INTER 111: Graduate Biochemistry

 Define electron transport chain, oxidative phosphorylation, and coupling  Know the locations of the participants of the system/pathways  Predict the flow of electrons under standard state conditions when given a redox half equation and know how to calculate the standard state free energy change given the proper equation and half reactions. Be able to predict the spontaneity of a reaction given the reduction potential.  List components of the respiratory chain and the electron carrying molecules. Know the differences between the hemes.  Outline the pathway of electron transport in mitochondria in terms of the transfer of electrons from the reducing equivalents to oxygen.

 Describe the mechanism of action of an uncoupler or inhibitor on the electron transfer chain or oxidative phosphorylation.  Recognize the site of inhibition of rotenone, carbon monoxide, antimycin A, and oligomycin and be able to describe the effect of these inhibitors.  Describe and understand the mechanism of how the F o F 1 ATPase complex forms ATP.  Estimate the net potential yield of ATP for each of the entry points into the electron transport system and know why there are discrepancies.

Glucose Acetyl CoA R5P Pyruvate NADH + H + and ATP Glycogen disaccharides NADH + H + and FADH 2 and CO 2 aerobic conditions citric acid cycle Electron transport Oxidative phosphorylation O2O2 H2OH2O AT P ADP + P i Acetyl CoA

Oxidative phosphorylation  is aerobic (i.e., in O 2 )  is a stepwise process  transfers electrons from reduced carriers to O 2  generates 3 moles ATP for every mole NADH Electron transfer chain  is a series of coupled oxidation-reduction reactions  is catalyzed by membrane-bound proteins on the inner membrane of mitochondria

An oxidation-reduction (redox) reaction involves an electron donor and an electron acceptor. The redox potential expresses the tendency of an electron donor to reduce its conjugate acceptor. Under standard conditions (25 o C, pH 7, [donor]=[acceptor]=1 M), the redox potential is E o ’ E o ’ is measured relative to the standard hydrogen electrode. Fe 2+ + Cu 2+ Fe 3+ + Cu + e - donor e - acceptor oxidized donor reduced acceptor

Compounds with a large negative E o are strong reducing agents. Compounds with a large positive E o are strong oxidizing agents. Redox pairE o ’ (V) NAD + / NADH FMN / FMNH 2 Cytochrome b Fe 3+ /Fe 2+ 1/2 O 2 / H 2 O

Eo’Eo’ NAD + / NADH FMN / FMNH 2 Fe 3+ S / Fe 2+ S H-Fe 3+ / H-Fe 2+ CoQ / CoQH 2 O 2 / H 2 O H-Fe 3+ / H-Fe 2+ Redox couples Q (mobile) (mobile) Cyto oxidase (Complex IV) Cyto bc 1 (Complex III) NADH-Q reductase (Complex I) NAD + FMN Fe-S centers Coenzyme Q Cyto b (Fe 3+ ) Fe-S centers Cyto c (Fe 3+ ) Cyto a (Fe 3+ ) Cyto a 3 (Fe 3+ ) O2O2 Cyto c (Fe 3+ ) 2 e - transfer

cristae matrix inner membrane outer membrane a a a3a3 a c b CoQ FMN NAD + Electron transport chain ATP synthase

The electron transport chain conducts a series of oxidation/reduction reactions. The components of the respiratory chain are flavoproteins, ubiquinone molecules, and cytochromes

 Formation of NADH  NAD + is reduced to NADH by dehydrogenases in the TCA cycle. Substrate (reduced) Product (oxidized) NAD + NADH + H +  NADH dehydrogenase  Coenzyme Q  Cytochromes

bHbH bLbL 2Fe-2S c1c1 Cyto b Cyto c 1 Cyto c Q 2Fe-2S QH 2 FMN 4Fe-4S NADH QH 2 matrix intermembrane space Cu A a a3a3 Cu B 1/2 O H + H2OH2O F 1 F O synthase Complex IComplex IIIComplex IV NADH dehydrogenase cytochrome bc 1 cytochrome c oxidase

NADH + H + NAD + FMN FMNH 2 Fe 2+ S Fe 3+ S CoQ CoQH 2 reduced oxidized reduced oxidized Q 2Fe-2S QH 2 FMN 4Fe-4S NADH QH 2 matrix intermembrane space

 Formation of NADH  NADH dehydrogenase  Coenzyme Q  A quinone derivative with long isoprenoid tail Mobile carrier that accepts hydrogens from FMNH2 (complex I) and FADH2 (Complex II). Transfers electrons to complex III  Cytochromes

Reduced form of coenzyme Q (QH 2, ubiquinol) Semiquinone intermediate (QH) Oxidized form of coenzyme Q (Q, ubiquinone)

matrix intermembrane space bHbH bLbL 2Fe-2S c1c1 Cyto b Cyto c 1 Cyto c QH 2 Heme

Cyto c matrix intermembrane space Cu A a a3a3 Cu B 1/2 O H + H2OH2O Cyt 2+ a 3 Cyt 3+ a 3 Cyt 3+ a Cyt 2+ a Cyt 2+ c Cyt 3+ c H2OH2O 1/2 O 2

Three elements for energy transduction: A cellular membrane Exergonic electron transport generates a proton gradient across a membrane Proton gradient furnishes energy for ATP production by ATP synthase Peter D. Mitchell

H+H+ H H+H+ H+H+ H+H+ bHbH bLbL 2Fe-2S c1c1 Cyto b Cyto c 1 Cyto c Q 2Fe-2S QH 2 FMN 4Fe-4S NADH QH 2 matrix intermembrane space Cu A a a3a3 Cu B 1/2 O H + H2OH2O ATP ADP + P i Complex V ATP synthase lower pH and greater positive charge

H+H+ H H+H+ H+H+ H+H+ bHbH bLbL 2Fe-2S c1c1 Cyto b Cyto c 1 Cyto c Q 2Fe-2S QH 2 FMN 4Fe-4S NADH QH 2 matrix intermembrane space Cu A a a3a3 Cu B 1/2 O H + H2OH2O ATP ADP + P i For 1 mol NADH oxidized, 3 mol ATP produced For 1 mol FADH 2 oxidized, 2 mol ATP produced Complex V ATP synthase

3H+3H+ ATP ADP + P i ATP ADP + P i When electrochemical H + gradient is favorable, F 1 F O ATPase complex catalyzes ATP synthesis. If no membrane potential or pH gradient exists to drive the forward reaction, K eq favors the reverse reaction (ATP hydrolysis). 3H+3H+ F1F1 F0F0

F 1 subunit present with stoichiometry      and   and  subunits (513 and 460 residues in E. coli) are homologous to one another 3 nucleotide-binding catalytic sites at  /  interface, but involving  residues Each subunit contains ATP, but is inactive in catalysis Mg 2+ binds with adenine nucleotides in both  and  subunits F 0 subunit present with stoichiometry a, b 2, and c 10 F1F1 F0F0       

Rotation of the  shaft relative to the ring of  and  subunits directly observed, by attaching fluorescent-labeled actin filament to the  subunit. Noji et al Nature 386, 299 The rotation rate is 100 Hz (revolutions/s) ATP-induced rotation occur in discrete 120 o steps. F 1 F O synthase

HEAT bHbH bLbL 2Fe-2S c1c1 Cyto b Cyto c 1 Cyto c Q 2Fe-2S QH 2 FMN 4Fe-4S QH 2 matrix intermembrane space Cu A a a3a3 Cu B Complex V ATP synthase 2,4-DNP