Electron paramagnetic resonance studies of Pseudomonas cytochrome c peroxidase

Abstract

The EPR spectrum at 15 K of Pseudomonas cytochrome $\text{c}$ peroxidase, which contains two hemes per molecule, is in the totally ferric form characteristic of low-spin heme giving two sets of $\text{g-}\text{values}$ with $\text{g}{}_{\mathrm{z}}$ 3.26 and 2.94. These vaues indicate an imidazole-nitrogen : heme-iron : methionine-sulfur and an imidazole-nitrogen : heme-iron : imidazole-nitrogen hemochrome structure, respectively. The spectrum is essentially identical at pH 6.0 and 4.6 and shows only a very small amount of high-spin heme iron ($\text{g 5–6}$) also at 77 K. Interaction between the two hemes is shown to exist by experiments in which one heme is reduced. This induces a change of the EPR signal of the other (to $\text{g}{}_{\mathrm{z}}\text{2.83, g}{}_{\mathrm{y}}\text{2.35}$ and $\text{g}{}_{\mathrm{x}}\text{1.54}$), indicative of the removal of a histidine proton from that heme, which is axially coordinated to two histidine residues. If hydrogen peroxide is added to the partially reduced protein, its EPR signal is replaced by still other signals ($\text{g}{}_{\mathrm{z}}\text{3.5}$ and 3.15). Only a very small free radical peak could be observed consistent with earlier mechanistic proposals. Contrary to the EPR spectra recorded at low temperature, the optical absorption spectra of both totally oxidized and partially reduced enzyme reveal the presence of high-spin heme at room temperature. It seems that a transition of one of the heme $\text{c}$ moieties from an essentially high-spin to a low-spin form takes place on cooling the enzyme from 298 to 15 K.