The Asp245Asn Mutant of Coprinus cinereus Peroxidase. Characterization by 1H-NMR Spectroscopy and Comparison with t

详细信息    查看全文

• 作者：Nigel C. Veitch ; Yuan Gao ; and Karen G. Welinder
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：November 12, 1996
• 年：1996
• 卷：35
• 期：45
• 页码：14370 - 14380
• 全文大小：828K
• 年卷期：v.35,no.45(November 12, 1996)
• ISSN：1520-4995

文摘

The resting, fluoride-ligated and cyanide-ligated states of theAsp245Asn mutant of Coprinuscinereus peroxidase (D245N CIP) have been characterized using¹H-NMR spectroscopy in conjunctionwith parallel studies of the wild-type enzyme. Analysis of thespectra of resting state D245N CIP overthe pH range 5-10 has uncovered the existence of three high-spinspecies in dynamic equilibrium witheach other. The predominant species at neutral pH issix-coordinate high-spin (6-c HS), with a distalwater molecule as the sixth ligand. This species is in slowexchange on the NMR time scale with asecond six-coordinate high-spin species (6-c HS*) and a five-coordinatehigh-spin species (5-c HS**),toward acidic and alkaline pH values, respectively. The 6-c HS*species appears to be unique and isproposed to differ from the 6-c HS species by protonation of theproximal His residue, whereas the 5-cHS** species lacks the proximal His ligand and is coordinated by ahydroxyl group. In sharp contrast,wild-type CIP is a five-coordinate high-spin (5-c HS) species over thesame pH range. The D245N CIPmutant also exhibits a greater affinity for fluoride than wild-typeCIP. The ¹H-NMR spectrum of cyanide-ligated D245N CIP, assigned using two-dimensional methods, differssignificantly from that of the wild-type enzyme. Perturbations to heme and heme-linked protonresonances are rationalised in terms of theloss or significant weakening of the hydrogen bond between His183N1H and the side-chain of residue245 when Asp is replaced by Asn. This subtle interaction directlyaffects the heme pocket structure ofCIP both proximal and distal to the heme plane.