PROTON NMR-STUDIES OF CYTOCHROME-C PEROXIDASE MUTANT N82A - HYPERFINERESONANCE ASSIGNMENTS, IDENTIFICATION OF 2 INTERCONVERTING ENZYME SPECIES, QUANTITATING THE RATE OF INTERCONVERSION, AND DETERMINATION OF EQUILIBRIUM-CONSTANTS

The cyanide-ligated form of the baker's yeast cytochrome c peroxidase mutant bearing the mutation Asn82 --> Ala82 ([N82A]CcPCN) has been stu died by proton NMR spectroscopy. This mutation alters an amino acid th at forms a hydrogen bond to His52, the distal histidine residue that i nteracts in the heme pocket with heme-bound ligands. His52 is a residu e critical to cytochrome c peroxidase's normal function. Proton hyperf ine resonance assignments have been made for the cyanide-ligated form of the mutant by comparison with 1-D and NOESY spectra of the wild-typ e native enzyme, For [N82A]CcPCN, proton NMR spectra reveal two signif icant phenomena, First, similar to results published for the related m utant [N82D]CcPCN [Satterlee, J.D., et al. (1994) Eur. J. Biochem. 224 , 81-87], the Ala82 mutation disrupts the hydrogen bond between His52 and the heme-ligated CN, Second, four of the 24 resolved hyperfine-shi fted resonances are doubled in the mutant enzyme's proton spectrum, le ading to the concept that the heme active site environment is dynamica lly microheterogeneous on a very localized scale. Two magnetically ine quivalent enzyme forms are detected in a pure enzyme preparation. Vary ing temperature causes the two enzyme forms to interconvert. Magnetiza tion transfer experiments further document this interconversion betwee n enzyme forms and have been used to determine that the rate of interc onversion is 250 (+/-53) s(-1). The equilibrium constant at 20 degrees C is 1.5. Equilibrium constants have been calculated at various tempe ratures between 5 and 29 degrees C leading to the following values: De lta H = 60 kJ mol(-1); Delta S = 0.20 kJ K-1 mol(-1).