Electron Transport Discovery Four Complexes

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BI/CH 422/622 OUTLINE: Pyruvate pyruvate dehydrogenase Krebs’ Cycle How did he figure it out? Overview 8 Steps Energetics Regulation Summary Oxidative Phosphorylation Energetics (–0.16 V needed for making ATP) Mitochondria Transport (2.4 kcal/mol needed to transport H+ out) Electron transport Discovery Four Complexes

Complex I: NADHà CoQH2

Complex II: Succinateà CoQH2 2+ Complex III: CoQH2à Cytochrome C (Fe ) 2+ Complex IV: Cytochrome C (Fe ) à H2O Chemiosmotic theory ATPase Mitchell Hypothesis Binding-Change Model Connection to the proton motive force

Electron Transport

TABLE The Protein Components of the 19-3 Mitochondrial Respiratory Chain

Reduction Enzyme Mass Number of Prosthetic Binding sites potential Inhibted by: complex/protein (kDa) subunitsa group(s) for: (DE’°V)

I NADH dehydrogenase 850 45 (14) FMN, Fe-S 0.36 NADH, CoQ amytal, rotenone II Succinate Succinate, 140 4 FAD-E, Fe-S 0.09 dehydrogenase CoQ III Ubiquinone: Hemes b, c , CoQ, cytochrome c 250 11 1 0.17 antimycin a Fe-S Cytochrome c oxidoreductaseb Cytochrome cc 13 1 Heme Hemes a, a ; Cytochrome c, Cyanide, azide, IV Cytochrome oxidaseb 204 13 (3–4) 3 0.57 CuA, CuB O2 CO aNumber of subunits in the bacterial complexes in parentheses. bMass and subunit data are for the monomeric form. cCytochrome c is not part of an enzyme complex; it moves between Complexes III and IV as a freely soluble protein.

1 Electron Transport • Mobile electron carrier; a peripheral membrane protein – Cytochrome c moves through the intermembrane space (IMS). 2 Cytochrome c Fe+3

CoQH2 Complex IV Complex III CoQ 2 Cytochrome c Fe+2 2H+

Electron Transport

TABLE The Protein Components of the 19-3 Mitochondrial Respiratory Chain

Reduction Enzyme Mass Number of Prosthetic Binding sites potential Inhibted by: complex/protein (kDa) subunitsa group(s) for: (DE’°V)

2 Electron Transport: Complex IV Cytochrome Oxidase, a.k.a. Complex IV CuA • Uses electrons from FOUR reduced cytochrome c’s to reduce dioxygen (O2) to waters (2). • This reaction is CuB among the most difficult chemically. • The bovine complex is a dimer of 13 subunits (26 proteins). Three main proteins for each of the redox centers. • Contains two copper proteins: one with a Cu-Cys cluster, and one with two different cytochrome

a’s (a & a3) and Cu. The third main protein moves protons from the matrix through the second and protein. Function of the other 10 are unknown, although undoubtedly involved as a proton pump.

Electron Transport: Complex IV Cytochrome Oxidase, a.k.a. Complex IV CuA • Uses electrons from FOUR reduced cytochrome c’s to reduce dioxygen (O2) to waters (2). • This reaction is CuB among the most difficult chemically. • The bovine complex is a dimer of 13 subunits (26 proteins). Three main proteins for each of the redox centers. • Contains two copper proteins: one with a Cu-Cys cluster, and one with two different cytochrome

a’s (a & a3) and Cu. The third main protein moves protons from the matrix through the second and protein. Function of the other 10 are unknown, although undoubtedly involved as a proton pump.

3 Electron Transport: Complex IV

+ 4H (matrix) Cytochrome Oxidase Passes Electrons to O2

CuA CuB

+ 4H (pumped)

• Contains 6 copper ions per complex (3/monomer13) • There are two types, sites for the copper:

– CuA: two ions that accept electrons from cyt c (looks like a 2Fe-2S cluster) – CuB: bonded to heme a3, forming a binuclear center that transfers four electrons to oxygen. • Four electrons are used to reduce one oxygen molecule into two water molecules. • Four protons are picked up from the matrix in this process. • Four additional protons are pumped from the matrix to the – intermembrane space. This scheme is for 4e coming down from 2 NADH or 2 FADH2

