Recent ENDOR and Pulsed Electron Paramagnetic Resonance Studies of Cytochrome c 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 compoundI: 1. Tryptophan-51~Phenylalanine; 2. Tryptophan-l91~Phenylalan-ine. Tryptophan-51 lies near the heme on the distal (H,.Oz-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 J measurements at liquid He temperatures showed the unusual temperature-dependent relaxation of compound I's axially symmetric signal (gil =2.035, gJ. =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~Phenyla-lanine does not perturb these ENDOR features. Pulse field-sweep EPR resolved for the first time distinct proton hyperfme 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 Phenylalanine mutant and was a minor feature in all samples. The Tryptophan191~Phenylalanine mutation does perturb or destabilize the majority radical species of compound I. Thus, Tryptophan-l91 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.

Charge Reversal of a Critical Active‐Site Residue of Cytochrome‐c Peroxidase

“…5 in nitrate containing buffers [2,32,331) or the Arg48-Leu variant (5.8 in phosphate and phosphate/ nitrate buffers [71).…”

“…Although the location of the free radical associated with compound I of wild-type cytochrome-c peroxidase has been identified as Trpl91 [4,5 ) and detailed characterization of the electronic properties of this radical has been reported [60], understanding the structural basis for stabilization of radical formation at this location remains incomplete. Investigation of cytochrome-c peroxidase variants has recently begun to provide some insight into these issues.…”

A cation binding motif stabilizes the compound I radical of cytochrome c peroxidase.

“…One oxidizing equivalent of peroxide is retained initially as a stable radical at by ferrocytochrome c. In addition, facile oxidation of Trp-191 is required for rapid electron transfer from cytochrome c to the compound I oxyferryl center (1,3,7,8). Thus, the radical constitutes an electron "gate" that allows the controlled reduction of peroxide, a two-electron oxidant, by cytochrome c, a one-electron reductant.…”

Characterisation of a haem active‐site mutant of horseradish peroxidase, Phe41 → Val, with altered reactivity towards hydrogen peroxide and reducing substrates

“…In cytochrome-c peroxidase, however, the iron of compound I is also in the ferryl state, but with the second unpaired electron residing on Trpl91 [4,5]. For both HRP and CCP, compound I reverts to the resting enzyme via two sequential one-electron reactions with reducing substrate.…”