INTERACTION DOMAIN FOR THE REACTION OF CYTOCHROME-C WITH THE RADICAL AND THE OXYFERRYL HEME IN CYTOCHROME-C PEROXIDASE COMPOUND-I

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 [Pelletie r and Kraut (1992) Science 258, 1748-1755]. In the crystalline CcP-yCC complex, two acidic regions of CcP contact lysine residues on yCC. Mu tants E32Q, D34N, E35Q, E290N, and E291Q were used to examine the effe ct of converting individual carboxylate side chains in the acidic regi ons to amides. The A193F mutant was used to test the effect of introdu cing a phenyl moiety at the point of closest contact between CcP and y CC in the crystal structure. Stopped-flow experiments carried out in 3 10 mM ionic strength buffer at pH 7 revealed that yCC initially reduce d the indole radical on Trp-191 of the parent CcP compound I with a bi molecular rate constant k(a), = 2.5 X 10(8) M(-1) s(-1). A second mole cule of yCC subsequently reduced the oxyferryl heme of compound II wit h a rate constant k(b) = 5 X 10(7) M(-1) s(-1). The bimolecular rate c onstants k(a) and k(b), were affected in parallel by each mutation exa mined. 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 pa rent. Mutants E32Q and E291Q that are removed from the interaction dom ain gave the same k(a) and k(b) values as the CcP parent. The A193F mu tant 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 w ith horse CC were nearly the same as on the reaction with yCC, indicat ing that both cytochromes use the same interaction domain. The rate co nstants k(et) for intracomplex electron transfer from the heme group o f ruthenium derivatives of horse CC to the Trp-191 radical of CMPI wer e 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 a re 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 doma in used for both electron transfer reactions in solution resembles the interaction domain in the crystalline CcP-yCC complex.