THE ASP245-]ASN MUTANT OF COPRINUS-CINEREUS PEROXIDASE - CHARACTERIZATION BY H-1-NMR SPECTROSCOPY AND COMPARISON WITH THE WILD-TYPE ENZYME

The resting, fluoride-ligated and cyanide-ligated states of the Asp245 -->Asn mutant of Coprinus cinereus peroxidase (D245N CIP) have been ch aracterized using H-1-NMR spectroscopy in conjunction with parallel st udies of the wild-type enzyme, Analysis of the spectra of resting stat e D245N CIP over the pH range 5-10 has uncovered the existence of thre e high-spin species in dynamic equilibrium with each other. The predom inant species at neutral pH is six-coordinate high-spin (6-c HS), with a distal water molecule as the sixth ligand. This species is in slow exchange on the NMR time scale with a second six-coordinate high-spin species (6-c HS) and a five-coordinate high-spin species (5-c HS**), toward acidic and alkaline pH values, respectively. The 6-c HS specie s appears to be unique and is proposed to differ from the 6-c HS speci es by protonation of the proximal His residue, whereas the 5-c HS* sp ecies lacks the proximal His ligand and is coordinated by a hydroxyl g roup. In sharp contrast, wild-type CIP is a five-coordinate high-spin (5-c HS) species over the same pH range. The D245N CIP mutant also exh ibits a greater affinity for fluoride than wild-type CIP. The H-1-NMR spectrum of cyanide-ligated D245N CIP, assigned using two-dimensional methods, differs significantly from that of the wildtype enzyme. Pertu rbations to heme and heme-linked proton resonances are rationalised in terms of the loss or significant weakening of the hydrogen bond betwe en His 183 N delta 1H and the side-chain of residue 245 when Asp is re placed by Asn. This subtle interaction directly affects the heme pocke t structure of CIP both proximal and distal to the heme plane.