Effects of surface amino acid replacements in cytochrome c peroxidase on intracomplex electron transfer from cytochrome c.
Transient absorption techniques were used to measure the intracomplex electron transfer rates between four recombinant yeast cytochrome c peroxidases and iso-1 cytochrome c (cytc). The binding affinities and catalytic activities with cytc were previously examined [Corin et al. (1991) Biochemistry 30, 11585]. The four include a wild-type peroxidase (ECcP) and three others, each of which has one surface aspartic acid converted to lysine at position 37, 79, or 217. These sites have been suggested to be within or proximal to the recognition site for cytc. These mutants conduct electron transfer with cytc but differ with respect to the ionic strength profiles of their limiting rate constants. At pH and mu = 114 mM, ECcP and D217K show similar limiting rate constants for electron transfer with cytc, k(lim), of ca. 2000 s-1. In the same peroxidase concentration range, the D37K mutant exhibits a k(obs) of ca. 100 s-1. Instability of the compound I form of D79K prevented a complete study of the intracomplex kinetics of this mutant by this technique. At pH 6 and low ionic strength (8 mM), D37K exhibits a dramatic increase in k(obs) to ca. 800 s-1 while the other two recombinants show a marked decrease to values < 150 s-1. D37K displays much lower affinity for cytc than do the other peroxidases at higher ionic strengths [Hake et al. (1992) J. Am. Chem. Soc. 114, 5442], thus preventing adequate complexation necessary for efficient electron transfer. Variations in binding affinity do not explain the more subtle ionic strength kinetic profile observed for D217K.(ABSTRACT TRUNCATED AT 250 WORDS)
NSF Center for Photoinduced Charge Transfer, University of Rochester, New York 14627.
A F
AF
Corin
R A
RA
Hake
G
G
McLendon
J T
JT
Hazzard
G
G
Tollin
D000595:Amino Acid Sequence; D003574:Cytochrome c Group / Q000378:metabolism*; D003578:Cytochrome-c Peroxidase / Q000235:genetics / Q000378:metabolism*; D045304:Cytochromes c; D004579:Electron Transport; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine; D010782:Photolysis; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c; D003578:Cytochrome-c Peroxidase; D008239:Lysine