Kyritsis P, Kümmerle R, Huber J G, Gaillard J, Guigliarelli B, Popescu C, Münck E, Moulis J M
Département de Biologie Moléculaire et Structurale, Laboratoire Métalloprotéines, CEA, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France.
Biochemistry. 1999 May 11;38(19):6335-45. doi: 10.1021/bi982894u.
The ferredoxin from Chromatium vinosum (CvFd) exhibits sequence and structure peculiarities. Its two Fe4S4(SCys)4 clusters have unusually low potential transitions that have been unambiguously assigned here through NMR, EPR, and Mössbauer spectroscopy in combination with site-directed mutagenesis. The [4Fe-4S]2+/1+ cluster (cluster II) whose coordination sphere includes a two-turn loop between cysteines 40 and 49 was reduced by dithionite with an E degrees ' of -460 mV. Its S = 1/2 EPR signal was fast relaxing and severely broadened by g-strain, and its Mössbauer spectra were broad and unresolved. These spectroscopic features were sensitive to small perturbations of the coordination environment, and they were associated with the particular structural elements of CvFd, including the two-turn loop between two ligands and the C-terminal alpha-helix. Bulk reduction of cluster I (E degrees ' = -660 mV) was not possible for spectroscopic studies, but the full reduction of the protein was achieved by replacing valine 13 with glycine due to an approximately 60 mV positive shift of the potential. At low temperatures, the EPR spectrum of the fully reduced protein was typical of two interacting S = 1/2 [4Fe-4S]1+ centers, but because the electronic relaxation of cluster I is much slower than that of cluster II, the resolved signal of cluster I was observed at temperatures above 20 K. Contact-shifted NMR resonances of beta-CH2 protons were detected in all combinations of redox states. These results establish that electron transfer reactions involving CvFd are quantitatively different from similar reactions in isopotential 2[4Fe-4S] ferredoxins. However, the reduced clusters of CvFd have electronic distributions that are similar to those of clusters coordinated by the CysIxxCysIIxxCysIII.CysIVP sequence motif found in other ferredoxins with different biochemical properties. In all these cases, the electron added to the oxidized clusters is mainly accommodated in the pair of iron ions coordinated by CysII and CysIV.
来自嗜硫小红卵菌的铁氧化还原蛋白（CvFd）表现出序列和结构上的独特之处。其两个Fe4S4(SCys)4簇具有异常低的电位跃迁，通过核磁共振（NMR）、电子顺磁共振（EPR）和穆斯堡尔光谱结合定点诱变，在此已明确确定了这些跃迁。[4Fe-4S]2+/1+簇（簇II）的配位球包括半胱氨酸40和49之间的一个双螺旋环，连二亚硫酸盐将其还原，E°'为-460 mV。其S = 1/2的EPR信号快速弛豫且因g应变而严重展宽，其穆斯堡尔光谱宽且无法分辨。这些光谱特征对配位环境的微小扰动很敏感，并且它们与CvFd的特定结构元件相关，包括两个配体之间的双螺旋环和C末端α螺旋。对于光谱研究，不可能对簇I进行整体还原（E°' = -660 mV），但由于电位大约正向移动60 mV，通过用甘氨酸取代缬氨酸13实现了蛋白质的完全还原。在低温下，完全还原的蛋白质的EPR光谱是两个相互作用的S = 1/2 [4Fe-4S]1+中心的典型光谱，但由于簇I的电子弛豫比簇II慢得多，在高于20 K的温度下观察到了簇I的分辨信号。在所有氧化还原状态组合中都检测到了β-CH2质子的接触位移核磁共振共振。这些结果表明，涉及CvFd的电子转移反应在数量上与等电位2[4Fe-4S]铁氧化还原蛋白中的类似反应不同。然而，CvFd的还原簇具有与在具有不同生化特性的其他铁氧化还原蛋白中发现的CysIxxCysIIxxCysIII.CysIVP序列基序配位的簇相似的电子分布。在所有这些情况下，添加到氧化簇的电子主要容纳在由半胱氨酸II和半胱氨酸IV配位的一对铁离子中。
