Lysine residues cytochrome

Reaction with Chemically Modified Cytochrome c. Chemically modified (CDNP) cytochrome c derivatives have been prepared by Margoliash and colleagues (22). Lysine residues react as in (17),... 

In one series of experiments the cytochrome c oxidase mutations replaced acidic residues by neutral ones, and some of them were thus expected to alter the nature of binding of the protein to cytochrome c. From the pattern of dependence of the heme c to Cua electron-transfer rate constant on these mutations it was deduced that the binding of cytochrome c to cytochrome c oxidase is mediated by electrostatic interactions between four specific acidic residues on cytochrome c oxidase and lysines on cytochrome c. In another series of experiments, tryptophan 143 of cytochrome c oxidase was mutated to Phe or Ala. These mutations had an insignificant effect on the binding of the two proteins, but they dramatically reduced the rate constant for electron transfer from heme c to Cua- It was concluded that electron transfer from... 

Cleavage of the oxirane C-0 bond produces a zwitterionic intermediate (Fig. 10.22), which that can undergo chloride shift (Pathway a) to 2,2-dich-loroacetyl chloride (10.90) followed by hydrolysis to 2,2-dichloroacetic acid (10.91). Furthermore, the zwitterionic intermediate reacts with H20 or H30+ (Pathway b) by pH-independent or a H30+-dependent hydrolysis, respectively. The pH-independent pathway only is shown in Fig. 10.22, Pathway b, but the mechanism of the H30+-dependent hydrolysis is comparable. Hydration and loss of Cl, thus, leads to glyoxylyl chloride (10.92), a reactive acyl chloride that is detoxified by H20 to glyoxylic acid (10.93), breaks down to formic acid and carbon monoxide, or reacts with lysine residues to form adducts with proteins and cytochrome P450 [157], There is also evidence for reaction with phosphatidylethanolamine in the membrane. 

The involvement of the lysine residues has been explored. Thus, trifluoroacetylation of lysine residues 13, 55 and 99 has been carried out,662 but only modification of residue 13 affects the reaction with cytochrome oxidase. Modification of lysine residues 13, 25, 27, 72 and 79 decreased the reaction rate of cytochrome c with cytochrome bs. It is possible that the lysine groups in unmodified cytochrome c interact with the carboxylate groups in cytochrome b5 (Asp-48, Glu-43, Glu-44 and Asp-60) and one of the heme propionate groups.663 In general, such studies support the proposal that these lysine groups represent binding sites for redox partners of cytochrome c. 

The importance of lysine groups around the heme-binding crevice in cytochrome c as binding groups for its redox partners has already been stressed, and has been demonstrated by chemical modification experiments.662 The question of whether there are different sites for binding of complex III and complex IV is not fully resolved. However, the same lysine residues are shielded from acetylation by complexing cytochrome c with either of these complexes,725 implying that they bind at the same site and that therefore entry and exit of the electron follows the same route. 

Based on these studies and the known chemical properties of these cytochromes, in 1976 Salemme proposed a novel model for the strong noncovalent c/b5 complex. i This model is graphically shown in fig. A. Key features Include an electrostatic binding region in which several lysine residues on cytc align with appropriate acidic amino acids on cytochrome b5 to form strong "salt-links". 

Fig. 14.36. Structure of horse cytochrome c obtained from the Brookhaven Protein Data Bank. Shown is the conventional front face view highlighting the exposed edge of the heme group (dark gray) located in a region of positive surface charge resulting from several lysine residues. (Reprinted from E. Bowden, Wiring Mother Nature, Interface 6(4) 40—45, Fig. 1, 1997. Reproduced by permission of the Electrochemical Society, Inc.)...

Guanidylated cytochrome c (G-cytc) was synthesized from native cytochrome c (N-cytc) and O-methylisourea (Figure 14.9) [19]. Almost all of the lysine residues of cytochrome c were substituted with a homoaiginine moiety as seen in Table 14.2, but... 

Carnitine is synthesized from lysine and methionine by the pathway shown in Figure 14.2 (Vaz and Wanders, 2002). The synthesis of carnitine involves the stepwise methylation of a protein-incorporated lysine residue at the expense of methionine to yield a trimethyllysine residue. Free trimethyllysine is then released by proteolysis. It is not clear whether there is a specific precursor protein for carnitine synthesis, because trimethyllysine occurs in a number of proteins, including actin, calmodulin, cytochrome c, histones, and myosin. 

Figure 5 A schematic diagram of horse heart c)ftochrome c viewed from the front of the heme crevice. The approximate positions of the /S-carbon atoms of the lysine residues are indicated by closed and dashed circles for residues located toward the front and back of Cc, respectively. The electrostatic free energy contribution of lysine i, Vi, is indicated by the number of diagonal marks in the circle, with —0.4kJM per hatch mark. The binding domain is essentially the same for CcO and cytochrome bc ...

Electron transport between cubane [Fe4S4] clusters, cytochrome c, or Cu + centers in blue copper proteins " and the periphery of the proteins has been examined by complexing ruthenium species to surface histidines. In the case of the iron sulfur cubane in Chromatium vinosum, four surface histidines served as points of ruthenium attachment. The rates of electron transport from the Fe4S4 core to ruthenium varied over two orders of magnitude and were used to diagnose the preferred channel for electron transport. Cysteine and lysine residues have also been used as binding sites in studies of cytochrome c and cytochrome P450 cam proteins. 

Concern may arise that elimination of the basic lysine residues, especially with amino terminally blocked peptides, might preclude detection by positive ion mode mass spectrometry. However, of the seven chymotryptic peptides from cytochrome-C which do not contain any basic residues other than lysine, all seven were detected in the LC-MS experiment, even after re-acetylation. Only one of these peptides (mass 2094) exhibited a weak signal. Ions with two and even three positive charges were visible with fliese peptides. 

Mitochondrial cytochrome c is perhaps the most widely studied of all metalloproteins with respect to its electrochemical properties. It is located in the inner-membrane space of mitochondria and transfers electrons between membrane-bound complex III and complex IV. The active site is an iron porphyrin with a redox potential (7) of -1-260 mV vs. NHE. The crystal structures of cytochrome c from tuna have been determined (8, 9) in both oxidation states at atomic resolution. It is found that the heme group is covalently linked to the protein via two thioether bridges, and part of its edge is exposed at the protein surface. Cytochrome c is a very basic protein, with an overall charge of -1-7/-l-8 at neutral pH. Furthermore, many of the excess basic lysine residues are clustered around the mouth of the heme crevice, giving rise to a pronounced charge asymmetry. 

These results support the electrode reaction mechanism originally proposed by Hill et al. (17), i.e., hydrogen bonding between the lysine residues surrounding the exposed heme edge of cytochrome c and the pyridyl nitrogens at the electrode surface stabilizes a transient protein-electrode complex oriented so as to allow rapid electron transfer to and from the heme group. 

Because lysine residues 13, 27, 72, and 79 on cytochrome c are supposed to be involved in binding to cytochrome bg, it was interesting to see how modification of these residues would affect the electrochemistry of the complex. Several mutated forms of yeast iso-l-cytochrome c have been studied electrochemically as described above, and it was now possible to carry out such studies. 

Dbpner, S., Hildebrandt, P., Resell, E.L, Mauk, A.G., Walter, M.V., Buse, G., and Soulimane, T. (1999) The structural and functional role of lysine residues in the binding domain of cytochrome c in the electron transfer to cytochrome c oxidase. European Journal of Biochemistry, 261, 379-391. 

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