Veitch N C, Gao Y, Welinder K G
Jodrell Laboratory, Royal Botanic Gardens, Richmond, Surrey, U.K.
Biochemistry. 1996 Nov 12;35(45):14370-80. doi: 10.1021/bi961582t.
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.
利用¹H-NMR光谱，并结合对野生型酶的平行研究，对灰盖鬼伞过氧化物酶（D245N CIP）的天冬氨酸245突变为天冬酰胺的突变体的静息态、氟结合态和氰结合态进行了表征。对pH范围为5至10的静息态D245N CIP的光谱分析揭示了三种高自旋物种彼此处于动态平衡状态。在中性pH下的主要物种是六配位高自旋（6-c HS），以一个远端水分子作为第六个配体。在NMR时间尺度上，该物种与第二个六配位高自旋物种（6-c HS*）和一个五配位高自旋物种（5-c HS**）缓慢交换，分别朝着酸性和碱性pH值变化。6-c HS*物种似乎是独特的，据推测它与6-c HS物种的区别在于近端组氨酸残基的质子化，而5-c HS**物种缺乏近端组氨酸配体，由一个羟基配位。与之形成鲜明对比的是，野生型CIP在相同pH范围内是一个五配位高自旋（5-c HS）物种。D245N CIP突变体对氟的亲和力也比野生型CIP更高。使用二维方法归属的氰结合态D245N CIP的¹H-NMR光谱与野生型酶的光谱有显著差异。根据天冬氨酸被天冬酰胺取代时，组氨酸183 Nδ¹H与残基245侧链之间氢键的丧失或显著减弱，对血红素和与血红素相连的质子共振的扰动进行了合理解释。这种微妙的相互作用直接影响了CIP在血红素平面近端和远端的血红素口袋结构。
