Smulevich G, Neri F, Marzocchi M P, Welinder K G
Dipartimento di Chimica, Università di Firenze, Firenze, Italy.
Biochemistry. 1996 Aug 13;35(32):10576-85. doi: 10.1021/bi9605898.
The pH dependence of the electronic absorption and resonance Raman (RR) spectra of FeIII and FeII forms of Coprinus cinereus peroxidase (CIP) and its Asp245-->Asn (D245N) mutant has been examined in detail. The spectral data were obtained in the pH range 3.8-12.0. These spectra were used to assess the spin and ligation states of the heme via the porphyrin marker band frequencies and the wavelengths of the absorption maxima, especially that of the band (CT1) due to the charge transfer from the porphyrin to the heme iron via the a' 2u(pi)-->eg (d pi) electronic transition. The RR spectra were obtained by using different excitation wavelengths and polarized light. The data obtained for ferric CIP show that two pH-induced structural transitions exist. At acid pH the Soret and the CT1 absorption maxima occur at 394 and 652 nm, respectively, compared with the values of 403 and 649 nm observed at neutral pH. The electronic data indicate that at acid pH the proximal Fe-Im bond might be weakened or ruptured, and the RR spectra show a new species (5-c HS) different from the normal neutral 5-coordinate high-spin (5-c HS) heme. At pH 12.0, the protein converts to a 6-coordinate low-spin (6-c LS) heme with a hydroxyl ligand coordinated in the sixth position of the heme iron and strongly hydrogen-bonded with the positively charged guanidinium group of the distal Arg51 residue. Replacement of the aspartate carboxylate group of Asp245, which acts as hydrogen-bond acceptor to the proximal His183 ligand of the heme Fe, with a carboxamide group of an asparagine residue has a profound influence on the heme coordination. The RR spectra of the Fe(II) form of this mutant at both neutral and alkaline pH values show a band at 204 cm-1 assigned to the Fe-His stretch associated with a fairly weak or non-hydrogen-bonded imidazole. The ferric form of the mutant shows a great variability in coordination and spin states upon pH titration. Between pH 8.8 and 3.8 the spectra are mainly characteristic of a 6-coordinate high-spin heme, presumably with a water molecule bound on the distal side of the Fe atom. The pKa of the alkaline transition of the mutant is much lower than that of the wild-type protein. At pH 10.0 the D245N mutant is in its final alkaline form, which markedly differs from that of the parent enzyme. The spectral data indicate that the majority of the protein has 5-coordinate high-spin heme (5-c HS), with the Fe-His 183 bond broken and the distal axial coordination site of the heme iron occupied by a hydroxyl group, which is strongly hydrogen-bonded with distal Arg51. Therefore, the Asp245-->Asn mutation on the proximal side results in the breakage of the Fe-His bond at alkaline pH.
已详细研究了灰盖鬼伞过氧化物酶（CIP）及其天冬氨酸245突变为天冬酰胺（D245N）的突变体的FeIII和FeII形式的电子吸收光谱和共振拉曼（RR）光谱的pH依赖性。光谱数据是在pH值3.8 - 12.0范围内获得的。这些光谱用于通过卟啉标记带频率和吸收最大值的波长，特别是由于通过a' 2u(π)→eg (d π)电子跃迁从卟啉到血红素铁的电荷转移产生的带（CT1）的波长，来评估血红素的自旋和配位状态。RR光谱是通过使用不同的激发波长和偏振光获得的。铁离子形式的CIP所获得的数据表明存在两个pH诱导的结构转变。在酸性pH下，Soret带和CT1吸收最大值分别出现在394和652 nm处，而在中性pH下观察到的值为403和649 nm。电子数据表明，在酸性pH下，近端Fe-Im键可能被削弱或断裂，并且RR光谱显示出一种不同于正常中性五配位高自旋（5-c HS）血红素的新物种（5-c HS）。在pH 12.0时，蛋白质转变为六配位低自旋（6-c LS）血红素，一个羟基配体配位在血红素铁的第六位，并与远端精氨酸51残基带正电荷的胍基形成强氢键。用天冬酰胺残基的羧酰胺基团取代作为血红素铁近端组氨酸183配体的氢键受体的天冬氨酸羧酸盐基团，对血红素配位有深远影响。该突变体的Fe(II)形式在中性和碱性pH值下的RR光谱在204 cm-1处显示出一个带，该带归属于与相当弱或非氢键咪唑相关的Fe-His伸缩振动。突变体的铁离子形式在pH滴定过程中配位和自旋状态表现出很大的变异性。在pH 8.8和3.8之间，光谱主要是六配位高自旋血红素的特征，推测在Fe原子的远端侧结合有一个水分子。突变体碱性转变的pKa远低于野生型蛋白质的pKa。在pH 10.0时，D245N突变体处于其最终碱性形式，这与亲本酶的形式明显不同。光谱数据表明，大多数蛋白质具有五配位高自旋血红素（5-c HS），Fe-His 183键断裂，血红素铁的远端轴向配位位点被一个羟基占据，该羟基与远端精氨酸51形成强氢键。因此，近端侧的天冬氨酸245突变为天冬酰胺导致在碱性pH下Fe-His键断裂。
