Staples C R, Ameyibor E, Fu W, Gardet-Salvi L, Stritt-Etter A L, Schürmann P, Knaff D B, Johnson M K
Department of Chemistry, University of Georgia, Athens 30602, USA.
Biochemistry. 1996 Sep 3;35(35):11425-34. doi: 10.1021/bi961007p.
Thioredoxin reduction in chloroplasts is catalyzed by a unique class of disulfide reductases which use a [2Fe-2S]2+/+ ferredoxin as the electron donor and contain an Fe-S cluster as the sole prosthetic group in addition to the active-site disulfide. The nature, properties, and function of the Fe-S cluster in spinach ferredoxin:thioredoxin reductase (FTR) have been investigated by the combination of UV/visible absorption, variable-temperature magnetic circular dichroism (MCD), EPR, and resonance Raman (RR) spectroscopies. The results indicate the presence of an S = 0 [4Fe-4S]2+ cluster with complete cysteinyl-S coordination that cannot be reduced at potentials down to -650 mV, but can be oxidized by ferricyanide to an S = 1/2 [4Fe-4S]3+ state (g = 2.09, 2.04, 2.02). The midpoint potential for the [4Fe-4S]3+/2+ couple is estimated to be +420 mV (versus NHE). These results argue against a role for the cluster in mediating electron transport from ferredoxin (Em = -420 mV) to the active-site disulfide (Em = -230 mV, n = 2). An alternative role for the cluster in stabilizing the one-electron-reduced intermediate is suggested by parallel spectroscopic studies of a modified form of the enzyme in which one of the cysteines of the active-site dithiol has been alkylated with N-ethylmaleimide (NEM). NEM-modified FTR is paramagnetic as prepared and exhibits a slow relaxing, S = 1/2 EPR signal, g = 2.11, 2.00, 1.98, that is observable without significant broadening up to 150 K. While the relaxation properties are characteristic of a radical species, MCD, RR, and absorption studies indicate at least partial cluster oxidation to the [4Fe-4S]3+ state. Dye-mediated EPR redox titrations indicate a midpoint potential of -210 mV for the one-electron reduction to a diamagnetic state. By analogy with the properties of the ferricyanide-oxidized [4Fe-4S] cluster in Azotobacter vinelandii 7Fe ferredoxin [Hu, Z., Jollie, D., Burgess, B. K., Stephens, P. J., & Münck, E. (1994) Biochemistry 33, 14475-14485], the spectroscopic and redox properties of NEM-modified FTR are interpreted in terms of a [4Fe-4S]2+ cluster covalently attached through a cluster sulfide to a cysteine-based thiyl radical formed on one of the active-site thiols. A mechanistic scheme for FTR is proposed with similarities to that established for the well-characterized NAD(P)H-dependent flavin-containing disulfide oxidoreductases, but involving sequential one-electron redox processes with the role of the [4Fe-4S]2+ cluster being to stabilize the thiyl radical formed by the initial one-electron reduction of the active-site disulfide. The results indicate a new biological role for Fe-S clusters involving both the stabilization of a thiyl radical intermediate and cluster site-specific chemistry involving a bridging sulfide.
叶绿体中的硫氧还蛋白还原由一类独特的二硫键还原酶催化，这类酶利用[2Fe-2S]2+/+铁氧还蛋白作为电子供体，除活性位点二硫键外，还含有一个铁硫簇作为唯一的辅基。通过紫外/可见吸收、变温磁圆二色性（MCD）、电子顺磁共振（EPR）和共振拉曼（RR）光谱的结合，研究了菠菜铁氧还蛋白：硫氧还蛋白还原酶（FTR）中铁硫簇的性质、特性和功能。结果表明存在一个S = 0的[4Fe-4S]2+簇，其具有完整的半胱氨酰-S配位，在低至-650 mV的电位下不能被还原，但可被铁氰化物氧化为S = 1/2的[4Fe-4S]3+状态（g = 2.09、2.04、2.02）。[4Fe-4S]3+/2+电对的中点电位估计为+420 mV（相对于标准氢电极）。这些结果反对该簇在介导电子从铁氧还蛋白（Em = -420 mV）传递到活性位点二硫键（Em = -230 mV，n = 2）中起作用。通过对活性位点二硫醇的一个半胱氨酸用N-乙基马来酰亚胺（NEM）进行烷基化修饰的酶的平行光谱研究，提出了该簇在稳定单电子还原中间体方面的另一种作用。制备的NEM修饰的FTR是顺磁性的，表现出缓慢弛豫的S = 1/2 EPR信号，g = 2.11、2.00、1.98，在高达150 K时观察到该信号且没有明显展宽。虽然弛豫特性是自由基物种的特征，但MCD、RR和吸收研究表明至少部分簇被氧化为[4Fe-4S]3+状态。染料介导的EPR氧化还原滴定表明单电子还原为抗磁性状态的中点电位为-210 mV。与棕色固氮菌7Fe铁氧还蛋白中被铁氰化物氧化的[4Fe-4S]簇的性质[Hu, Z., Jollie, D., Burgess, B. K., Stephens, P. J., & Münck, E. (1994) Biochemistry 33, 14475 - 14485]类似，NEM修饰的FTR的光谱和氧化还原性质被解释为一个[4Fe-4S]2+簇通过簇硫化物共价连接到在活性位点硫醇之一上形成的基于半胱氨酸的硫自由基。提出了一个FTR的机制方案，与已确定的特征明确的依赖NAD(P)H的含黄素二硫键氧化还原酶的机制方案相似，但涉及连续的单电子氧化还原过程，其中[4Fe-4S]2+簇的作用是稳定由活性位点二硫键的初始单电子还原形成的硫自由基。结果表明铁硫簇具有一种新的生物学作用，涉及硫自由基中间体的稳定以及涉及桥连硫化物的簇位点特异性化学。
