title: Charge reversal of a critical active-site residue of cytochrome-c peroxidase: characterization of the Arg48-->Glu variant.
abstract: A new variant of cytochrome-c peroxidase in which the positively charged Arg48 present in the distal heme-binding pocket has been replaced with a Glu residue has been prepared and characterized to explore, in part, the possibility that a negative charge close to the heme could contribute to stabilization of a porphyrin-centered pi-cation radical in the compound I derivative of the variant. Between pH 4 and 8, this variant forms three pH-linked spectroscopic species. The electronic absorption and 1H-NMR spectra of the predominant form at low pH (HS1) are indicative of a high-spin, pentacoordinate heme iron system. Near neutral pH, a second high-spin species (HS2) is dominant, in which the heme iron center is hexacoordinated, with a water molecule as the sixth axial ligand. At high pH, the third form (LS) exhibits the spectroscopic characteristics of a low-spin, hexacoordinate heme center with bishistidine axial ligation. The apparent pKa values for these transitions are 4.4 and 7.4, respectively, in phosphate buffers and 5.0 and 7.1, respectively, in phosphate/nitrate buffers. Replacement of Arg48 with Glu reduces the thermal stability of the enzyme and also decreases the Fe(III)/Fe(II) reduction potential of the enzyme by approximately 50 mV relative to that of the wild-type enzyme. The stability of compound I formed by the variant is decreased although the rate at which it forms is just one order of magnitude less than that of the wild-type enzyme, thus confirming previous results which indicate that the function of residue 48 in the wild-type peroxidase is more related to the stability of compound I than to its formation [Erman, J. E., Vitello, L. B., Miller, M. A. & Kraut, J. (1992) J. Am. Chem. Soc. 114, 6592-6593; Vitello, L. B., Erman, J. E., Miller, M. A., Wang, J. & Kraut, J. (1993) Biochemistry 32, 9807-9818]. Stopped-flow studies failed to detect even transient formation of a porphyrin-centered radical following addition of hydrogen peroxide to the Fe(III)-enzyme. The consequences of this drastic electrostatic modification of the active site on the steady-state kinetics of the variant are relatively minor.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Bujons
authors.forename: A
authors.initials: A
authors.lastname: Dikiy
authors.forename: J C
authors.initials: JC
authors.lastname: Ferrer
authors.forename: L
authors.initials: L
authors.lastname: Banci
authors.forename: A G
authors.initials: AG
authors.lastname: Mauk
mesh_terms: D001665:Binding Sites; D003578:Cytochrome-c Peroxidase / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D005656:Fungal Proteins / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D006420:Hemeproteins / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae; D013057:Spectrum Analysis; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D006420:Hemeproteins; D006418:Heme; D006861:Hydrogen Peroxide; D003578:Cytochrome-c Peroxidase