4.1 Oxidative Phosphorylation
Oxidative phosphorylation takes place in the inner mitochondrial membrane (Figure 32).1,2 Mitochondria are oval-shaped organelles, typically about 2um in length and 0.5 um in diameter, and are located outside of the nucleus of the cell. Mitochondria are the site of aerobic respiration within the cell. Bounded by an inner and outer phospholipid bilayer membrane, mitochondria are the powerhouse of energy for the cell. The inner membrane is an extensive, highly folded membrane with many convolutions and a series of internal ridges. Furthermore, the inner membrane has a high protein content that includes the proteins of the electron transport chain. The area between the inner and outer membrane is known as the inter-membrane space. The area bounded by the inner membrane is known as the mitochondrial matrix. Most of the reactions of the citric acid cycle and fatty acid oxidation occur in the matrix.
Figure 33 is an illustration of the oxidative phosphorylation chain found in the inner membrane of the mitochondria.3 I is NADH dehydrogenase, II is succinate dehydrogenase, C is cytochrome c, Fo and F1 belong to the ATPase, cytochrome c oxidase is shown where oxygen is reacting, and cytochrome c reductase is between II and cytochrome c oxidase. Two electrons are transferred from NADH to NADH dehydrogenase. Likewise, succinate provides reducing equivalents to succinate dehydrogenase. Ubiquinone or coenzyme Q collects reducing equivalents and shuttles them to cytochrome c reductase through a process known as the Q cycle. Incidentally, the Q cycle is believed to serve the purpose of transferring electrons as well as transporting protons from the matrix side of the inner membrane to the cytosolic side of the membrane. Cytochrome c is reduced by cytochrome c reductase on the outer face of the cytosolic side of the inner membrane. The reduced cytochrome c is then oxidized by cytochrome c oxidase. Finally, Cytochrome c oxidase catalyzes the transfer of electrons to molecular oxygen. Four electrons are funneled into oxygen to reduce it to water, and concurrently protons are pumped from the matrix to the cytosolic side of the inner mitochondrial membrane. This chain of enzyme complexes produces a pH difference across the membrane of approximately one pH unit. This electromotive gradient is called the proton-motive force and is responsible for the driving of protons back across the inner membrane and into the matrix. Since the membrane is impermeable to ions, the protons must flow through specialized channels of the enzyme complexes called ATP synthetases. As the protons pass through the ATP synthetases, energy is released to promote the phosphorylation of ADP to ATP.
ADP + Pi + H+ Û ATP + H2O D G° ¢ = +7.3 kcal/mol
The phosphorylation of ADP to ATP provides energy for endergonic reactions including muscle contraction, motility, and the active transport of substances.
Oxidative phosphorylation involves the oxidation of NADH and the phosphorylation of ADP. ATP, or adenosine triphosphate (Figure 34), serves as the principal source of energy in biological systems. ATP is composed of the nitrogenous base adenine, the sugar ribose, and three weakly linked phosphate groups.
Mitochondrial cytochromes c are the most extensively studied electron-transfer proteins (Figure 35).4-6 For example, the cytochrome c amino acid sequences have been determined for organisms including humans, chimps, rhesus monkeys, spider monkeys, horses, donkeys, zebras, cows, pigs, sheep, camels, great whales, elephant seals, dogs, hippos, bats, rabbits, bull frogs, starfish and fruit flies. Cytochrome c is easily separated from its mitochondrial environment because of its solubility in water. Furthermore, cytochrome c is weakly associated with the inner membrane space. Cytochrome c is readily available in pure and native form, although it is expensive. For one gram of cytochrome c: $108.60 from bovine heart to $27,700 from rat heart. The crystal structures of mitochondrial cytochrome c from several sources have been determined to atomic resolution. The interpretation of the physiochemical properties of cytochrome c is facilitated by the knowledge of conformation.
