Cytochrome lysine modification

Figure 10. View of cytochrome c with positions of exposed heme edge (block) and lysine modifications (sequence numbers for a-carbons) shown. The smaller circles indicate the relative effectiveness of modifications on rate constants for the reaction of Cyt c(II) + PCu(II) ( see Table III). Preferential interaction with PCu(II) in the direction 25,27,13,87 is indicated.

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. 

H. Cai, F. P. Guengerich, Acylation of Protein Lysines by Trichloroethylene Oxide , Chem. Res. Toxicol. 2000,13, 327 - 335 H. Cai, F. P. Guengerich, Reaction of Trichloroethylene and Trichloroethylene Oxide with Cytochrome P450 Enzymes Inactivation and Sites of Modification , Chem. Res. Toxicol. 2001, 14, 451 - 458. 

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. 

The copper metalloenzymes are involved in oxygen-using reactions. These enzymes include cytochrome c oxidase (respiratory chain), lysyl oxidase (collagen synthesis), and dopamine [3-hydroxylase (neurotransmitter synthesis). Lysyl oxidase is a small protein with a molecular weight of 32 kDa. This enzyme contains an unusual modification, namely cross-linking between two different parts of its polypeptide chain. The cross-linked region consists of a structure called lysine tyrosylquinone (Klinman, 1996). Two amino acids are involved in this cross-linked structure, and these are Lys 314 and Tyr 349. Lysine tyrosylquinone is used as a cofactor and is necessary for the catalytic activity of the enzyme. Other copper metalloenzymes contain a related cofactor, namely 2,4,5-tiihydrox5q5henylalanine (topaquinone, TPQ). Serum amino oxidase is a copper metalloenzyme that contains TPQ. TPQ consists of a modified residue of phenylalanine. The copper in the active site of the enzyme occurs immediately adjacent to the TPQ cofactor. 

The e-N-methyllysines have been found in histones (see DeLange and Smith, 1971, 1974), cytochromes c (see DeLange et al. 1970), flagellin (see Glazer et al. 1969), ribosoraal proteins (Comb et al. 1966) and muscle proteins (see Paik and Kim, 1971 for a review). In some proteins (e.g. calf thymus histone III) e-N-monomethyllysine, e-N-dimethyl-lysine and e-N-trimethyllysine are all three present, whereas in other proteins only one derivative (e.g. cytochromes c) or two of the derivatives (e.g. calf thymus histone IV) are present. Methylated lysines can also be formed in proteins by chemical modification in vitro ( 3.1.1.3). 

The involvement of lysine amino acid residues on cytochrome c in the heterogeneous reactions with functionalized electrodes seems to have been established. Importantly, it is now thought that the proposed protein-promoter complex is more likely to be dynamic, as revealed by the results of a recent investigation (28) of site-specific 4-chloro-3,5-dinitrophenyl (CDNP)-substituted cytochrome c. It was found that monosubstitution of either Lys 13 or Lys 72 did not result in any significant change in its electrochemical response, whereas two modifications greatly decreased the heterogeneous rate constant, and complete loss of electrochemical activity was observed upon modification of more lysines. It was proposed that the electrode reaction occurred in numerous rotational conformations. Therefore, for the mono-... 

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. 

The useful lysine derivatives for cytochrome c are shown in Fig. 17. Chemical modifications of the lysine side chains which remove the positive charge or replace it with a negative charge destroy the interaction with cytochrome oxidase (213-215). Results with acetylation, succinyla-tion, trinitrophenylation, and guanidination are given in Table XIV. Suc-... 

Fig. 17. Chemical modifications of lysine side chains which have proved useful in studying cytochrome c mechanisms. See also M. J. Wimmer, M. Foster, K. T. Mo, D. L. Sawyers, and J. H. Harrison, Fed. Proc., Fed. Amer. Soc. Exp. Biol. Abstr. 34, 630 (1975).

Halpert, J. (1981). Covalent modification of lysine during the suicide inactivation of rat liver cytochrome P-450 by chloramphenicol. Biochem. Pharmacol. 30, 875-881. 

CHEMICAL MODIFICATION OF THE LYSINE RESIDUES ON PLASTOCYANIN AND THE EFFECT ON CYTOCHROME f. 

Adamovich TB, Pikuleva lA, Chashchin VF, Us-anov SA (1989) Selective chemical modification of cytochrome P-450SCC lysine residues. Identification of lysines involved in the interaction with ad-renodoxin. Biochim Biophys Acta 996 247-253... 

Theodorakis JL, Garber EAE, McCracken J, Peisach J, Schejter A, Margoliash E. 1995. A chemical modification of cytochrome-c lysines leading to changes in heme iron ligation. Biochim Biophys Acta 1252(1) 103-113. 

Covalent modifications of proteins serve many purposes (1-4). Some are structural and affect the three dimensional structure of proteins, such as disulfide bonds or cross linking of collagen chains via allysine side chains. There are a many different modifications that allow for the attachment of a variety of nonpeptide prosthetic groups to the protein. The attachment of the heme group to cysteine in c type cytochromes and that of biotin or pyridoxal phosphate to lysine are but a few examples. Some processes, such as cysteine isoprenylation or N myristoylation, allow proteins to become tightly associated with membranes. In other situations, a protein may be regulated by a reversible reaction, such as phosphorylation. The best know examples of this are serine, threonine, and tyrosine phosphate esters. In many other cases, the function of a particular modification is less evident. 

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