Effect of active site and surface mutations on the reduction potential of yeast cytochrome c peroxidase and spectroscopic properties of the oxidized and reduced enzyme.
The reduction potentials of 22 yeast cytochrome c peroxidase (CcP) mutants were determined at pH 7.0 in order to determine the effect of both heme pocket and surface mutations on the Fe(III)/Fe(II) redox couple of CcP, as well as to determine the range in redox potentials that could be obtained through point mutations in the enzyme. Spectroscopic properties of the Fe(III) and Fe(II) forms of the mutant enzymes are also reported. The mutations include variants in the distal and proximal heme pockets as well as on the enzyme surface and involve single, double, and triple point mutations. A spectrochemical redox titration technique used in this study gave an E(0') value of -189 mV for yeast CcP compared to a previously reported value of -194 mV determined by potentiometry [C.W. Conroy, P. Tyma, P.H. Daum, J.E. Erman, Biochim. Biophys. Acta 537 (1978) 62-69]. Both positive and negative shifts in the reduction potential from that of the wild-type enzyme were observed, spanning a range of 113 mV. The His-52-->Asn mutation gave the most negative potential, -259 mV, while a triple mutant in which the three distal pocket residues, Arg-48, Trp-51, and His-52, were all converted to leucine residues gave the most positive potential, -146 mV.
DiCarlo
Cory M
CM
Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115, USA.
Vitello
Lidia B
LB
Erman
James E
JE
D001665:Binding Sites; D020134:Catalytic Domain; D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005290:Ferric Compounds / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D017354:Point Mutation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship
D005290:Ferric Compounds; D005296:Ferrous Compounds; D006418:Heme; D003578:Cytochrome-c Peroxidase