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.
Veitch
N C
NC
Jodrell Laboratory, Royal Botanic Gardens, Richmond, Surrey, U.K. n.veitch@rbgkew.org.uk
Gao
Y
Y
Welinder
K G
KG
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