Cytochrome c (MW 12, 400) consists of a single polypeptide chain of 104 amino acid residues and covalently attached to a heme group.7-11 They are roughly spherical with a diameter of 34Å. The heme is surrounded by many tightly packed hydrophobic side chains. Only one edge of the planar heme ring is accessible to the surface. Small channels on either side of the ring permit only small hydrophobic molecules to the heme iron. The mechanism of electron transfer is unclear; however, it is believed that features on the surface of the molecule near the heme may be involved in electron-transfer activities. The cationic side chains of several lysine and arginine residues are clustered at the surface on one face of the molecule and are thought to provide a binding site for anionic groups on cytochrome oxidase. Likewise, on the opposite face of the molecule, a cluster of anionic residues including glutamic and aspartic acids may provide a binding site for a reductase or other components of the electron transport systems. Cytochrome c has 19 positively charged lysine residues, plus two arginines also positively charged, but only 12 acidic residues (aspartic or glutamic acids). Cytochrome c is very basic with an isoelectric point near pH 10. Isoelectric point is the pH at which the number of positive charges and the number of negative charges of a compound are equal.
Heme is an important prosthetic group in which the ferrous iron, Fe (II), is coordinated with the four nitrogen atoms of a type of tetracyclic aromatic substance known as a porphyrin.1 This non-polypeptide unit is essential for the physiochemical activity of cytochrome c (Figure 36). The iron atom is the redox component of cytochrome c and is low spin in an octahedral environment. Furthermore, iron oscillates between the ferrous [Fe(II)] and ferric [Fe(III)] states. The axial coordination sites are occupied by two strong field ligand ( acceptors) - the imidazole nitrogen atom of histidine 18 and the sulfur atom of methionine 80. The porphyrin ring occupies the equatorial ligand sites.
The exact Ferricytochrome c structure has not yet been determined conclusively. At high pH, the Met80 ligand to the iron is replaced by some other amino acid residue. The most frequent suggestion concerning the identity of new ligand at high pH is that it is an epsilon-amino group from a lysine residue a substitution that would require a major conformational change in the protein.
4.3 Conformations of Cytochrome
Throrell and Akesson12-14 (1941) showed that horse ferricytochrome c has 5 pH dependent conformational states and ferrocytochrome c has three as proposed on the basis of spectrophotometric measurements (Figure 37). Additional states of ferricytochrome c were reported by Myer et al.15 using Resonance Raman spectroscopic measurements. Subsequent work has revealed that there are more conformational states at the extremes of pH, and four states were confirmed electrochemically by Ikeshoji et al. (Figure 38).16 Other concepts to consider include conformational differences between two redox states, dynamic fluctuations in the cytochrome c structure, the physiological relevance of conformational transitions, structural and functional effects of chemical modifications on mitochondrial cytochrome c, and conformational differences and functional significance of pH dependent transitions. Most effort has been directed at understanding the structural and functional properties of the form of the protein that exists in the form of the protein that exists at neutral pH (the native form).
Greenwood and Palmer17 and Wilson and Greenwood18 showed the existence of two functionally distinct forms of ferricytochrome c at alkaline pH. Only one form was capable of reacting with ascorbate or tetrachlorohydroquinone. They are linked by a pH-dependent equilibrium (Figure 39)17 where A (ferricytochrome c) is irreducible by ascorbate, B (ferricytochrome c) is reducible by ascorbate, and C is ferrocytochrome c.
A and B are in equilibrium. K1 and K-1 are first order rate constants governing the interconversion of the conformers. K2 is the pseudo first order rate constant for the reduction at a fixed ascorbate concentration. The ascorbate concentration is considered to be very much higher than the total cytochrome c concentration and the back reaction C Þ B is neglected.
Figure 4018 is a minimal scheme describing the proton and conformational equilibria involved in the alkaline conversion of both ferri- and ferrocytochrome c. The pH-linked alkaline conformational change for ferrocytochrome c and the reoxidation of the alkaline form of ferrocytochrome c is suggested. N is the native Met/His ligation. A is the proposed alkaline Lys/His ligation. O and R are the oxidized and reduced forms of iron. * is the singly deprotonated state with respect to the native structure. K01, K02, KR1, KR2 are rates describing conformational changes. This scheme omits the changes in protonation accompanying the change of redox state in each conformational state.
