Xia B, Cheng H, Bandarian V, Reed G H, Markley J L
Department of Biochemistry, University of Wisconsin-Madison 53706, USA.
Biochemistry. 1996 Jul 23;35(29):9488-95. doi: 10.1021/bi960467f.
Human ferredoxin, the human equivalent of bovine adrenodoxin, is a small iron-sulfur protein with one [2Fe-2S] cluster. It functions, as do other vertebrate ferredoxins, to transfer electrons during the processes of steroid hormone synthesis. A DNA fragment encoding the mature form of human ferredoxin was cloned into an expression vector under control of the T7 RNA polymerase/promoter system. The protein was overproduced in Escherichia coli, and the [2Fe-2S] cluster was incorporated into the protein by in vitro reconstitution. The overall yield was approximately 30 mg of purified, reconstituted ferredoxin per liter of culture. Four of the five cysteines in human ferredoxin are coordinated to the iron-sulfur cluster. First, the non-ligand cysteine (cysteine-95) was mutated to alanine, and then double mutants were created in which each of the other four cysteines (at positions 46, 52, 55, and 92) were mutated individually to serine. The wild-type ferredoxin and each of the five mutant proteins were studied by UV-visible spectroscopy and electron paramagnetic resonance spectroscopy. The EPR gav values of all five mutants were very similar to that of wild-type human ferredoxin. In the reduced state, three of the cysteine-to-serine mutants exhibited axial EPR spectra similar to that of wild-type, but one of the double mutants (C52S/C95A) exhibited a rhombic EPR spectrum. The UV-visible spectroscopic properties of the wild-type and the C95A mutant ferredoxins were identical, but those of the other cysteine-to-serine mutant proteins of human ferredoxin were quite different from those of the wild-type protein and each other. These results, along with those from cysteine-to-serine mutations in other ferredoxins, provide the basis for a more comprehensive theoretical and practical understanding of the features important to the ligation of [2Fe-2S] clusters, although they do not yet permit determination of which two cysteines ligate Fe(II) and which ligate Fe(III) in the reduced protein.
人铁氧化还原蛋白，相当于牛肾上腺铁氧化还原蛋白，是一种含有一个[2Fe-2S]簇的小铁硫蛋白。它与其他脊椎动物铁氧化还原蛋白一样，在类固醇激素合成过程中发挥传递电子的作用。将编码人铁氧化还原蛋白成熟形式的DNA片段克隆到受T7 RNA聚合酶/启动子系统控制的表达载体中。该蛋白在大肠杆菌中过量表达，并通过体外重组将[2Fe-2S]簇整合到蛋白中。每升培养物的总产率约为30毫克纯化的、重组的铁氧化还原蛋白。人铁氧化还原蛋白的五个半胱氨酸中有四个与铁硫簇配位。首先，将非配体半胱氨酸（半胱氨酸-95）突变为丙氨酸，然后创建双突变体，其中其他四个半胱氨酸（位于46、52、55和92位）分别突变为丝氨酸。通过紫外可见光谱和电子顺磁共振光谱对野生型铁氧化还原蛋白和五个突变蛋白进行了研究。所有五个突变体的电子顺磁共振gav值与野生型人铁氧化还原蛋白的非常相似。在还原状态下，三个半胱氨酸到丝氨酸的突变体表现出与野生型相似的轴向电子顺磁共振光谱，但其中一个双突变体（C52S/C95A）表现出菱形电子顺磁共振光谱。野生型和C95A突变体铁氧化还原蛋白的紫外可见光谱性质相同，但人铁氧化还原蛋白的其他半胱氨酸到丝氨酸突变蛋白的紫外可见光谱性质与野生型蛋白以及彼此之间有很大不同。这些结果，连同其他铁氧化还原蛋白中半胱氨酸到丝氨酸突变的结果，为更全面地从理论和实践上理解[2Fe-2S]簇连接的重要特征提供了基础，尽管它们还不能确定在还原蛋白中哪两个半胱氨酸连接Fe(II)以及哪两个连接Fe(III)。
