Requirements for oxidative phosphorylation Requirements for the production of ATP 1. An ion impermeable membrane A mechanism for moving protons (H+) across the membrane to produce an energy-rich proton gradient A mechanism to capture the energy made available as protons move down the proton gradient

Requirements for oxidative phosphorylation 1. An ion impermeable membrane See Fig 14.6 in Horton

A typical representation of an electron transport chain A typical representation of an electron transport chain. Images from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.

Requirements for oxidative phosphorylation A mechanism for moving protons (H+) across the membrane to produce an energy-rich proton gradient Electron transport chain 4 2 2H+

NMN AMP nicotinamide H+ H

Isoalloxazine FMNH FMNH2 (semiquinone) (hydroquinone) 2H+ -H+, -e- 2e- 1 5 Isoalloxazine -H+, -e- 2H+ 2e- H FMNH (semiquinone) H FMNH2 (hydroquinone) -H+, -e-

H FADH2

+ 1 e- - + 1 e-, +2H+ Isoprenoid unit Semiquinone ( Q- ) H Ubiquinol ( QH2 )

Non-heme iron sulfur proteins (Fe-S clusters) 2S -2Fe Cys Cys S S S Fe Fe S S S Cys Cys 4S -4Fe S Cys Fe Cys S S Fe S Fe S S Fe Cys Cys S S

Cytochromes Heme-containing proteins Classified as a, b or c based on absorption spectrum Electron transport has: a and a3, b566 (bL) and b562 (bH). and c and c1 carry 1 electron per heme iron a, a3, b566,,, b562,, and c1 are integral membrane proteins c is a peripheral membrane protein on outer surface of inner mitochondrial membrane

Heme group in cytochromes methyl proprionate Heme group of Cytochrome b See fig. 7-34 in Horton

Heme group in cytochromes a and c See fig. 7-34 in Horton

ca 550-558 nm ba 555-567 nm aa 592-604 nm See Fig 7-35 -Horton

Difference spectra b c a O2 = spectra of experimental– spectra of fully oxidized mito b c a O2 Normal Blocked between Cyt b and c1 a and c fully oxidised b fully reduced

Standard redox potentials of mitochondrial oxidation-reduction components Substrate or complex Eo’ (V) NADH -0.32 Complex I FMN Fe-S clusters   -0.30 -0.25 - -0.05 Succinate + 0.03 Complex II FAD 0.0 -0.26 – 0.00 QH2/Q +0.04 Complex III Cyt B560 Cyt b566 Cyt c1 +0.28 - 0.1 + 0.05 + 0.22 Cytochrome c + 0.23 Complex IV Cyt a CuA Cyt a3 CuB + 0.21 + 0.24 + 0.39 + 0.34 O2 + 0.82

NADH + H+ + CoQox NADH + CoQH2 Electron transport complexes   Complex I: NADH-Q reductase DE0' = + 0.32 V DG = -70 kJ/2e- Contains at least 34 polypeptides FMN, 2Fe-2S and 4Fe-4S clusters, tightly bound CoQ Mr = 880kD Result: 2 e- from NADH to CoQ 4 H+ from matrix to intermembrane space NADH + H+ + CoQox NADH + CoQH2 Intermembrane space 4H+ 2H+ QH2 FMNH2 Fe-S 2e- 2e- FMN Q 4H+ matrix NAD NADH + H+

Intermembrane space 2 x e- 2e- matrix QH2 Fe-S Q FAD Fumarate + 2H+ Complex II: succinate dehydrogenase/ succinate – Q reductase   DE0' = + 0.015 V DG = -2.9 kJ/2e- Contains: FAD, Fe_S Result: 2 e- from FADH2 to CoQ No protons translocated FADH2 + CoQox FADH + CoQH2 Intermembrane space FAD Fe-S Q QH2 2e- 2 x e- 2H+ succinate Fumarate + matrix

CoQH2 + 2Cyt c (Fe+3) CoQ + 2Cyt c (Fe+2) Complex III: Cytochrome C reductase   DE0' = + 0.25 V DG = --37 kJ/2e- Contains: Cyt b (bL and bH), Cyt c1, Fe-S protein, several additional proteins Result: 2 e- from CoQH2 to Cyt c 2 H+ taken up from matrix, 4 H+ to intermembrane space CoQH2 + 2Cyt c (Fe+3) CoQ + 2Cyt c (Fe+2) Intermembrane space 2H+ 2 x e- c 4H+ QH2 Q matrix

CoQ cycle C1 Fe-S bL bH C1 C1 Fe-S Fe-S bL bL bH bH C1 Fe-S bL bH

4Cyt c (Fe+2) + 4 H+ + O2 4Cyt c (Fe+3) + 2 H2O Complex IV: Cytochrome oxidase   DE0' = + 0.57 V DG = -110 kJ/2e- Contains: 10 subunits, Cyt a and Cyt a3, 2 Cu (A , B) Result: 4 e- from 4 Cyt c to form 2 H2O 4 H+ taken up from matrix, 2 H+ to intermembrane space 4Cyt c (Fe+2) + 4 H+ + O2 4Cyt c (Fe+3) + 2 H2O Intermembrane space a - CuA 1/2O2 2 x e- 2H+ c a3 - CuB H2O matrix

Cytochrome oxidase: electron transfer to O2 CuB Cu2+ Fe3+ OH- a3 Cu2+ . Fe3+ . Cu+ Fe2+ Cu2+ Fe3+ O- H Cu2+ Fe3+ O- Cu+ Fe3+ O- See Horton, fig 14.16

Electron transport chain Cplx I Cplx III Cyt c Cyt c Cplx IV CoQ CoQ e- H2O NADH + H+ H+ H+ 2H+ + ½ O2 H+ NAD Summary of protons translocated per 2 e- Complex Matrix Intramembrane space I -5 +4 III -2 IV -4 +2 II

1997 Nobel Prize for Chemistry Paul D. Boyer John E. Walker Jens C. Skou "for their elucidation of the enzymatic mechanism underlying the synthesis of adenosine triphosphate (ATP)" "for the first discovery of an ion-transporting enzyme, Na+, K+ -ATPase"

ATP synthase These and the following images can be found at the home page of Boris A. Feniouk (http://www.biologie.uni-osnabrueck.de/biophysik/Feniouk/Home.html) that contains a wealth of additional information on ATP synthesis. See also Horton p 450 and 451.

Binding-change mechanism (See page 451 in Horton) From: http://www.cse.ucsc.edu/~hongwang/ATP_synthase.html

For movies and details go to: http://www.res.titech.ac.jp/~seibutu/

H+ H+ Cplx I Cplx III c c Cplx IV ATP synthase CoQ CoQ H2O e- H+ H+ H+ NADH + H+ 2H+ + ½ O2 ADP + PO4 H+ ATP NAD