Iismaa S E, Vázquez A E, Jensen G M, Stephens P J, Butt J N, Armstrong F A, Burgess B K
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
J Biol Chem. 1991 Nov 15;266(32):21563-71.
We have used site-directed mutagenesis to obtain two variants of Azotobacter vinelandii ferredoxin I (AvFdI), whose x-ray structures are now available. In the C20A protein, a ligand to the [4Fe-4S] cluster was removed whereas in the C24A mutant a free cysteine next to that cluster was removed. Like native FdI, both mutants contain one [4Fe-4S] cluster and one [3Fe-4S] cluster. The structure of C24A is very similar to that of native FdI, while the structure of C20A is rearranged in the region of the [4Fe-4S] cluster to allow it to use the free Cys-24 as a replacement ligand. Here we compare the properties of the native, C20A, and C24A proteins. Although all three proteins are O2 stable in vitro, the C20A protein is much less stable toward proteolysis than the other two in vivo. Spectroscopic results show that all three proteins exhibit the same general redox behavior during O2-oxidation and dithionite reduction. Electrochemical data show that the [3Fe-4S] clusters in all three proteins have the same pH-dependent reduction potentials (-425 mV versus SHE, pH 7.8), whereas the [4Fe-4S] cluster potentials vary over a approximately 150 mV range from -600 mV (C24A) to -647 mV (native) to -746 mV (C20A). Despite this variation in potential both the C20A and C24A proteins appear to be functional in vivo. Native FdI reacts with three equivalents of Fe(CN)3-(6) to form a paramagnetic species previously proposed to be a cysteinyl-disulfide radical. Neither the C20A nor the C24A variant undergoes this reaction, strongly suggesting that it involves the free Cys-24.
我们利用定点诱变技术获得了两种维涅兰德固氮菌铁氧化还原蛋白I（AvFdI）变体，其X射线结构现已可得。在C20A蛋白中，[4Fe-4S]簇的一个配体被去除，而在C24A突变体中，该簇旁边的一个游离半胱氨酸被去除。与天然FdI一样，这两种突变体都含有一个[4Fe-4S]簇和一个[3Fe-4S]簇。C24A的结构与天然FdI的结构非常相似，而C20A的结构在[4Fe-4S]簇区域发生了重排，使其能够利用游离的Cys-24作为替代配体。在此，我们比较了天然、C20A和C24A蛋白的特性。尽管这三种蛋白在体外对O2都稳定，但在体内，C20A蛋白对蛋白水解的稳定性远低于其他两种蛋白。光谱结果表明，这三种蛋白在O2氧化和连二亚硫酸盐还原过程中表现出相同的一般氧化还原行为。电化学数据表明，这三种蛋白中的[3Fe-4S]簇具有相同的pH依赖性还原电位（相对于标准氢电极，pH 7.8时为-425 mV），而[4Fe-4S]簇的电位在大约150 mV范围内变化，从-600 mV（C24A）到-647 mV（天然）再到-746 mV（C20A）。尽管电位存在这种变化，但C20A和C24A蛋白在体内似乎都具有功能。天然FdI与三当量的Fe(CN)3-(6)反应形成一种顺磁性物质，先前认为该物质是半胱氨酰二硫自由基。C20A和C24A变体都不发生这种反应，这强烈表明该反应涉及游离的Cys-24。
