The effect of the Asn82-->Asp mutation in yeast cytochrome c peroxidase studied by proton NMR spectroscopy.

Proton NMR studies of the mutant of baker's yeast cytochrome c peroxidase-cyanide with the Asn 82-->Asp mutation ([N82D]cytochrome c peroxidase-CN) are presented and compared to the wild-type enzyme. This mutation alters an amino acid that forms a hydrogen bond to His52, the distal histidine residue that interacts in the heme pocket with heme-bound ligands. His52 is an important participant in the initial hydrogen peroxide decomposition step of cytochrome c peroxidase. In wild-type cytochrome c peroxidase-CN, His52 hydrogen bonds to the neighboring Asn82 peptide carbonyl group and to heme-coordinated cyanide. His52 thus manifests itself as an extensively hydrogen bonded histidinium moiety. The principal result from this study is the observation that three hyperfine-shifted resonances disappear from the spectrum of [N82D] cytochrome c peroxidase-CN compared to the wild-type enzyme. All three absent resonances in [N82D]cytochrome c peroxidase-CN belong to His52 and this leads to the conclusion that the result of the mutation has been elimination of the His52-Asn82 and His52-heme-coordinated cyanide hydrogen bonds.