The function and properties of the iron-sulfur center in spinach ferredoxin: thioredoxin reductase: a new biological role for iron-sulfur clusters.
The function and properties of the iron-sulfur center in spinach ferredoxin: thioredoxin reductase: a new biological role for iron-sulfur clusters.
复制标题
菠菜铁氧还蛋白铁硫中心的功能和性质：硫氧还蛋白还原酶：铁硫簇的新生物学作用。
DOI：
10.1021/bi961007p
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Johnson,MK
中科院分区：
文献类型：
--
作者：
Staples,CR;Ameyibor,E;Fu,W;Gardet-Salvi,L;Stritt-Etter,AL;Schurmann,P;Knaff,DB;Johnson,MK
叶绿体中硫氧还蛋白的还原是由一类独特的二硫还原酶催化的，这些二硫还原酶以[2Fe-2S]2+/+铁氧还蛋白为电子供体，除了活性位点二硫外，还含有一个Fe-S簇作为唯一的假基。采用紫外/可见吸收、变温磁圆二色性（MCD）、EPR和共振拉曼（RR）光谱等方法研究了菠菜铁氧还蛋白：硫氧还蛋白还原酶（FTR）中Fe-S簇的性质、性质和功能。结果表明，anS= 0 [4Fe-4S]2+具有完全半胱氨酸配位，在−650 mV电位下不能被还原，但可以被铁氰化物氧化成anS=1/2[4Fe-4S]3+态（g= 2.09, 2.04, 2.02）。[4Fe-4S]3+/2+对的中点电位估计为+420 mV（相对于nhe）。这些结果反驳了簇在介导电子从铁氧还蛋白（Em= - 420 mV）到活性位点二硫化物（Em= - 230 mV,n= 2）的传递中的作用。通过对活性位点二硫醇的一个半胱氨酸在n-乙基马来酰亚胺（NEM）中烷基化的酶的修饰形式的平行光谱研究，提出了该簇在稳定单电子还原中间体中的另一种作用。制备的nem修饰的FTR呈顺磁性，表现出缓慢的弛豫，S=1/2EPR信号，g= 2.11, 2.00, 1.98，在150 K以下没有明显的展宽。虽然弛豫性质是自由基的特征，但MCD， RR和吸收研究表明，至少部分团簇氧化为[4Fe-4S]3+态。染料介导的EPR氧化还原滴定表明，单电子还原到抗磁状态的中点电位为−210 mV。与偶氮细菌vinelandii7Fe铁氧还蛋白中铁氰化物氧化的[4Fe-4S]簇的性质类似[Hu， Z., Jollie， D., Burgess, b.k ., Stephens, p.j ., & m<s:1> nck， E.（1994）生物化学33,14475−14485]，nem修饰的FTR的光谱和氧化还原性质解释为[4Fe-4S]2+簇通过簇硫共价连接到一个活性位点硫醇上形成的半胱氨酸基噻基自由基。提出了一种FTR的机制方案，类似于已被充分表征的NAD(P) h依赖的含黄素二硫氧化还原酶，但涉及顺序的单电子氧化还原过程，其中[4Fe-4S]2+簇的作用是稳定活性位点二硫的初始单电子还原形成的硫基自由基。结果表明，Fe-S簇具有新的生物学作用，包括噻基自由基中间体的稳定和涉及桥接硫化物的簇位点特异性化学。
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 anS= 0 [4Fe-4S]2+cluster with complete cysteinyl-Scoordination that cannot be reduced at potentials down to −650 mV, but can be oxidized by ferricyanide to anS=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 (versusNHE). 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 withN-ethylmaleimide (NEM). NEM-modified FTR is paramagnetic as prepared and exhibits a slow relaxing,S=1/2EPR 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 inAzotobacter vinelandii7Fe ferredoxin [Hu, Z., Jollie, D., Burgess, B. K., Stephens, P. J., & Münck, E. (1994)Biochemistry33, 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.