TCA Cycle Voet Biochemistry 3e
Voet Biochemistry 3e
The Electron Transport System (ETS) and Oxidative Phosphorylation (OxPhos) We have seen that glycolysis, the linking step, and TCA generate a large number of reduced cofactors, mostly NADH. This will later be seen to be true for β-oxidation of lipids as well. The paths we discussed now pass those electrons to an eager receptor, oxygen, and the released energy is converted to the more common ATP coinage. Standard state free energies show: Voet Biochemistry 3e C 6 H 12 O 6 + 6 O 2 6 CO 2 + 6 H 2 O G o = -2823 kj/mol And we want to see how much of that we can get back. Recall, anaerobic glycolysis only gave us 2 ATPs/glucose
Figure 22-1 The sites of electron transfer that form NADH and FADH 2 in glycolysis and the citric acid cycle. 2 ATP C 6 H 12 O 6 Voet Biochemistry 3e Page 798 + 6 H 2 O 6 CO 2 + 10 NADH + 2 FADH 2 + 2 ATP + 2 GTP 2 GTP
Porins O 2, CO 2 H 2 O Rough ER Voet Biochemistry 3e Page 799 Figure 22-2a Mitochondria. (a) An electron micrograph of an animal mitochondrion. (b) Cutaway diagram. Matrix: PDC / TCA / β-oxid. Inner Membrane: ET / OxPhos
Voet Biochemistry 3e Mitochondria are selectively permeable to a range of chemicals. The outer membrane passes most <10K, but the inner membrane is more selective; it has a range of transporters and carriers.
Converting cytoplasmic NADP to ATP A cytosolic transport system, the glycerophosphate shuttle. Voet Biochemistry 3e Page 802
Transport of cytosolic NADH Figure 22-7 The malate aspartate shuttle. Voet Biochemistry 3e Page 801
Overview of the Electron Transport System Voet Biochemistry 3e
Figure 22-13 Determination of the stoichiometry of coupled oxidation and phosphorylation (the P/O ratio) with different electron donors. Page 807 Voet Biochemistry 3e This classic experiment uses an oxygraph to measure lose of O 2. At time 0, 90 umoles ADP and excess substrate are added. It runs until substrate is exhausted and we observer 15 umoles O 2, ie 30 umoles O are consumed. P/O ration is thus 90/30 = 3. C1 is blocked by rotenone and ADP + succinate added. 90 umoles ATP are produced from 22.5 umoles O 2, so P/O =2. Etc. In each case, ADP is limiting, not the organic substrate.
Voet Biochemistry 3e Researchers spent years searching for direct phosphorylation, creation of ATP, at each of the 3 major complexes. That is, they THOUGHT the ETS created ATP in the way glycolysis made it, by enzymatic transfer of phosphate to ADP.
43 proteins 900 kda 11 proteins 250 kda 13 proteins 160 kda Voet Biochemistry 3e Page 808 Complex II Figure 22-14 The mitochondrial electron-transport chain. The purpose of an ETS is to take electrons from an electron rich reduced compound, and hand them to an oxidizing agent, O 2, tapping off the difference in energy.
Figure 22-9 The mitochondrial electron-transport chain. Voet Biochemistry 3e Page 803 There is a sufficient voltage drop at each complex to generate ATP.
