Hurley J K, Weber-Main A M, Stankovich M T, Benning M M, Thoden J B, Vanhooke J L, Holden H M, Chae Y K, Xia B, Cheng H, Markley J L, Martinez-Júlvez M, Gómez-Moreno C, Schmeits J L, Tollin G
Department of Biochemistry, University of Arizona, Tucson, Arizona 85721, USA.
Biochemistry. 1997 Sep 16;36(37):11100-17. doi: 10.1021/bi9709001.
A combination of structural, thermodynamic, and transient kinetic data on wild-type and mutant Anabaena vegetative cell ferredoxins has been used to investigate the nature of the protein-protein interactions leading to electron transfer from reduced ferredoxin to oxidized ferredoxin:NADP+ reductase (FNR). We have determined the reduction potentials of wild-type vegetative ferredoxin, heterocyst ferredoxin, and 12 site-specific mutants at seven surface residues of vegetative ferredoxin, as well as the one- and two-electron reduction potentials of FNR, both alone and in complexes with wild-type and three mutant ferredoxins. X-ray crystallographic structure determinations have been carried out for six of the ferredoxin mutants. None of the mutants showed significant structural changes in the immediate vicinity of the [2Fe-2S] cluster, despite large decreases in electron-transfer reactivity (for E94K and S47A) and sizable increases in reduction potential (80 mV for E94K and 47 mV for S47A). Furthermore, the relatively small changes in Calpha backbone atom positions which were observed in these mutants do not correlate with the kinetic and thermodynamic properties. In sharp contrast to the S47A mutant, S47T retains electron-transfer activity, and its reduction potential is 100 mV more negative than that of the S47A mutant, implicating the importance of the hydrogen bond which exists between the side chain hydroxyl group of S47 and the side chain carboxyl oxygen of E94. Other ferredoxin mutations that alter both reduction potential and electron-transfer reactivity are E94Q, F65A, and F65I, whereas D62K, D68K, Q70K, E94D, and F65Y have reduction potentials and electron-transfer reactivity that are similar to those of wild-type ferredoxin. In electrostatic complexes with recombinant FNR, three of the kinetically impaired ferredoxin mutants, as did wild-type ferredoxin, induced large (approximately 40 mV) positive shifts in the reduction potential of the flavoprotein, thereby making electron transfer thermodynamically feasible. On the basis of these observations, we conclude that nonconservative mutations of three critical residues (S47, F65, and E94) on the surface of ferredoxin have large parallel effects on both the reduction potential and the electron-transfer reactivity of the [2Fe-2S] cluster and that the reduction potential changes are not the principal factor governing electron-transfer reactivity. Rather, the kinetic properties are most likely controlled by the specific orientations of the proteins within the transient electron-transfer complex.
结合野生型和突变型鱼腥藻营养细胞铁氧化还原蛋白的结构、热力学及瞬态动力学数据，来研究导致电子从还原态铁氧化还原蛋白转移至氧化态铁氧化还原蛋白：NADP⁺还原酶（FNR）的蛋白质 - 蛋白质相互作用的本质。我们测定了野生型营养铁氧化还原蛋白、异形胞铁氧化还原蛋白以及营养铁氧化还原蛋白七个表面残基处的12个位点特异性突变体的还原电位，以及FNR单独存在时和与野生型及三种突变型铁氧化还原蛋白形成复合物时的单电子和双电子还原电位。已对六个铁氧化还原蛋白突变体进行了X射线晶体学结构测定。尽管电子转移反应性大幅降低（对于E94K和S47A）且还原电位显著升高（E94K升高80 mV，S47A升高47 mV），但没有一个突变体在[2Fe - 2S]簇紧邻区域显示出明显的结构变化。此外，在这些突变体中观察到的Cα主链原子位置的相对小变化与动力学和热力学性质不相关。与S47A突变体形成鲜明对比的是，S47T保留了电子转移活性，其还原电位比S47A突变体负100 mV，这表明S47侧链羟基与E94侧链羧基氧之间存在的氢键很重要。其他改变还原电位和电子转移反应性的铁氧化还原蛋白突变是E94Q、F65A和F65I，而D62K、D68K、Q70K、E94D和F65Y的还原电位和电子转移反应性与野生型铁氧化还原蛋白相似。在与重组FNR形成的静电复合物中，三个动力学受损的铁氧化还原蛋白突变体与野生型铁氧化还原蛋白一样，使黄素蛋白的还原电位产生了大幅（约40 mV）正移，从而使电子转移在热力学上变得可行。基于这些观察结果，我们得出结论，铁氧化还原蛋白表面三个关键残基（S47、F65和E94）的非保守突变对[2Fe - 2S]簇的还原电位和电子转移反应性具有很大的平行影响，并且还原电位变化不是控制电子转移反应性的主要因素。相反，动力学性质很可能由瞬态电子转移复合物中蛋白质的特定取向控制。
