Effect of serinate ligation at each of the iron sites of the [Fe4S4] cluster of Pyrococcus furiosus ferredoxin on the redox, spectroscopic, and biological properties
Effect of serinate ligation at each of the iron sites of the [Fe4S4] cluster of Pyrococcus furiosus ferredoxin on the redox, spectroscopic, and biological properties
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DOI：
10.1021/bi990671d
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发表时间：
1999-08-10
期刊：
影响因子：
2.9
通讯作者：
Adams, MWW
中科院分区：
文献类型：
--
作者：
Brereton, PS;Duderstadt, RE;Adams, MWW
激烈热球菌铁氧还蛋白（Fd）含有由三个半胱氨酸（在位置11、17和56处）和一个天冬氨酸配体（在位置14处）配位的单个[Fe 4S 4]簇。在这项研究中，光谱，氧化还原，和D14 C，D14 C/C11 S，D14 S，D14 C/C17 S和D14 C/C56 S突变的功能后果进行了研究。四个丝氨酸变体各自含有一个丝氨酸和三个半胱氨酸残基的潜在簇配位球，在[Fe 4S 4]簇的四个Fe位点中的每一个处具有丝氨酸连接。所有五种变体都在大肠杆菌中表达，并且每个变体都含有[Fe 4S 4]（2+，+）簇，如通过氧化蛋白的UV-可见吸收和共振拉曼研究以及制备的连二亚硫酸盐还原蛋白的EPR和变温磁性圆二色性（VTMCD）研究所示。吸收和共振拉曼光谱的变化与从D14 C变体中的完全半胱氨酸簇连接到四种丝氨酸变体中的每一种中的三个半胱氨酸和一个含氧配体的变化一致。EPR和VTMCD研究表明，这些变体蛋白中的每一个中的顺磁性[Fe 4S 4]（+）中心具有独特的基态和激发态性质，其中D14 C和D14 C/C11 S变体具有均匀的S = 1/2基态，而D14 S、D14 C/C17 S和D14 C/C56 S变体具有混合自旋，S = 1/2和3/2基态。在实验误差范围内，D14 C/C11 S和D14 C/C17 S变体的中点电位（pH 7.0，23 ℃）与D14 C变体的中点电位（E-m = -427 mV）相比没有变化，但D14 C/C56 S和D14 S变体的电位分别负49 mV和78 mV。由于VTMCD光谱表明在所有五种变体中存在价离域Fe2.5+Fe2.5+对，因此中点电位被解释为Cys 11和Cys 17连接D14 C中的不可还原价离域对。只有D14 S变体表现出在3.5-10范围内的pH依赖性氧化还原电位;这归因于丝氨酸配体质子化为还原簇（pK（a）= 4.75）。所有五种变体在偶联测定中具有相似的K-m和Vm值，其中Fd被丙酮酸铁氧还蛋白氧化还原酶（POR）还原，并被铁氧还蛋白NADP氧化还原酶（FNOR）氧化，两者都从激烈毕赤酵母中纯化。因此，[Fe_4S_4]团簇中每个Fe原子的配位方式对Fd与FNOR之间的相互作用和电子转移影响不大。
Pyrococcus furiosus ferredoxin (Fd) contains a single [Fe4S4] cluster coordinated by three cysteine (at positions 11, 17, and 56) and one aspartate Ligand (at position 14). In this study, the spectroscopic, redox, and functional consequences of D14C, D14C/C11S, D14S, D14C/C17S, and D14C/C56S mutations have been investigated. The four serine variants each contain a potential cluster coordination sphere of one serine and three cysteine residues, with serine ligation at each of the four Fe sites of the [Fe4S4] cluster. All five variants were expressed in Escherichia coli, and each contained a [Fe4S4](2+,+) cluster as shown by UV-visible absorption and resonance Raman studies of the oxidized protein and EPR and variable-temperature magnetic circular dichroism (VTMCD) studies of the as-prepared, dithionite-reduced protein. Changes in both the absorption and resonance Raman spectra are consistent with changing from complete cysteinyl cluster ligation in the D14C variant to three cysteines' and one oxygenic ligand in each of the four serine variants. EPR and VTMCD studies show distinctive ground and excited state properties for the paramagnetic [Fe4S4](+) centers in each of these variant proteins, with the D14C and D14C/C11S variants having homogeneous S = 1/2 ground states and the D14S,D14C/C17S,and D14C/C56S variants having mixed-spin, S = 1/2 and 3/2 ground states. The midpoint potentials (pH 7.0, 23 degrees C) of the D14C/C11S and D14C/C17S variants were unchanged compared to that of the D14C variant (E-m = -427 mV) within experimental error, but the potentials of D14C/C56S and D14S variants were more negative by 49 and 78 mV, respectively. Since the VTMCD spectra indicate the presence of a valence-delocalized Fe2.5+Fe2.5+ pair in all five variants, the midpoint potentials are interpreted in terms of Cys11 and Cys17 ligating the nonreducible valence-delocalized pair in D14C. Only the D14S variant exhibited a pH-dependent redox potential over the range of 3.5-10; and this is attributed to protonation of the serinate ligand to the reduced cluster (pK(a) = 4.75). All five variants had similar K-m and Vm values in a coupled assay in which Fd was reduced by pyruvate ferredoxin oxidoreductase (POR) and,oxidized by ferredoxin NADP oxidoreductase (FNOR), both purified from P. furiosus. Hence, the mode of Ligation at each Fe atom in the [Fe4S4] cluster appears to have little effect on the interaction and the electron transfer between Fd and FNOR.