Chapter 14 (Part 1) Electron transport

Chemiosmotic Theory Electron Transport: Electrons carried by reduced coenzymes are passed through a chain of proteins and coenzymes to drive the generation of a proton gradient across the inner mitochondrial membrane Oxidative Phosphorylation: The proton gradient runs downhill to drive the synthesis of ATP Electron transport is coupled with oxidative phosphorylation It all happens in or at the inner mitochondrial membrane

Outer Membrane – Freely permeable to small molecules and ions. Contains porins with 10,000 dalton limit Inner membrane – Protein rich (4:1 protein:lipid). Impermeable. Contains ETR, ATP synthase, transporters. Cristae – Highly folded inner membrane structure. Increase surface area. Matrix- “cytosol” of the mitochondria. Protein rich (500 mg/ml) Contains TCA cycle enzymes, pyruvate dehydrogenase, fatty and amino acid oxidation pathway, DNA, ribosomes Intermembrane Space – composition similar to cytosol

Reduction Potentials High E o ' indicates a strong tendency to be reduced Crucial equation:  G o ' = -nF  E o '  E o ' = E o '(acceptor) - E o '(donor) NADH + ½ O 2 + H +  NAD + + H + + H 2 O NAD + + H + + 2e -  NADH E o’ = ½ O 2 + 2e - + 2H +  H 2 O E o’ =  G o ‘= -nF(E o '(O 2 ) - E o '(NADH))  G o ‘= -nF(0.82 –(-0.32)) = -nF(1.14) = -2(96.5 kJ mol -1 V -1 )(1.136) = -220 kJ mol -1

Electron Transport Four protein complexes in the inner mitochondrial membrane A lipid soluble coenzyme (UQ, CoQ) and a water soluble protein (cyt c) shuttle between protein complexes Electrons generally fall in energy through the chain - from complexes I and II to complex IV

Standard reduction potentials of the major respiratory electron carriers.

Complex I NADH-CoQ Reductase Electron transfer from NADH to CoQ More than 30 protein subunits - mass of 850 kD 1 st step is 2 e - transfer from NADH to FMN FMNH 2 converts 2 e - to 1 e - transfer Four H + transported out per 2 e- NADH + H + FMN Fe 2+ S CoQ NAD + FMNH 2 Fe 3+ SCoQH 2

Succinate FAD Fe 2+ S CoQ FumarateFADH 2 Fe 3+ SCoQH 2 Complex II Succinate-CoQ Reductase aka succinate dehydrogenase (from TCA cycle!) four subunits Two largest subunits contain 2 Fe-S proteins Other subunits involved in binding succinate dehydrogenase to membrane and passing e - to Ubiquinone FAD accepts 2 e - and then passes 1 e - at a time to Fe-S protein No protons pumped from this step

Q-Cycle Transfer from the 2 e - carrier ubiquinone (QH2) to Complex III must occur 1 e - at a time. Works by two single electron transfer steps taking advantage of the stable semiquinone intermediate Also allows for the pumping of 4 protons out of mitochondria at Complex III Myxothiazol (antifungal agent) inhibits electron transfer from UQH 2 and Complex III. UQ UQ.- UQH 2

Complex III CoQ-Cytochrome c Reductase CoQ passes electrons to cyt c (and pumps H + ) in a unique redox cycle known as the Q cycle Cytochromes, like Fe in Fe-S clusters, are one- electron transfer agents cyt c is a water-soluble electron carrier 4 protons pumped out of mitochondria (2 from UQH 2 ) CoQH 2 cyt b ox Fe 2+ S cyt c 1 ox cyt c red CoQcyt b red Fe 3+ S cyt c 1 red cyt c ox

cyt c red cyt a ox cyt a 3 red O2O2 cyt c ox cyt a red cyt a 3 ox 2 H 2 O Complex IV Cytochrome c Oxidase Electrons from cyt c are used in a four- electron reduction of O 2 to produce 2H 2 O Oxygen is thus the terminal acceptor of electrons in the electron transport pathway - the end! Cytochrome c oxidase utilizes 2 hemes (a and a 3 ) and 2 copper sites Complex IV also transports H + (2 protons)

Inhibitors of Oxidative Phosphorylation Rotenone inhibits Complex I - and helps natives of the Amazon rain forest catch fish! Cyanide, azide and CO inhibit Complex IV, binding tightly to the ferric form (Fe 3+ ) of a 3 Oligomycin and DCCD are ATP synthase inhibitors

Shuttling Electron Carriers into the Mitochondrion The inner mitochondrial membrane is impermeable to NADH. Electrons carried by NADH that are created in the cytoplasm (such as in glycolysis) must be shuttled into the mitochondrial matrix before they can enter the ETS

Glycerol phosphate shuttle

malate/aspartate shuttle system

Electron transport is coupled to oxidative phosphorylation

Uncouplers Uncouplers disrupt the tight coupling between electron transport and oxidative phosphorylation by dissipating the proton gradient Uncouplers are hydrophobic molecules with a dissociable proton They shuttle back and forth across the membrane, carrying protons to dissipate the gradient w/o oxidative-phosphorylation energy lost as heat Dinitrophenol once used as diet drug, people ran 107 o F temperatures