Upon binding to its redox partners, cytochrome c may undergo some of the same or related conformational changes. Furthermore, site-specific variants of cytochrome c have been described that are drastically altered in their conformational stability.19,20 Amino acid substitution at certain positions in cytochrome c yield proteins in which the alkaline form is the most thermodynamically stable form of cytochrome c at neutral pH. Amino acid substituents have been made to alter the ligands to the heme iron, in various species of cytochrome c and resultant proteins exhibited multiple conformers. An understanding of the structure and properties will greatly enhance the ability to design novel heme-containing proteins rationally.21-29
1. Stryer, L. Biochemistry. New York: W.H. Freeman and Company, 1988.
2. Lehinger, A.L. Principles of Biochemistry. New York: Worth Publishers, 1982.
3. Palmer,G. Pure and Appl. Chem. 1987, 59, 749-757.
4. Bowden, E.F.; Hawkridge, F.M.; Blount, H.N. J. Electroanal. Chem. 1984, 161, 355- 376.
5. Funk, W.D.; Lo, T.P.; Mauk, M.R.; Brayer, G.D.; MacGillivray, R.T.A.; Mauk, A.G.
Biochemistry. 1990, 29, 5500-5508.
6. Caffrey, M.S.; Daldal, F.; Holden, H.M.; Cusanovich, M.A. Biochemistry. 1991, 30, 4119-4125.
7. Harbury, H.A.; Loach, P.A. J. Biol. Chem. 1960, 235, 3640-3645.
8. Dickerson, R.E.; Timkovich, R. The Enzymes. (P. Boyer Ed.) New York: Academic Press, 1975.
9. Wuthrich, K. Q. Rev. Biophys. 1985, 18, 111-134.
10. Marchon, J.C.; Mashiko, T.; Reed, C.A. Electron-Transport and Oxygen Utilization. (C.H.O. Ed.) North Holland, New York: Elsevier, 1982.
11. Raphael, A.L.; Gray, H.B. J. Am. Chem. Soc. 1991, 1038-1040.
12. Theorell, H.; Akesson, A. J. Am. Chem. Soc. 1941, 63, 1812-1818.
13. Theorell, H.; Akesson, A. Science. 1939, 67.
14. Pettigrew, G.W.; Moore, G.R. Cytochromes c: Evolutionary, Structural and Physiochemical Aspects. Berlin: Springer-Verlag, 1990.
15. Myer, Y.P.; Srivastava, R.B.; Kumar, S.; Raghavendra, K. J. Protein Chem. 1950, 182, 17.
16. Ikeshoji, T.; Taniguchi, I.; Hawkridge, F.M. J. Electroanal. Chem. 1989, 270, 297- 308.
17. Greenwood, C.; Palmer, G. J. Biol. Chem. 1965, 240, 3660-3663.
18. Wilson, M.T.; Greenwood, C. Eur. J. Biochem. 1971, 22, 11-18.
19. Barker, P.D.; Mauk, G. J. Am. Chem. Soc. 1992, 114, 3619-3624.
20. Nall, B.T.; Zuniga, E.H.; White, T.B.; Wood, L.C.; Ramdas, L. Biochemistry. 1989, 28, 9834-9839.
21. Sorrell, T.N.; Martin, P.K. J. Am. Chem. Soc. 1989, 111, 766-767.
22. Casmiro, D.R.; Wong, L.L.; Colon, J.L.; Zewert, T.E.; Richards, J.H.; Chang, I.J.; Winkler, J.R.; Gray, H.B. J. Am. Chem. Soc. 1993, 115, 1485-1489.
23. Sun, S.C.; Reed, D.E.; Cullison, J.K.; Rickard, L.H.; Hawkridge, F.M. Mikrochim. Acta. 1988, III, 97-104.
24. Reed, D.E.; Hawkridge, F.M.; Anal. Chem. 1987, 59, 2334-2339.
25. Koller, K.B.; Hawkridge, F.M.; Fauque, G.; LeGall, J. Biochem. Biophys. R. Comm. 1987, 145, 619-624.
26. Hildebrandt, P.; Heimburg, T.; Marsh, D.; Powell, G.L. J. Am. Chem. Soc. 1990, 29, 1661-1667.
27. Pearce, L.L.; Gartner, A.L.; Smith, M.; Mauk, A.G. Biochemistry. 1989, 28, 3152- 3156.
28. Armstrong, F.A.; Bond, A.M.; Hill, A.O.; Oliver, B.N.; Psalti, I.S.M. J. Am. Chem. Soc. 1989, 111, 9185-9189.
29. Long, R.C.; Hawkridge, F.M.; Hartzell, C.R.J. J. Electroanal. Chem. 1986, 198, 89- 98.