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 [Pelletier & 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 ka = 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 kb = 5 x 10(7) M-1 s-1. The bimolecular rate constants ka and kb 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 ka and kb, 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 ka and kb values as the CcP parent.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville 72701.
M A
MA
Miller
R Q
RQ
Liu
S
S
Hahm
L
L
Geren
S
S
Hibdon
J
J
Kraut
B
B
Durham
F
F
Millett
D000818:Animals; D018360:Crystallography, X-Ray; D003574:Cytochrome c Group / Q000737:chemistry*; D003578:Cytochrome-c Peroxidase / Q000737:chemistry*; D005290:Ferric Compounds / Q000737:chemistry; D005609:Free Radicals; D005656:Fungal Proteins / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D006736:Horses; D007700:Kinetics; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship
D003574:Cytochrome c Group; D005290:Ferric Compounds; D005609:Free Radicals; D005656:Fungal Proteins; D011994:Recombinant Proteins; D006418:Heme; D003578:Cytochrome-c Peroxidase