Electron Transport: Complex IV Electron Flow Through Complex IV 4H+

4H+ This scheme is for 4e– coming down 4H+ 2 from 2 NADH or 2 FADH2

4 Electron Transport: Complex IV Electron Flow Through Complex IV: Chemistry

Oxygen binding A R O2 (peroxy) OH | O=O Fe+2•Cu+1•Tyr OH H2O (oxy) | Second water/ Fe+2•Cu+1•Tyr reduction of Fe H+ Splitting of dioxygen – – OH e from –2 – •O OH cyto-a+2 O OH FH | | (ferryl) Fe+3•Cu+1•Tyr Fe+4•Cu+2•Tyr PM – – e from e from + +2 +2 Reduction of Cu H cyto-a cyto-a Quenching radical – – – e from OH O cyto-a+2 –2 – | O OH O– EH Fe+3•Cu+2•Tyr | +4 +2 PR –2 Fe •Cu •Tyr O O– | H+ H+ Fe+4•Cu+2•Tyr H2O Reduction of Fe First water/from Cu OH

Electron Transport Multiple Complexes Associate Together to Form a “Respirasome”

Complex III and Complex IV

5 Electron Transport Summary of the Electron Flow in the Respiratory Chain

Difference in number of protons transported reflects differences in ATP – This scheme is for 2e coming down from 1 NADH or 1 FADH2 synthesized=P/O ratios: Complex I à Complex IV Substrate P/O ratio + + + 1NADH + 11H (N) + ½O2 à NAD + 10H (P) + H2O Pyruvate 3 Acetyl CoA 3 Complex II à Complex IV NADH 3 FADH + 6H+ + ½O à FAD + 6H+ + H O 2 (N) 2 (P) 2 Succinate 2

Phosphorylation

(The Chemiosmotic Theory)

Dr. Kornberg: Lecture 02.17.17 (45:00-48:25)- mitochondria coupling (3.5 min)

6 Oxidative Phosphorylation

Glycogenolysis

Glycolysis Pyruvate Oxidation

Krebs' Cycle

Phosphorylation Oxidative

Oxidative Phosphorylation

Relationship of ETC and ATP Synthesis

• As described, ATP synthesis requires electron transport. • But electron transport does not requires ATP synthesis.

7 Oxidative Phosphorylation

Relationship of ETC and ATP Synthesis

• As described, ATP synthesis requires electron transport. • But electron transport does not requires ATP synthesis.

Phosphorylation How Does Oxidative Phosphorylation Form ATP?

• Before the 1970’s, it was thought that the ET chain used the energy of redox to make a ”high-energy” intermediate for “substrate-level phosphorylation, as occurs in glycolysis and TCA cycle. • It was clear that one of the most abundant proteins V on the inner-mitochondrial membrane had a curious mushroom-shaped structure. • It was purified, along with the other Complexes I-IV, initially called Complex V.

8 Phosphorylation How Does Oxidative Phosphorylation Form ATP?

• Before the 1970’s, it was thought that the ET chain used the energy of redox to make a ”high-energy” intermediate for “substrate-level phosphorylation, as occurs in glycolysis and TCA cycle. • It was clear that one of the most abundant proteins on the inner-mitochondrial membrane had a curious mushroom-shaped structure. • It was purified, along with the other Complexes I-IV, initially called Complex V.

Phosphorylation

How Does Oxidative Phosphorylation Form ATP? • Complex V, when purified separately from the F1-ATPase membrane is an effective ATPase enzyme, hence it was initially called the F1- ATPase. • The F1 part had 9 subunits F0 (a3b3gde) • When more careful purifications were performed, it was clear that F1 its activity was closely coupled to an intact inner membrane with little activity. • When membrane proteins were isolated, the F0 part • Inhibited by had 15 subunits of 3 venturicidin or different proteins (a, b2, c10). oligomycin

9 Phosphorylation

How Does Oxidative Phosphorylation Form ATP? • Complex V, when purified separately from the membrane is an effective ATPase enzyme, hence it was initially called the F1- ATPase.

• The F1 part had 9 subunits (a3b3gde) • When more careful purifications were performed, it was clear that its activity was closely coupled to an intact inner membrane with little activity. • When membrane proteins were isolated, the F0 part • Inhibited by had 15 subunits of 3 venturicidin or different proteins (a, b2, c10). oligomycin

Phosphorylation

Contains two functional units: •F1 • soluble complex in the matrix • individually catalyzes the hydrolysis of ATP •F0 • integral membrane complex • transports protons from IMS to matrix, dissipating the proton gradient

• energy transferred to F1 to catalyze phosphorylation of ADP. • This mechanism was only fully accepted in late 1970s.