Time-resolved resonance Raman study on the binding of carbon monoxide to recombinant human myoglobin and its distal histidine mutants.
Time-resolved resonance Raman study on the binding of carbon monoxide to recombinant human myoglobin and its distal histidine mutants.
复制标题
一氧化碳与重组人肌红蛋白及其远端组氨酸突变体结合的时间分辨共振拉曼研究。
DOI：
10.1021/bi00073a014
复制
发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Ikeda-Saito,M
中科院分区：
文献类型：
--
作者：
Sakan,Y;Ogura,T;Kitagawa,T;Fraunfelter,FA;Mattera,R;Ikeda-Saito,M
本文报道了光解CO与重组人肌红蛋白（Mb）和几种E7突变体（H64 G、H64 Q、H64 A、H64 I、H64 V、H64 L和瓦尔）的时间分辨共振拉曼光谱。在脉冲激发下，当激光功率大大降低时，成功地观察到了Fe-CO伸缩（ype-co）RR带。H64 H、H64 G和H64 Q在其稳定状态下在505-510 cm-1处给出vFe-co谱带。在光解离后1-100 μ s的恢复过程中，在比H64 G和H64 Q的平衡结构略低的频率处观察到宽的瞬态带，但在490 cm™ 1附近没有发现对应于所谓的“开放”形式的瞬态γ Fe-co带。第二组H64 A、H64 I、H64 V和H64 L在静止状态下在490-495 cm™ 1处产生主要pFe-co谱带，肩部约为510 cm™ 1（H64 L除外），并且表现出比第一组快得多的恢复。这后四种物质在100-1000 ns的时间范围内在492-500 cm™ 1附近产生宽的瞬态带，而约510 cm™ 1的肩部出现得晚得多。在500 ms时，两个谱带的相对强度达到平衡，表明两种形式之间的相互转换慢于100 ms。对于所有的MbCO，由vFe-co谱带的面积强度确定的回收率，无论是否存在一个或两个pFe_co谱带，都呈现两个阶段。vFe-co频率与E7残基的亲水指数相关，但与其物理尺寸无关。据推断，CO周围更疏水的环境一方面降低了CO的极化并将yFe-co频率降低至~ 490 cm™ 1，另一方面稳定了称为蛋白质分离对的中间状态。后者增加了该中间体的重组贡献，导致CO更快的重组。肌红蛋白（Mb），一种储氧蛋白，及其CO复合物（MbCO）1的分子结构已被X射线晶体学测定，分辨率为1.5 μ m。并且已经注意到配体没有从溶剂迁移到血红素口袋中的埋藏结合位点的途径（Kuriyan埃塔尔，1986年）。至关重要的是
Time-resolved resonance Raman (RR) spectra of the recombined species of photodissociated CO with recombinant human myoglobin (Mb) and several E7 mutants, in whichdistal His was replaced by Gly (H64G), Gin (H64Q), Ala (H64A), lie (H64I), Val (H64V), and Leu (H64L) through site-directed mutagenesis, were observed in the time range-20 ns to 1 ms following photolysis. The Fe-CO stretching (ype-co) RR band was observed successfully with pulse excitation when thelaser power was greatly reduced. H64H, H64G, and H64Q gave the vFe-co band at 505-510 cm™ 1 in their stationary states. In their recovery processes 1-100 µ& after photodissociation, a broad transient band was observed at slightly lower frequencies than those of their equilibrium structures for H64G and H64Q, but a transient yFe-co band corresponding to the so-called “open” form was not identified around490 cm™ 1 for any of the three species. A second group, H64A, H64I, H64V, and H64L, gave the main pFe-co band at 490-495 cm™ 1 with a shoulder around 510 cm™ 1 (except for H64L) in the stationary state and exhibited a much faster recovery than the first group. These latter four species gave a broad transient band around 492-500 cm™ 1 in the time range of 100-1000 ns, while the~ 510 cm™ 1 shoulder appeared much later. The equilibrium relative intensity of the two bands was attained at500 ms, suggesting that the interconversion between the two forms is slower than 100 ms. For all MbCO examined here, the recovery, determinedfrom the area intensity of the vFe-co band, exhibited two phases irrespective of the presence of one or two pFe_co bands. The vFe-co frequencies could be correlated with the hydropathy index of the E7 residue but not with its physical size. It is inferred that the more hydrophobic environment around CO reduces the polarization of CO and lowers the yFe-co frequency to~ 490 cm™ 1 on the one hand and stabilizes the intermediate state called the protein-separated pair on the other. The latter increases the contribution of recombination from this intermediate, resulting in faster recombination of CO.Molecular structures of myoglobin (Mb), an oxygen storage protein, and its CO complex (MbCO) 1 havebeen determined by X-ray crystallography at a level of 1.5-Á resolution, and it has beennoted that there is no pathway for the migration of a ligand from solvent to the buried binding site in the heme pocket (Kuriyan etal., 1986). It is of fundamental importance