Recent ENDOR and Pulsed Electron Paramagnetic Resonance Studies of Cytochrome <i>c</i> Peroxidase ‐ Compound I and Its Site‐Directed Mutants

Abstract

Abstract EPR, ENDOR and pulsed EPR were applied to the radical signals of yeast cytochrome c peroxidase‐compound I from this protein expressed in E. coli and two variants prepared after site‐directed mutagenesis. The compound I complexes studied were first, that of the parent peroxidase expressed in E. coli , and next, those of the following amino acid mutants as specific probes for the radical in compound I: 1. Tryptophan‐51 → Phenylalanine; 2. Tryptophan‐191 → Phenylalanine. Tryptophan‐51 lies near the heme on the distal (H 2 O 2 ‐binding) side of cytochrome c peroxidase, while Tryptophan‐191 lies in contact with the proximal heme ligand, Histidine‐175. EPR above 20 K and saturation recovery T 1 measurements at liquid He temperatures showed the unusual temperature‐dependent relaxation of compound I's axially symmetric signal ( g __ = 2.035, g⟂ = 2.00), which has comprised the major radical species in bakers' yeast wildtype compound I and here in all but the Tryptophan‐191 → Phenylalanine mutant. The ENDOR of the major radical species was characterized by the substantial hyperfine couplings previously proposed to be at a sulfur‐based radical. The mutation Tryptophan‐51 → Phenylalanine does not perturb these ENDOR features. Pulse field‐sweep EPR resolved for the first time distinct proton hyperfine couplings due to the features observed by ENDOR. A narrow, more typical organic free radical signal at g = 2.001 with resolved hyperfine structure was the only species observed from the Tryptophan‐191 → Phenylalanine mutant and was a minor feature in all samples. The Tryptophan‐191 → Phenylalanine mutation does perturb or destabilize the majority radical species of compound I. Thus, Tryptophan‐191 is either intimately involved with the kinetics of radical formation or participates intimately in the majority radical site, despite not being a sulfur‐containing amino acid.