Effect of electrostatics on the stability of the compound I radical in heme peroxidases

The effect of electrostatics on the stability of the compound I radical was examined in the heme peroxidases cytochrome c peroxidase, CcP, and ascorbate peroxidase, APX. Site-directed-mutagenesis was used in CcP and APX to investigate the role that Met residues play in stabilizing the cation radical of compound I in these enzymes. Removing three Met residues from the proximal pocket of a previously reported potassium binding mutant of CcP causes a drastic destabilization of the Trp cation radical in compound I. Electron paramagnetic resonance spectra suggested that the Trp radical had been reduced by a Tyr residue, and transient-state kinetic experiments estimate that this occurs within 25 ms of formation of compound I. Attempts were made at determining which of the 14 native Tyr residues in CcP were responsible for the reduction of the Trp cation radical without much success. One such mutant was notable. CcPK2M3 Y236F resulted in the temporary recovery of wild-type rates of substrate oxidation in single-turnover experiments, but as the age of compound I increased both the rate and level of turn-over quickly declined to near zero. This indicates that by far the easiest source of a reducing equivalent to the unstable Trp radical in CcPK2M3 is Tyr236.; Similar experiments were carried out on APX, with the introduction of three Met residues into the proximal pocket. When introduced into APX the Met residues destabilize the native compound I radical, a porphyrin-pi cation radical. Transient-state kinetics show a drastic increase in the rate of spontaneous decay of the porphyrin-pi cation radical, and the electron paramagnetic resonance spectra suggest that the native radical may be reduced by a Trp residue. It is possible that the Trp residue responsible is Trp179 which is analogous to the active Trp residue in CcP. The nature of this radical was examined by the further mutation Trp179Phe which yielded an electron paramagnetic resonance spectrum resembling that of the equivalent CcP mutant, CcP Trp191Gly.