Electron Transfer Properties and Hydrogen Peroxide Electrocatalysis of Cytochrome c Variants at Positions 67 and 80
Electron Transfer Properties and Hydrogen Peroxide Electrocatalysis of Cytochrome c Variants at Positions 67 and 80
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DOI：
10.1021/jp9090365
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发表时间：
2010-02-04
影响因子：
3.3
通讯作者：
Sola, Marco
中科院分区：
文献类型：
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作者：
Casalini, Stefano;Battistuzzi, Gianantonio;Sola, Marco
用非配位Ala残基取代电极固定的细胞色素c中轴向的Met80血红素配体，以及取代Tyr67后附近区域氢键网络的变化，作为蛋白质-电极电子转移(ET)和血红素介导的H_2O_2电催化还原的热力学和动力学的影响因素。为此，在不同温度和过氧化氢浓度下，测定了酵母iso-1-细胞色素c的Met80Ala、Met80Ala/Tyr67His和Met80Ala/Tyr67Ala变异体在不同温度和过氧化氢浓度下的伏安曲线。热力学研究表明，His和Ala残基取代Tyr67主要导致血红素缝隙处蛋白质-溶剂相互作用的差异，而对pH为7时的E度没有相关影响，单变异体和双变异体的E度变化范围约为-0.200~-0.220 V(Vs Se)。油相反，这两个双变异体显示出比Met80Ala低得多的ET率，很可能是ET途径的结果或改变。在目前的非变性固定化条件下，过氧化氢浓度在微摩尔范围内，变异体催化电极上的过氧化氢还原，而野生型细胞色素c不催化。H_2O_2发生电催化，所有有效的机制可能包括快速的类似过氧化氢酶的过程，然后在电极上产生的氧被电催化还原。Met80Ala/Tyr67His与Met80Ala/Tyr67Ala的比较表明，在远端的血红素位点上存在一个用于识别和通过氢键结合过氧化氢的酸碱残基，这是该底物还原周转的关键必要条件。
Replacement of the axial Met80 heme ligand in electrode-immobilized cytochrome c with a noncoordinating Ala residue and alteration of the hydrogen bonding network in the region nearby following substitution of Tyr67 were investigated as effectors of the thermodynamics and kinetics of the protein-electrode electron transfer (ET) and the heme-mediated electrocatalytic reduction of H2O2. To this end, the voltammetry of the Met8OAla, Met8OAla/Tyr67His, and Met80Ala/Tyr67Ala variants of yeast iso-1-cytochrome c chemisorbed on carboxyalkanethiol self-assembled monolayers was measured at varying temperature and hydrogen peroxide concentration. The thermodynamic Study shows that insertion of His and Ala residues in place of Tyr67 results mainly in differences in protein-solvent interactions at the heme crevice with no relevant effects on the E degrees' values at pH 7, which for single and double variants range from approximately -0.200 to -0.220 V (vs SHE). Oil the contrary, both double variants show much lower ET rates compared to Met8OAla, most likely as a consequence or a change in the ET pathways. In the present nondenaturing immobilizing conditions, and with hydrogen peroxide concentrations in the micromolar range, the variants catalyze H2O2 reduction at the electrode, whereas wild-type cytochrome c does not. H2O2 electrocatalysis Occurs with all efficient mechanism likely involving I fast catalase-like process followed by electrocatalytic reduction of the resulting dioxygen at the electrode. Comparison of Met80Ala/Tyr67His with Met80Ala/Tyr67Ala shows that the presence of a general acid-base residue for H2O2 recognition and binding through H-bonding in the distal heme site is a key requisite for the reductive turnover of this substrate.