Resonance Raman detection of two conformers for the cyanide adduct of cytochrome c peroxidase and their pH dependence

The resonance Raman spectra of the adducts of cytochrome c peroxidase with four cyanide isotopomers (12C14N−, 13C14N−, 12C15N− and 13C15N−) are presented. The spectra reveal the presence of two conformers, an essentially linear conformer whose v(Fe—C) and δ(FeCN) modes occur at 445 cm−1 and 407 cm−1, respectively (at pH 10) and a bent conformer which exhibits two modes at 355 cm−1 and 445 cm−1 (at pH 10). At pH values below 7, the linear form v(Fe—C) shifts to ∼455 cm−1 in response to protonation of the distal histidyl imidazole (which strengthens the Fe—C bond). The stretching mode of the bent form experiences only a slight shift. This smaller shift of the bent form is consistent with the proposal that the bent form originates as a consequence of hydrogen bond formation with the off‐axis proton donor group of arginine (which is protonated at both pH values). The overall spectral response to pH changes reflects interactions with the bound ligand which may be important for heterolytic bond cleavage.