Interaction Domain for the Reaction of Cytochrome-C with the Radical and the Oxyferryl Heme in Cytochrome-C Peroxidase Compound-i
by Miller, Mark A.; Liu, Rui-Qin; Hahm, Seung W.; Geren, Lois M.; Hibdon, Sharon A.; Kraut, Joseph; Durham, Bill; Millett, Francis Spencer
Site-directed mutants of cytochrome c peroxidase (CcP) were created to modify the interaction domain between CcP and yeast iso-1-cytochrome c (yCC) seen in the crystal structure of the CcP-yCC complex [Pelletier and Kraut (1992) Science 258, 1748-1755]. In the crystalline CcP-yCC complex, two acidic regions of CcP contact lysine residues on yCC. Mutants E32Q, D34N, E35Q, E290N, and E291Q were used to examine the effect of converting individual carboxylate side chains in the acidic regions to amides. The A193F mutant was used to test the effect of introducing a phenyl moiety at the point of closest contact between CcP and yCC in the crystal structure. Stopped-flow experiments carried out in 310 mM ionic strength buffer at pH 7 revealed that yCC initially reduced the indole radical on Trp-191 of the parent CcP compound I with a bimolecular rate constant k(a), = 2.5 X 10(8) M(-1) s(-1). A second molecule of yCC subsequently reduced the oxyferryl heme of compound II with a rate constant k(b) = 5 X 10(7) M(-1) s(-1). The bimolecular rate constants k(a) and k(b), were affected in parallel by each mutation examined. CcP mutants D34N and E290N that are closest to a complementary yCC lysine residue in the crystalline CcP-yCC complex gave the lowest values for k(a) and k(b) which were 25-50% of the values of the CcP parent. Mutants E32Q and E291Q that are removed from the interaction domain gave the same k(a) and k(b) values as the CcP parent. The A193F mutant gave k(a) and k(b) values that were 25-45% of the values for the CcP parent. The effects of the mutations on the bimolecular reaction with horse CC were nearly the same as on the reaction with yCC, indicating that both cytochromes use the same interaction domain. The rate constants k(et) for intracomplex electron transfer from the heme group of ruthenium derivatives of horse CC to the Trp-191 radical of CMPI were measured by flash photolysis at low ionic strength. The values of k(et) varied with the mutant CcP enzymes, as well as the position of the ruthenium label on horse CC. The results of all the kinetic studies are consistent with the following conclusions: (1) the same interaction domain is used for the reactions of CC with the radical in compound I, and with the oxyferryl heme in compound II; (2) the interaction domain used for both electron transfer reactions in solution resembles the interaction domain in the crystalline CcP-yCC complex.
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