Molecular Biochemistry I

Oxidative Phosphorylation: Chemiosmotic Coupling

Reading:

Textbook Reading: Biochemistry, 3rd Edition, by Voet & Voet, Chapter 22, especially p. 820-827.

Some recent articles (optional reading): 
T. Friedrich & B. Böttcher (2004) "The gross structure of the respiratory complex I: a Lego system," Biochim. Biophys. Acta 1608: 1-9.
C. Hunte, H. Palsdottir & B. L. Trumpower (2003) "Protonmotive pathways and mechanisms in the cytochrome bc₁ complex," FEBS Lett. 545: 39-46.
M. Wikström & M. I. Verkhovsky (2002) "Proton translocation by cytochrome c oxidase in different phases of the catalytic cycle," Biochim. Biophys. Acta 1555: 128-132.
C. R. D. Lancaster (2003) "The role of electrostatics in proton-conducting membrane protein complexes," FEBS Lett. 545: 52-60.
P. Brzezinski (2004) "Redox-driven membrane-bound proton pumps," Trends in Biochem. Sci. 29: 380-387.
A. R. Crofts (2004) "The cytochrome bc₁ complex: Function in the context of structure," Annu. Rev. Physiol. 66: 689-733.
A. Osyczka, C. C. Moser & L. P. Dutton (2005) "Fixing the Q cycle," Trends in Biochem. Sci. 30: 176-182.
S. Papa, N. Capitanio, G. Capitanio & L. L. Palese (2004) "Protonmotive cooperativity in cytochrome c oxidase," Biochim. Biophys. Acta 1658: 95-105.
A. Namslauer & P. Brzezinski (2004) "Structural elements involved in electron-coupled proton transfer in cytochrome c oxidase," FEBS Lett. 567: 103-110.
P. C. Hinkle (2005) "P/O ratios of mitochondrial oxidative phosphorylation," Biochim. Biophys. Acta 1706: 1-11.
H. Tedeschi (2005) "Old and new data, new issues: the mitochondrial DY," Biochim. Biophys. Acta 1709: 195-202.
J. P. Hosler, S. Ferguson-Miller & D. A. Mills (2006) "Energy transduction: proton transfer through the respiratory complexes," Annu. Rev. Biochem. 75: 165-187.
G. Brändén, R. B. Gennis & P. Brzezinski (2006) "Transmembrane proton translocation by cytochrome c oxidase," Biochim. Biophys. Acta 1757: 1052-1063.
A. Y. Mulkidjanian (2005) "Ubiquinol oxidation in the cytochrome bc₁ complex: Reaction mechanism and prevention of short-circuiting," Biochim. Biophys. Acta 1709: 5-34.
P. Brzezinski & P. Adelroth (2006) "Design principles of proton-pumping haem-copper oxidases," Current Opin. Struct. Biol. 16: 465-472.
A. R. Crofts, S. Lhee, S. B. Crofts, J. Cheng  & S. Rose (2006) "Proton pumping in the bc₁ complex: A new gating mechanism that prevents short circuits," Biochim. Biophys. Acta 1757: 1019-1034.
A. Y. Mulkidjanian, J. Heberle & D. A. Cherepanov (2006) "Protons @ interfaces: Implications for biological energy conversion," Biochim. Biophys. Acta 1757: 913-930.

Potential Test Questions:

1.a. Summarize the Chemiosmotic theory of oxidative phosphorylation. What is the nature of the coupling between electron transfer and ATP synthesis? Why is an intact membrane required for oxidative phosphorylation?
  b.  Describe and explain the effects of uncoupling reagents on mitochondrial respiration (oxygen consumption), on ATP synthesis, and on ATP hydrolysis. What is the mechanism of action of uncouplers?
  c. Write out an equation for the overall reaction catalyzed by respiratory chain complex III, including net inputs and outputs of the Q cycle.

2. Diagram and describe the effect of ADP addition on oxygen consumption by mitochondria in the presence of an excess of inorganic phosphate and an electron source (e.g., succinate). Emphasizing thermodynamic relationships (the spontaneity of coupled reactions), explain in relation to the Chemiosmotic theory the dependence of mitochondrial respiration on availability of ADP. Why is respiration (e.g., electron transfer from succinate to oxygen) inhibited in the absence of ADP? Why does addition of ADP in the presence of P_i stimulate respiration?

Studio activity: Problems

1. Predictions regarding respiratory control and P/O ratio.  

Based on these assumptions, what would be the predicted P/O ratio, i.e., the number of  ATP molecules synthesized per 2 e^- transferred from NADH to O₂? _________  

Complex II of the respiratory chain (Succinate Dehydrogenase) catalyzes transfer of 2 e^- from succinate to coenzyme Q (via FAD and Fe-S centers). What would be the predicted P/O ratio with succinate as electron source instead of NADH? _________     

2. ATP production with aerobic metabolism of glucose

For this calculation, assume that oxidative phosphorylation in mitochondria yields 2.5 ~P bonds of ATP per NADH oxidized (except 1.5 ~P for the NADH from Glycolysis), or 1.5 ~P bonds of ATP per coenzyme QH₂ oxidized.
Fill in the number of NADH, QH₂, and ~P bonds of ATP produced per glucose at each stage of the process, and add up the total. 
Question: How does the yield of ~P bonds in aerobic metabolism compare to the yield of ~P bonds from catabolism of glucose in an anaerobic organism?

3. Determination of respiratory control and P/O ratios

An oxygen electrode recording generated by an RPI undergraduate student is provided below. In this experiment, isolated rat liver mitochondria were suspended in buffered medium containing 200 mM sucrose (for osmotic support), 7.5 mM inorganic phosphate, and 7.5 mM succinate (for input of electrons to the respiratory chain via Succinate Dehydrogenase).
When indicated, 0.6 or 1.5 mmole ADP was added.
Assume that the 2.2 ml total volume was equilibrated with air at the outset, so the initial [O₂] was 0.24 mM. P/O ratios are calculated per oxygen atom (or per 2 electrons transferred), so the total oxygen present from the beginning of the recording is 2.2 ml X 0.48 mmol/ml of ½ O₂, or 1.06 mmol O.
For scaling, you need to estimate how many divisions (or mm) across the chart paper corresponds to this amount of O.

What is the respiratory control ratio, based on the ratio of slopes b/a during phosphorylation of ADP (b), and before addition of ADP (a). Respiratory control ratio: _____________

Some questions to be answered:

• Why is the respiration rate very low in the absence of ADP? What might be the basis for the finding that the rate of oxygen consumption is not zero in the absence of ADP?
• How might you explain the gradual change of slope (non-linearity) when ADP is nearly exhausted?
• How could you estimate the K_M for oxygen of cytochrome oxidase (complex IV) from the recording given?

Calculate the P/O ratio from the amount of ADP added, divided by the amount of oxygen used up while phosphorylating the ADP to ATP. Since the rate of oxygen consumption slows as [ADP] becomes low, you must use a ruler to estimate the intersection of the slopes before and after exhaustion of ADP.
P/O ratio estimated with succinate as e^- source (molecules of ATP synthesized per 2e^- transferred from succinate to ½O₂): _____________

Copyright © 1998-2007 by Joyce J. Diwan. All rights reserved.

Lecture notes on Oxidative Phosphorylation

Interactive Quiz on   Ox. Phosphorylation