General Biochemistry 2: Lecture 8 (Electron Transport Chain, Complexes, Inhibitors)

LECTURE 8: ELECTRON TRANSPORT CHAIN, COMPLEXES, INHIBITORS

A. NADH oxidation- is highly exergonic- -218 kJ/mol

-NAD⁺ + H⁺ + 2e à NADH (-.315 V)

-oxygen + 2H+ + 2e à water (0.815 V)

Overall: oxygen + NADH + H⁺à water + NAD⁺

ATPàADP: -30.5 kJ/mol

ATPàAMP: -32.2 kJ/mol

2. Efficiency of ATP Synthesis- (3 ATP per NADH) ÷ (total energy per NADH) = 42%

-70% in mitochondria

B. ET Carriers- NADH; FMN; Q; FeS; Cyt a+a3,bl+bh,c,c1; Cua; Cub; O₂

1.Hydrogen carriers- FMN; NAD⁺; Q;

-FMN- add 2H⁺ and 2e, get FMNH2. (1H and 1 e= FMNH*)

-NAD+ - add 2H+ and 2e, get NADH + H⁺.

-Q- add 2H+ and 2e, get QH2.

2. Electron carriers- FeS, cytochromes (ET heme proteins), Cu proteins

-Fe3+ à Fe2+

-Cu2+àCu+

C. Complexes that make up ET chain

1. Complex I- NADH-CoenzQ Oxidoreductase;

-NADH à [[[FMN à2 FeS]]]à Q (passes 2e from NADH to Q)

-inside: 8-9 iron-sulfur clusters and 1 FMN

-4H+ go outside

-possible mechanism: differential H⁺ binding and release caused by protein shape change; H bonded groups in proteins and water are like a proton wire.

AH+BàA+BH. Proton moves from AH to B.

-bacteriorhodopsin-model for proton pump through proton wire

a.Cycle 1- QH₂ + Cyt c1 (Fe3+) à Q*- + Cyt c1(Fe2+) + 2H+ (cystol)

-QH2 from complex 1; pump 2H+ to intermembrane space, then to cytosol;

-donate 1e to ISP, which goes to c1;

-makes 1 Q-*

b.Cycle 2- QH₂ + Q-* + Cytc1(Fe3+) + 2H⁺(mito) à Q + QH₂+Cytc1(Fe2+)

-obtain QH₂ from complex I; pump 2H⁺ to cyto;

-Donate 1e to ISP, goes to c1. Take up 2 H⁺ from mito.

-Generate 1 QH₂, not net gain or loss of QH₂.

Net: QH₂ + 2 Cytc1(Fe3+) + 2H⁺ (mitochondria) à Q + 2 Cytc1(Fe2+) + 4 H⁺ (cystol)

-4 H⁺ pumped out, 2 c1 reduced, use 2 H⁺ from matrix.

2.Complex II- Succinate-CoenzQ Oxidoreductase

-Succinateà[[[FAD (succinate DH, 2H+ released)à3 iron-sulfur clusters à Cyt b560]]]à Q

-I and II not in series

-inside: 3 FeS clusters, succinate dehydrogenase FAD, and cyt b560

-2H+ go outside

3.Complex III- CoenzQ-Cytochrome C Oxidoreductase

-QH2à[[[CytB or FeSàCyt c1]]]à Cyt c

-inside: 2 Cyt b (bL for low and bH for high potential), 1 Cyt c1, and 1 Fe-S cluster.

-4H+ go outside, 2H+ inside

4. Complex IV- Cytochrome c oxidase

-2 Cytc(Fe2+) + 2H⁺ + .5O₂ à[[[CuAàCytaàCyta3-CuB]]]à 2 Cytc(Fe3+) + H₂O

-has cyt a, cyt a3, cua, vub

-high potential- (CuA-Cyta) lower potential than (Cyta3-CuB)

-2H⁺ go to cytosol

- Cyt c- between III and IV. Loosely bound to outer surface of inner membrane. Shuttles e⁻ between Cyt c1 and cyt c oxidase.

D. Donaters/Acceptors

1.Physio e⁻ donating systems:

-β-hydroxybutyrate dehydrogenase- β-HOButyr+NAD⁺àAcetoacetate+NADH

-Succinate DH- Succinate+FADàFumarate+FADH₂

2. Artifical e⁻ donating systems: ascorbateàtetramethyl-p-phenylenediamine (TMPD)àCyt C

3. Physio e⁻ acceptor: O₂ as acceptor of e from Complex IV

4. Artifical e⁻ acceptor: Fe(CN6)3- accepts e from Cyt c_red

E. ΔG=-nFΔE, where F=96494 J/mol*V

1.Site 1(Complex 1)- ΔG=-69.5, ΔE=.36V

-NADH+H⁺+QàNAD⁺+QH₂

-reduce NAD=-.315V; reduce Q=.045V

2.Complex II- no phosphorylation

-FADH₂+QàFAD+QH₂

-ΔE=.085 V, ΔG=-16.4 kJ/mol

3.Site 2 (Complex III)- ΔG=-36.7 kJ/mol, ΔE=0.19V

-QH₂+2Cytc_oxàQ+2Cytc_red
4.Site 3 (Complex IV)- ΔG=-112kJ; ΔE=.58V;

-2CytRED+ 2H⁺+ .5O₂à2CytOX+ H₂O

F.Inhibtors

Rotenone and Amytal- block complex I

Antimycin A- blocks complex III

CN^- -blocks complex IV

G.P/O value- ratio of ATP made over oxygens used

- β-HOButyr/NADH and O₂= 3

- Succinate/FADH₂ and O₂= 2

- β-HOButyr/NADH and Fe(CN6)-3= 2

- Succinate/FADH₂ and Fe(CN6)-3= 1

- ascorbate and O₂= 1

-phosphorylation and oxidation tightly coupled, NADH+P+O₂ wont happen until ADP is added.

-Reconsitution- isolated components and phospholipid vesicles remake active sites.