CHARGE REVERSAL OF A CRITICAL ACTIVE-SITE RESIDUE OF CYTOCHROME-C PEROXIDASE - CHARACTERIZATION OF THE ARG48-]GLU VARIANT

A new variant of cytochrome-c peroxidase in which the positively charg ed Arg48 present in the distal heme-binding pocket has been replaced w ith a Glu residue has been prepared and characterized to explore, in p art, the possibility that a negative charge close to the heme could co ntribute to stabilization of a porphyrin-centered pi-cation radical in the compound I derivative of the variant. Between pH 4 and 8, this va riant forms three pH-linked spectroscopic species. The electronic abso rption and H-1-NMR spectra of the predominant form at low pH (HS1) are indicative of a high-spin, pentacoordinate heme iron system. Near neu tral pH, a second high-spin species (HS2) is dominant, in which the he me iron center is hexacoordinated, with a water molecule as the sixth axial ligand. At high pH, the third form (LS) exhibits the spectroscop ic characteristics of a low-spin, hexacoordinate heme center with bish istidine axial ligation. The apparent pK(a) values for these transitio ns are 4.4 and 7.4, respectively, in phosphate buffers and 5.0 and 7.1 , respectively, in phosphate/nitrate buffers. Replacement of Arg48 wit h Glu reduces the thermal stability of the enzyme and also decreases t he Fe(III)/Fe(II) reduction potential of the enzyme by approximately 5 0 mV relative to that of the wild-type enzyme. The stability of compou nd 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 e nzyme, thus confirming previous results which indicate that the functi on of residue 48 in the wild-type peroxidase is more related to the st ability of compound I than to its formation [Erman, J. E., Vitello, L. B., Miller, M. A. & Kraut, J. (1992) J. Am. Chem. Sec. 114, 6592-6593 ; Vitello, L. B., Erman, J. E., Miller, M. A., Wang, J. & Kraut, J. (1 993) Biochemistry 32, 9807-9818]. Stopped-flow studies failed to detec t even transient formation of a porphyrin-centered radical following a ddition of hydrogen peroxide to the Fe(III)-enzyme. The consequences o f this drastic electrostatic modification of the active site on the st eady-state kinetics of the variant are relatively minor.