A multigeneration analysis of cytochrome b(562) redox variants: evolutionary strategies for modulating redox potential revealed using a library approach.
A multigeneration analysis of cytochrome b(562) redox variants: evolutionary strategies for modulating redox potential revealed using a library approach.
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细胞色素 b(562) 氧化还原变体的多代分析：使用库方法揭示调节氧化还原电位的进化策略。
DOI：
10.1021/bi012066s
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发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
McLendon,GeorgeL
中科院分区：
文献类型：
--
作者：
Springs,StacyL;Bass,SusannaE;Bowman,Greg;Nodelman,Ilana;Schutt,CE;McLendon,GeorgeL
细胞色素的氧化还原电位决定了代谢过程中可能产生的能量，也是氧化还原反应进行速度的关键决定因素。在这里，血红素蛋白cytochromeb562被用于研究含有相同结构原型（四螺旋束）的变异体文库中氧化还原电位的体外进化。介绍了细胞色素b562活性位点的多位点变异。建立了包含R98和R106随机突变的变异文库，并测量了该文库的统计抽样的氧化还原电位。该程序适用于先前研究的F61X/F65X的低电位和高电位变体，第一代文库[Springs， S. L., Bass， S. E., and McLendon， G. L. (2000)Biochemistry39, 6075]。本文报道的第二代文库的氧化还原电位范围大于具有相同轴向配体（但折叠不同）的细胞色素之间已知可达电位的40% (160 mV)，并且超过了内部保持相同轴向连接和折叠的细胞色素家族在系统发育上所显示的范围。对文库的统计分析显示，WT cytb562的氧化还原电位位于分布的高电位极值处，表明该蛋白明显进化为差异稳定还原蛋白。求解了F61I/F65Y/R106L（第二代库的低电位变体）的2.7 Å晶体结构，并与野生型结构和F61I/F65Y变体（第一代库的低电位变体）的2.2 Å分辨率结构进行了比较。结构表明，电荷偶极子效应是导致F61I/F65Y和F61I/F65Y/R106L变异体氧化还原平衡向氧化态转移的原因。具体来说，由于F65Y突变，血红素微环境中引入了一个新的蛋白质偶极子，发现了两个新的内部水分子（一个在Y65的氢键距离上），并且在F61I/F65Y/R106L （ΔEm= 158 mV vs NHE）的情况下，由于R106L取代，血红素的溶剂暴露增加。
The redox potential of cytochromes sets the energy yield possible in metabolism and is also a key determinant of the rate at which redox reactions proceed. Here, the heme protein, cytochromeb562, is used to study the in vitro evolution of redox potential within a library of variants containing the same structural archetype, the four-helix bundle. Multisite variations in the active site of cytochromeb562were introduced. A library of variants containing random mutations in place of R98 and R106 was created, and the redox potentials of a statistical sampling of this library were measured. This procedure was carried out for both the low- and high-potential variants of a previously studied F61X/F65X, first-generation library [Springs, S. L., Bass, S. E., and McLendon, G. L. (2000)Biochemistry39, 6075]. The second-generation library reported here has a range of redox potentials which is greater than 40% (160 mV) of the known accessible potential among cytochromes with identical axial ligands (but different folds) and exceeds the range exhibited phylogenetically by the cytochromec‘ family which internally maintains the same axial ligation and fold. A statistical analysis of the libraries examined reveals that the redox potential of WT cytb562is found at the high-potential extremum of the distribution, indicating that this protein apparently evolved to differentially stabilize the reduced protein. The 2.7 Å crystal structure of F61I/F65Y/R106L (low-potential variant of the second-generation library) was solved and is compared to the wild-type structure and the 2.2 Å resolution structure of the F61I/F65Y variant (low-potential variant of the first-generation library). The structures indicate that charge−dipole effects are responsible for shifting the redox equilibrium toward the oxidized state in both the F61I/F65Y and F61I/F65Y/R106L variants. Specifically, a new protein dipole is introduced into the heme microenvironment as a result of the F65Y mutation, two new internal water molecules (one in hydrogen-bonding distance of Y65) are found, and in the case of F61I/F65Y/R106L (ΔEm= 158 mV vs NHE), increased solvent exposure of the heme as a result of the R106L substitution is identified.