title: Reduction potential of yeast cytochrome c peroxidase and three distal histidine mutants: dependence on pH.
abstract: The pH dependence of the Fe(III) reduction potential, E(0)', for yeast cytochrome c peroxidase (yCcP) and three distal pocket mutants, CcP(H52L), CcP(H52Q), and CcP(R48L/W51L/H52L), has been determined between pH 4 and 8. E(0)' values at pH 7.0 for the yCcP, CcP(H52L), CcP(H52Q), and CcP(R48L/W51L/H52L) are -189, -170, -224, and -146mV, respectively. A heme-linked ionization in the reduced enzyme affects the reduction potential for yCcP and all three mutants. Apparent pK(A) values for the heme-linked ionization are 7.5±0.2, 6.5±0.3, 6.4±0.2, and 7.0±0.3 for yCcP and the H52L, H52Q, and R48L/W51L/H52L mutants, respectively. A cooperative, two-proton ionization causing a spectroscopically-detectable transition was observed in the ferrous states of yCcP, CcP(H52L) and CcP(H52Q), with apparent pK(A) values of 7.7±0.2, 7.4±0.1 and 7.8±0.1, respectively. These data indicate that: (1) the distal histidine in CcP is not the site of proton binding upon reduction of the ferric CcP, (2) the distal histidine is not one of the two groups involved in the cooperative, two-proton ionization observed in ferrous CcP, and (3) the proton-binding site is not involved in the cooperative, two-proton ionization observed in the reduced enzyme.
authors.affiliation: Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115, USA.
authors.forename: Cory M
authors.initials: CM
authors.lastname: DiCarlo
authors.forename: Lidia B
authors.initials: LB
authors.lastname: Vitello
authors.forename: James E
authors.initials: JE
authors.lastname: Erman
mesh_terms: D001665:Binding Sites; D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism
chemical_list: D006418:Heme; D006639:Histidine; D003578:Cytochrome-c Peroxidase