COMPOUND-I RADICAL IN SITE-DIRECTED MUTANTS OF CYTOCHROME-C PEROXIDASE AS PROBED BY ELECTRON-PARAMAGNETIC RESONANCE AND ELECTRON NUCLEAR DOUBLE-RESONANCE

The reaction of ferric cytochrome c peroxidase (CcP) from Saccharomyces cerevisiae with peroxide produces compound I, characterized by both an oxyferryl iron center and a protein-based free radical. The electron paramagnetic resonance (EPR) signal of the CcP compound I radical can be resolved into a broad majority component which accounts for approximately 90% of the spin intensity and a narrow minority component which accounts for approximately 10% of the integrated spin intensity [Hori, H., & Yonetani, T. (1985) J. Biol. Chem. 260, 3549-3555]. It was shown previously that the broad component of the compound I radical signal is eliminated by mutation of Trp-191 to Phe [Scholes, C. P., Liu, Y., Fishel, L. F., Farnum, M. F., Mauro, J. M., & Kraut, J. (1989) Isr. J. Chem. 29, 85-92]. The present work probed the effect of mutations in the vicinity of this residue by EPR and electron-nuclear double resonance (ENDOR). These mutations were obtained from a plasmid-encoded form of S. cerevisiae expressed in Escherichia coli [Fishel, L. A., Villafranca, J. E., Mauro, J. M., & Kraut, J. (1987) Biochemistry 26, 351-360]. The EPR line shape and ENDOR signals of the compound I radical were perturbed only by mutations that alter Trp-191 or residues in its immediate vicinity: namely, Met-230 and Met-231, which have sulfur atoms within 4 angstrom of the indole ring, and Asp-235, which forms a hydrogen bond with the indole nitrogen of Trp-191. Mutations of other potential oxidizable sites (tryptophan, tyrosine, methionine, and cysteine) did not alter the EPR line shapes of the compound I radical, although the integrated spin intensities were weaker in some of these mutants. Mutations at Met-230 and/or -231 perturbed the EPR line shapes of the compound I radical signal but did not eliminate it. ENDOR of these two methionine mutants showed alteration to the hyperfine couplings of several strongly coupled protons, which are characteristic of the majority compound I radical electronic structure, and a change in weaker hyperfine couplings, which suggests a different orientation of the radical with respect to its surroundings in the presence of these methionine mutations. Besides the Trp-191 --> Phe mutation, only the Asp-235 --> Asn mutation eliminated the broad component of the compound I signal. Loss of the broad compound I EPR signal coincides with both the loss of the Asp --> Trp-191 hydrogen-bonding interaction and alteration of the position of the indole ring of Trp-191. These results argue against the involvement of a Trp other than Trp-191 in the formation of the compound I radical. In the Trp-191 --> Phe and Asp-235 --> Asn mutants, the narrow component of the EPR radical signal was detected, despite the loss of the broad component. The line shape of the narrow signals in these mutants, as well as the line shape of the narrow minority radical of CcP compound I with no site-directed mutations, resembled that observed for the Tyr radical of photosystem II of green plants. The presence of a narrow EPR signal in Phe-191 and Asn-235 mutants may indicate that replacement of Trp-191 or perturbation to its interaction with Asp-235 favors oxidation of tyrosine over tryptophan. The radical signal of the Asn-235 mutant differed from that of the Phe-191 mutant in EPR line shape, spin relaxation time, and solvent exposure. This, together with small differences in the narrow radical EPR line shapes among several other mutants, suggests that the narrow radical signal could arise from any of several sites. Such a conclusion is further supported by the failure to date of any single-site mutation to eliminate the narrow radical signal.