The Asp245-->Asn mutant of Coprinus cinereus peroxidase. Characterization by 1H-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 characterized using 1H-NMR spectroscopy in conjunction with parallel studies of the wild-type enzyme. Analysis of the spectra of resting state D245N CIP over the pH range 5-10 has uncovered the existence of three high-spin species in dynamic equilibrium with each other. The predominant 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* species appears to be unique and is proposed to differ from the 6-c HS species by protonation of the proximal His residue, whereas the 5-c HS** species lacks the proximal His ligand and is coordinated by a hydroxyl group. 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 exhibits a greater affinity for fluoride than wild-type CIP. The 1H-NMR spectrum of cyanide-ligated D245N CIP, assigned using two-dimensional methods, differs significantly from that of the wild-type enzyme. Perturbations to heme and heme-linked proton resonances are rationalised in terms of the loss or significant weakening of the hydrogen bond between His183 N delta 1H and the side-chain of residue 245 when Asp is replaced by Asn. This subtle interaction directly affects the heme pocket structure of CIP both proximal and distal to the heme plane.
Jodrell Laboratory, Royal Botanic Gardens, Richmond, Surrey, U.K. n.veitch@rbgkew.org.uk
N C
NC
Veitch
Y
Y
Gao
K G
KG
Welinder
D001120:Arginine / Q000737:chemistry; D003302:Coprinus / Q000201:enzymology*; D003486:Cyanides / Q000737:chemistry; D005459:Fluorides / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D006418:Heme / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D010544:Peroxidases / Q000737:chemistry / Q000378:metabolism*; D017354:Point Mutation
D003486:Cyanides; D005656:Fungal Proteins; D006418:Heme; D006639:Histidine; D001120:Arginine; D010544:Peroxidases; D005459:Fluorides