respectively), 1 Cyt c₁ and 1 Fe-S cluster.

4 H⁺ (to cytosol via Q cycle)

↑

· QH₂ ® ® ® ® Cyt c₁ ® Cyt c

↑

2 H⁺ (Matrix)

Q Cycle

See Fig. 17-15.

Cycle 1: QH₂ + Cyt c₁ (Fe³⁺) ® Q^-^• + Cyt c₁ (Fe²⁺) + 2 H⁺ (cytosolic)

· Obtain 1 QH₂ from Complex I.

· Pump 2 H⁺ to cytosol.

· Donate 1e to ISP and in turn to c₁.

· Generate 1 Q^-^•.

Cycle 2:

QH₂ + Q^-^• + Cyt c₁ (Fe³⁺) + 2 H⁺ (mitochondrial) ® Q + QH₂ + Cyt c₁ (Fe²⁺) + 2 H⁺ (cytosolic)

· Obtain another QH₂ from Complex I.

· Pump 2 H⁺ to cytosol.

· Donate 1e to ISP and in turn to c₁.

· Taking up 2 H⁺ from mitochondrial matrix.

· Generate 1 QH₂. No net gain or loss of QH₂.

NET OUTCOME

QH₂ + 2 Cyt c₁ (Fe³⁺) + 2 H⁺ (mitochondrial) ® Q+ 2 Cyt c₁ (Fe²⁺) + 4 H⁺ (cytosolic)

· Obtain 1 QH₂ from Complex I.

· 4 H⁺ pumped to cytosol.

· 2 c₁ reduced.

· Uptake 2 H⁺ from mitochondrial matrix.

Between Complex III and Complex IV

· Cyt c.

· Loosely bound to the outer surface of the inner mitochondrial membrane.

· Shuttles electrons between Cyt c₁ and Cyt c oxidase (Complex IV).

Complex IV (Cytochrome c Oxidase)

· Contains Cyt a, Cyt a₃, Cu_A, and Cu_B.

· (Cu_ACyt a) are of a lower potential. (Cyt a₃Cu_B) are of a higher potential.

· 2 Cyt c(Fe²⁺) + 2 H⁺ + 0.5 O₂ ® 2 Cyt c(Fe³⁺) + H₂O

· As shown in Fig. 17-8, 2H⁺ are transferred to cytosol by complex IV by unknown mechanism.

Physiological E-Donating Systems

· b-Hydroxybutyrate Dehydrogenase

b-Hydroxybutyrate + NAD⁺ ® Acetoacetate + NADH

· Succinate DH

Succinate + FAD ® Fumarate + FADH₂

Artificial E-Donating Systems

· Ascorbate ® Tetramethyl-p-phenylenediamine (TMPD) ® Cyt c

Physiological E Acceptor

· O₂ as the acceptor of e from Complex IV.

Artificial E Acceptors

· 2. Fe(CN₆)³^- accepts e from Cyt c_red.

THREE SITES OF PHOSPHORYLATION

I. Thermodynamic Consideration (Fig. 17-7)

· Site 1 (Complex I)

NAD⁺ + 2H⁺ + 2e ® NADH + H⁺ E°’ = -0.315 V (1)

Q + 2H⁺ + 2e ® QH₂ E°’ = 0.045 V (2)

(2 ) – (1) NADH + H⁺ + Q ® NAD⁺ + QH₂

DE°’ = 0.045 + 0.315 = 0.36 V

DG°’ = -69.5 kJ mol^-¹

· Complex II Is Not a Site for Phosphorylation

FADH₂ + Q ® FAD + QH₂

DE°’ = 0.045 + 0.04 = 0.085 V

DG°’ = -16.4 kJ mol^-¹

· Site 2 (Complex III)

QH₂ + 2 Cyt c_ox ® Q + 2 Cyt c_red

DE°’ = 0.235 - 0.045 = 0.19 V

DG°’ = -36.7 kJ mol^-¹

· Note: The number of electrons involved is taken into consideration when DE°’ is converted to DG°’ (DG°’ = -nFDE°’ where n is the number of electrons and F = 96,494 J V^-¹ mol^-¹).

· Site 3 (Complex IV)

2 Cyt c_red + 2 H⁺ + 0.5 O₂ ® 2 Cyt c_ox + H₂O

DE°’ = 0.815 - 0.235 = 0.58 V

DG°’ = -112 kJ mol^-¹

II. Specific Inhibitors (Fig. 17-7)

· Rotenone and Amytal block Complex I.

· Antimycin A blocks Complex III.

· CN^- blocks Complex IV.

III. Partial Reactions and P/O (or P/2e) Ratio (Fig. 17-7)

P/O (or P/2e) ratio is the ratio of the number of ATP synthesized over the number of oxygen ATOM (= 0.5 O₂) reduced to water.

E Donor	E Acceptor	P/O or P/2e
b-Hydroxybutyrate (or NADH)	1/2 O₂	3
Succinate (or FADH₂)	1/2 O₂	2
b-Hydroxybutyrate (or NADH)	Fe(CN₆)³^-	2
Succinate (or FADH₂)	Fe(CN₆)³^-	1
Ascorbate	1/2 O₂	1

Note:

· Phosphorylation and oxidation are tightly coupled. [Substrate (e.g. b-hydroxybutyrate or succinate) + Pi + O₂] does not lead to continuous oxygen consumption until ADP is added.

· P/O ratios may not be exactly integers.

IV. Reconstitution

Reconstitution of active individual sites using isolated components and phospholipid vesicles has been done.