Oxidative Phosphorylation General Considerations How do we define

Oxidative Phosphorylation

General Considerations • How do we define oxidative phosphorylation? – formation of ATP using the energy released by the transfer of electrons from NADH and FADH 2 through a series of electron carriers • What couples the formation of ATP to the transfer of electrons? – a proton gradient

General Considerations • Where in the cell does oxidative phosphorylation take place? – inner mitocondrial membrane • What do we know about the mitocondrial membranes? – outer membrane – reasonably permeable • contains porins – VDAC – inner membrane – relatively impermeable

Origin of Mitocondria • What is the believed origin of mitocondria? – endosymbiosis • What evidence supports this idea? – mitcondrial DNA – machinery for transcription and translation – similarity of genome to bacteria

Redox Potentials and Free Energy Changes • How does one determine the redox potential of a substance? sample half-cell standard reference half-cell

Redox Potentials and Free Energy Changes • What is the relationship between change in redox potential and change in free energy? – G 01 = -n. F E 10 • n = number of electrons transferred • F =faraday (constant, 23. 06 kcal/mole/volt) – Can calculate free energy change from reduction potentials of the reactants • By knowing the electron transfer potential of NADH relative to O 2 one can calculate the amount of free energy released when O 2 is reduced by NADH.

Redox Potentials and Free Energy Changes • One can also quantify the energy associated with a proton gradient. – G = RTln(c 2/c 1) + ZF V • • c 2 = concentration on one side of membrane c 1 = concenetration on other side of membrane Z = electrical charge of transported material F = Faraday constant (23. 06 kcal/mole/volt)

Electron Transport • What determines the rate of electron transport? – distance between donor and acceptor

Electron Transport – driving force or free energy change

Electron Transport • What are the complexes making up the respiratory chain? – three proton pumps – one link to citric acid cycle

Electron Transport • What is the role of ubiquinone or coenzyme Q?

Electron Transport • What happens to the electrons from NADH? – enter ETS at NADH-Q oxidoreductase

Electron Transport • Initial step is transfer of electrons to FMN a prosthetic group of the enzyme

Electron Transport • Electrons are then transferred to iron-sulfur clusters another prosthetic group

Electron Transport • Electrons from clusters transferred to coenzyme Q – as a result of electron transfer four protons are pumped out of mitocondrial matrix • Reaction summarized: – NADH + Q + 5 H+matrix NAD+ + QH 2 + 4 H+cytosol

Electron Transport • Coenzyme Q also serves as entry point for electrons from FADH 2 from oxidation of succinate – succinate-Q reductase complex • inner mitocondrial membrane • FADH 2 transfers electrons to iron-sulfur clusters then to Q – no protons are pumped

Electron Transport • Q-cytochrome c oxidoreductase catalyzes the transfer of electrons from Q to cytochrome c – What is a cytochrome? • electron transferring protein with heme prosthetic group • transfers only electrons • iron in heme goes between Fe+2 and Fe+3

Electron Transport • Q-cytochrome c oxidoreductase contains 3 hemes and a iron-sulfur cluster

Electron Transport • What is the Q cycle? – mechanism of coupling of electron transfer from Q to cytochrome c to proton transport

Electron Transport • What is the function of cytochrome c oxidase? – reduction of oxygen to water • What are the major prosthetic groups of this complex? – Cu. A/Cu. A – heme a 3 -Cub

Cytochrome c Oxidase

Cytochrome c Oxidase • Mechanism of action

Cytochrome c Oxidase • cytochrome c oxidase pumps four additional protons from matrix for a total of eight protons removed from matrix

Electron Transport System

Electron Transport System

Electron Transport • Toxic derivatives of molecular oxygen may be formed by partial reduction _ O 2 superoxide anion _ O 2 2 peroxide

Electron Transport • How does the cell protect itself against these reactive oxygen species? – makes use of superoxide dismutase and catalase _ – 2 O 2 + 2 H+ O 2 + H 2 O 2 – 2 H 2 O 2 + 2 H 2 O

ATP Synthesis • What is the chemiosmotic hypothesis? – ATP synthesis and electron transport are coupled by proton gradient across mitocondrial membrane

ATP Synthesis • What is ATP synthase and what do we know about its structure? – consists of F 1 and F 0 – F 1 has 5 types of polypeptide chains • 3, 3, , , – F 0 contains proton channel • 10 -14 c subunits • a, b 2 subunits

ATP Synthesis • How is ATP synthesized?

ATP Synthesis • What is the role of the proton gradient in ATP synthesis? – part of binding-change mechansm • 3 subunits promote ADP & P binding, ATP synthesis, ATP release

ATP Synthesis • How does proton flow through F 0 drive the rotation of the subunit? – each c subunits consists of 2 helices with one helix containing an aspartic acid residue – a subunit contains two proton half channels

ATP Synthesis • Proton enters half-channel, neutralizes charge on aspartate • c can rotate clockwise • proton can move into matrix

ATP Synthesis • Since c ring is linked to and subunits, as c turns these subunits rotate – rotation protmotes synthesis of ATP via binding -change mechanism – each 3600 rotation of subunit leads to synthesis of 3 ATP’s • 10 protons generate 3 ATP’s • each ATP requires transport of about 3 protons

Mitocondrial Shuttles • Reoxidation of cytosolic NADH requires shuttle mechanism – glycerol 3 -phosphate shuttle • found in muscle

Mitocondrial Shuttles • malate-aspartate shuttle – heart and liver

Mitocondrial Shuttles • What is an ATP-ADP translocase? – transport protein allowing ATP to exit mitocondrion and ADP to enter – result in moving one negative charge out of matrix • decreases proton motive force

Mitocondrial Shuttles • Other mitocondrial transport proteins act as shuttles

Regulation of Respiration • Energy formed from oxidation of glucose • 3 protons = 1 ATP • 1 proton used to move ATP • one pair of electons from NADH = 2. 5 molecules of ATP

Regulation of Respiration • What controls rate of electron transport?

Regulation of Respiration • Oxidative phosphorylation can be inhibited by many substances

Regulation of Respiration • What are uncoupling agents? – transport protons across mitocondrial membrane

Regulation of Respiration • Does uncoupling serve any useful purpose? – body heat generation