Biochemistry I Fall Term, 2004

December 3 & 6, 2004

Lecture 32: Oxidative Phosphorylation

Assigned reading in Campbell: Chapter 17 Key Terms: ATP synthase Chemiosmotic coupling Coenzyme Q (CoQ <=> CoQH2) Membrane potential Links: (I) Review Quiz on Lecture 32 concepts (I) ATP Synthesis: Flash tutorial of the FoF1 ATP synthase (S) Synthesis of ATP from the Proton Gradient: animation of the FoF1 ATP synthesis cycle. (S) ATP Synthase: Overall architecture of the FoF1 complex (Chime). (O) ATP Synthase: A detailed lecture by Antony Crofts, University of Illinois. (Be sure to watch the movie of the rotating F1 ATPase.) The overall reactions are: 1. O2 + 4 H+ + 4e- --> 2 H2O 2. nADP + nPi --> nATP Four Key Complexes in Electron Transport The following complexes are localized to the inner mitochondrial membrane. Complex I: NADH-CoQ oxidoreductase An integral membrane protein complex. Contains flavin mononucleotide (FMN) covalently attached to proteins; also contains iron-sulfur proteins. Two electrons from NADH are transferred to Coenzyme Q (CoQ). a. CoQ is mobile within the inner membrane. b. Cycles between CoQ <=> CoQH2. Two protons are pumped from the inside (matrix) to the intermembrane space. Proton gradient P/O ratios Respiratory inhibitors & uncouplers

Complex II: Succinate-CoQ oxidoreductase An integral membrane protein complex. Succinate dehydrogenase of the citric acid cycle is part of this complex. Two electrons from FADH2 are transferred to CoQ. Electrons are passed to cyctochromes. Does not pump any protons.

Complex III: CoQH2-cytochrome c oxidoreductase An integral membrane protein complex. Transfers electrons from CoQ to cytochrome c one electron at a time. Four protons are pumped for each pair of electrons transferred.

Complex IV: Cytochrome c oxidase An integral membrane protein complex. Contains cyt a and cyt a3 Accepts one electron at a time from cytochrome c. Donates a total of four electrons/O2. Site of oxygen reduction to water. Produces 2 water molecules/O2 molecule. Pumps an additional two protons across the membrane.

ATP Synthesis ATP synthesis occurs in the mitochondrial matrix. The endergonic formation of ATP from ADP + Pi is driven by two factors related to the electron transport described above. 1. The proton gradient. The transport of electrons caused protons to be pumped out of the matrix space. The resulting difference in pH can be coupled to ATP synthesis. 2. The membrane potential. The transport of H+ out leaves the matrix at a negative electrical potential relative to the cytosol. The resulting difference in membrane potential can be coupled to ATP synthesis.

The free energy available for ATP synthesis can be calculated by combining the terms for the above contributions: G = 2.3RTpH + ZF, where: pH = pHout - pHin; Z = the proton charge; F = the Faraday constant (96,494 J/volt-mol); = the membrane potential (volts). For typical values of these terms (pH = 1.0, and = -150 mV at 37C) G = -5.9 kJ/mol - 14.5 kJ/mol G = -20.4 kJ/mol Thus, both features of the proton gradient contribute to the energy available to synthesize ATP. The "ATP synthase motor" (FoF1 ATPase) converts the free energy of the proton gradient to chemical energy in the form of ATP. The Fo Complex Membrane-spanning, multiprotein complex (13 subunits: a, b2, and c10). Responsible for coupling the movement of three protons to 120 rotations of the csubunit ring and the subunit of the F1 complex. The antibiotic, oligomycin B, binds to Fo and prevents H+ transport. Hence the name, Fo.

The F1 Complex Five different subunits: 33 Attached to Fo, it protrudes into the mitochondrial matrix. The subunits are asymmetric due to their interactions with the Fo. 1. One subunit has very low affinity for both ADP and ATP. 2. One subunit has high affinity for ADP and Pi. 3. One subunit has high affinity for ATP. The subunit is the rotating shaft at the center of the 33 disk.

How the motor works (See the tutorial linked at the top of this page.) 3

Every time three protons are pumped, the F1 subunit rotates 120. The actual synthesis (formation of the bond between ADP and Pi is catalyzed by conformational changes of the enzyme that occur as a consequence of the rotation. The key point is that the rotation changes the subunit that contains ADP + Pi to a new conformation. In this new conformation the subunit would rather bind ATP, and thus catalyzes the formation of a ATP from the bound ADP and Pi. The newly-formed ATP is released with the transport of three additional protons. ATP can only be released from the low affinity subunit. Three protons must be transported to make one ATP.

The net gains by oxidative phosphorylation are, with (Campbell's "consensus values"): Compound NADH FADH2 Protons Pumped ~8 ~6 Maximum ATP Synthesized ~3 (2.5) ~2 (1.5)

Coupling Electron Transport to ATP Synthesis Mitchell's chemiosmotic theory. Requirement for an intact (i.e. ion-impermeable) membrane-enclosed space. Requirement for a proton concentration gradient (pH). The membrane potential is a driving force. ATP synthase is physically separate from electron flow (and proton pumping out of the mitochondrion). a. Intact mitochondria. b. Inside-out vesicles. c. Reconstituted vesicles with bacteriorhodopsin as H+ pumps. The "Racker-Stoeckenius Experiment". Uncouplers permit protons to flow in without ATP synthesis. a. Dinitrophenol (DNP) shuttles H+ across the membrane. 4

b. Some specific antibiotics create ion channels, e.g. gramicidin A. c. Beneficial uncouplers result in heat production where it is needed. Respiratory Inhibitors Act at Selected Sites. Complex I: Rotenone, a fish poison, is harmless to humans. Complex II: Antimycin A. Complex IV: CN-, N3-, and CO.

Inhibition at one site results in a highly reduced electron transport chain upstream and an oxidized chain downstream.

12.7.04