A worked example Note: written as oxidation. In the first stage of the ETS, electrons from NADH are passed through complex 1 to CoQ. NADH is oxidized and CoQ reduced. The overall reaction, and subsequent voltage drop is: NADH + H + NAD + + 2e - +2H + E 0 = 0.315 V CoQ + 2e - + 2H + CoQH 2 E 0 = 0.045 V NADH + H + + CoQ NAD + + CoQH 2 E 0 = 0.36 V In terms of more conventional free energy measures: Voet Biochemistry 3e G 0 = -nfe 0 = -2 x 96 KJ/molV x 0.36 V = -69.5 KJ/mol Or: G 0 = -nfe 0 = -2 x 23 Kcal/molV x 0.36 V = -16.6 Kcal/mol
Energetics of the ETS We want to take electrons from NADH and drop them in energy to oxygen. Voet Biochemistry 3e
ETS energy retrieval Voet Biochemistry 3e
Electron transport involves metals and other redox susceptible chemicals. Voet Biochemistry 3e
Figure 22-17 (a) FMN (b) CoQ Voet Biochemistry 3e Page 810
Figure 22-15a Structures of the common iron sulfur clusters. (a) [Fe S] cluster. (b) [2Fe 2S] cluster (c) [4Fe 4S] cluster Voet Biochemistry 3e Page 808 ferredoxin
Figure 22-22a Porphyrin rings in cytochromes. Voet Biochemistry 3e Page 813
The complex contains a flavoprotein (blue), an iron-cluster protein (red) and a membrane spanning proteins (green and purple) Voet Biochemistry 3e Page 811 Figure 22-19a X-Ray structure of E. coli quinol fumarate reductase (QFR) in complex with its inhibitor oxaloacetic acid (OAA). (a) Ribbon diagram. (b) QFR s redox cofactors (Homolog of Complex II Succinate CoQ reductase)
Complex III 11 Subunits Voet Biochemistry 3e Page 814 Figure 22-23a X-ray structures of cytochrome bc 1
Figure 22-23b X-ray structures of cytochrome bc 1. (b) The yeast enzyme in complex with cytochrome c and the inhibitor stigmatellin viewed with a ~90 rotation about its 2-fold axis. Voet Biochemistry 3e Page 814 Complex III 11 Subunits
Complex III is known in some detail Voet Biochemistry 3e
The Q cycle of complex III reveals the details of the proton pump. Voet Biochemistry 3e
Complex IV Voet Biochemistry 3e Page 816 Figure 22-25c X-Ray structure of fully oxidized bovine heart cytochrome c oxidase. (c) A protomer viewed similarly to Part a showing the positions of the complex s redox centers.
Complex IV Figure 22-26 The redox centers in the X-Ray structure of bovine heart cytochrome c oxidase. Voet Biochemistry 3e Page 818
The exact mechanism of O 2 reduction to water is uncertain. The trick is to provide 4 electrons sequentially and maintain stable intermediates. Voet Biochemistry 3e Page 819 Figure 22-28 Proposed reaction sequence for the reduction of O 2 by the cytochrome a 3 Cu B binuclear complex of cytochrome c oxidase.
Oxidative phosphorylation Voet Biochemistry 3e
Coupling of Electron Transport with ATP Synthesis Chemiosmotic Hypothesis Proton Gradient Voet Biochemistry 3e
Walker won the 1997 Nobel Prize in Chemistry for this work. Voet Biochemistry 3e
Voet Biochemistry 3e Page 828 Figure 22-38a X-Ray structure of F 1 ATPase from bovine heart mitochondria. (a) A ribbon diagram. (b) Cross section through the electron density map of the protein.
Architecture of the ATP Synthase F 1 α3β3 γ1δ1ε1 F 0 a1b2c9-11 Voet Biochemistry 3e
Mechanism of ATP Synthesis. Voet Biochemistry 3e
Energetics of ATP Synthesis Voet Biochemistry 3e
Voet Biochemistry 3e
Voet Biochemistry 3e Page 832 Figure 22-44b Rotation of the c- ring in E. coli F 1 F 0 ATPase. (a) The experimental system used to observe the rotation.(b) The rotation of a 3.6-µm-long actin filament in the presence of 5 mm MgATP as seen in successive video images taken through a fluorescence microscope.
Figure 22-42 Energy-dependent binding change mechanism for ATP synthesis by proton-translocating ATP synthase. Voet Biochemistry 3e Page 831
Membrane transporters complement the synthesis of ATP in the mitochondria Voet Biochemistry 3e
DNP Figure 22-46 Uncoupling of oxidative phosphorylation. Voet Biochemistry 3e Page 834
Page 835 Figure 22-47 Mechanism of hormonally induced uncoupling of oxidative phosphorylation in brown fat mitochondria. Voet Biochemistry 3e