Studies of the radical species in compound ES of cytochrome c peroxidase altered by site-directed mutagenesis.

Abstract

Yeast cytochrome c peroxidase reacts with hydrogen peroxide to form an intermediate, compound ES, in which the heme iron atom is converted to a ferryl function (Fe4" =O) and a radical center is generated on a reversibly oxidizable amino acid residue of uncertain identity. As methionine-172 is a possible site of this radical, we have constructed specific variants of cytochrome c peroxidase in which methionine-172 is replaced by serine or cysteine. These mutants and the wild-type enzyme have been expressed in Saccharomyces cerevisiae, purified, and crystallized. Both mutant enzymes are fully active. A stable, reversible, peroxide-induced intermediate with optical properties characteristic of compound ES is observed for the three forms of the enzyme. The electron paramagnetic resonance spectrum of this intermediate at 93 K for the serine mutant exhibits the narrow free-radical signal and hyperfine structure observed for the wild-type enzyme. However, a broader component of the signal that is observed for the wild-type enzyme at this temperature is absent from the spectrum observed with the serine mutant. These results demonstrate that the narrow component of the free-radical signal observed at 93 K cannot reside at methionine-172. The absence of the broader component of the signal for the serine mutant may reflect the loss of spin density on methionine or, alternatively, could arise from conformationally induced changes in the properties of the radical. The results are consistent with a heterogeneity of radical species in the ES complex.

The Rise of Radicals in Bioinorganic Chemistry

“…[12] The advent of site-directed mutagenesis helped resolve some of the uncertainty surrounding the identity of the CCP ES radical. Dave Goodin, Grant Mauk, and Michael Smith demonstrated that Met 172 could not be the site of this radical, [13] and measurements from Kraut’s laboratory on the CCP Trp 51 Phe mutant demonstrated that this residue was not the source of the radical signal. [14] The X-ray crystal structure of CCP ES identified a new possible locus for the radical site, near a cluster composed of the Met 230 , Met 231 , and Trp 191 sidechains, about 10 Å from the proximal side of the heme.…”

EPR characterization of ascorbyl and sulfur dioxide anion radicals trapped during the reaction of bovine Cytochrome c Oxidase with molecular oxygen

“…Protein-based radicals are essential to a variety of enzymatic systems [3], such as ribonucleotide reductases [4,5] and heme peroxidases [6][7][8][9][10][11][12]. Cytochrome c Oxidase (CcO), which catalyzes the reduction of oxygen to water, has been proposed to involve a protein-based radical [13][14][15] in its catalytic chemistry.…”

Resonance Raman spectroscopy of oxoiron(IV) porphyrin π-cation radical and oxoiron(IV) hemes in peroxidase intermediates

“…Myoglobin [61][62][63][64][65][66][67][68][69][70] and its site-directed mutants [71,72], when oxidized to high oxidation states, are investigated as important protein models for compounds I and II. Cytochrome c peroxidase (CcP) was the first peroxidase for which a crystal structure became available [73], and was the first to be extensively studied by site-directed mutagenesis [74,75]. In contrast to HRP, the two-electron oxidized compound I analogue of CcP (also called compound ES) [47,[76][77][78] is red colored and contains a tryptophan cation radical [77,79,80] rather than a porphyrin p-cation radical.…”