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 peroxid ase-cyanide with the Asn 82-->Asp mutation ([N82D]cytochrome c peroxid ase-CN) are presented and compared to the wild-type enzyme. This mutat ion alters an amino acid that forms a hydrogen bond to His52, the dist al histidine residue that interacts in the heme pocket with heme-bound ligands. His52 is an important participant in the initial hydrogen pe roxide decomposition step of cytochrome c peroxidase. In wild-type cyt ochrome c peroxidase-CN, His52 hydrogen bonds to the neighboring Asn82 peptide carbonyl group and to heme-coordinated cyanide. His52 thus ma nifests itself as an extensively hydrogen bended histidinium moiety. T he principal result from this study is the observation that three hype rfine-shifted resonances disappear from the spectrum of [N82D] cytochr ome c peroxidase-CN compared to the wild-type enzyme. All three absent resonances in [N82D]cytochrome c peroxidase-CN belong to His52 and th is leads to the conclusion that the result of the mutation has been el imination of the His52-Asn82 and His52-heme-coordinated cyanide hydrog en bonds.