INVESTIGATION OF METAL-ION UPTAKE REACTIVITIES OF [3FE-4S] CLUSTERS IN PROTEINS - VOLTAMMETRY OF COADSORBED FERREDOXIN AMINOCYCLITOL FILMS AT GRAPHITE-ELECTRODES AND SPECTROSCOPIC IDENTIFICATION OF TRANSFORMED CLUSTERS
INVESTIGATION OF METAL-ION UPTAKE REACTIVITIES OF [3FE-4S] CLUSTERS IN PROTEINS - VOLTAMMETRY OF COADSORBED FERREDOXIN AMINOCYCLITOL FILMS AT GRAPHITE-ELECTRODES AND SPECTROSCOPIC IDENTIFICATION OF TRANSFORMED CLUSTERS
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DOI：
10.1021/ja00017a045
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发表时间：
1991-08-14
影响因子：
15
通讯作者：
HATCHIKIAN, EC
中科院分区：
文献类型：
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作者：
BUTT, JN;ARMSTRONG, FA;HATCHIKIAN, EC
用一种简便、经济的伏安法研究了蛋白质中Fe-S簇的易转化过程，即[3Fe-4S {0 + M2 +]可逆[M3Fe-4S] 2+，其中金属离子M进入3Fe簇的空位形成立方烷型结构.该技术扩展了最近的发现，铁氧还蛋白与氨基环醇共吸附在热解石墨"'边缘-（PGE）电极，得到一个稳定的电活性膜，并证明了由非洲脱硫弧菌铁氧还蛋白III（Fd 111）的集群相互转换的调查。转移到pH 7的含EGTA的缓冲溶液中的预成型膜的循环伏安扫描（通常在0至-850 mV vs SHE的区域内）揭示了由于三个氧化还原对而产生的明确的伏安信号。其中一个（B '）对应于稳定的[4Fe-4S] 2 +/+团簇;另外两个（A'和C '）与[3Fe-4S]团簇相关，并分别指定为正常的1 +/0对和化学可逆的双电子过程，但尚未建立公式。如果然后将涂覆的电极转移到不含EGTA但含有低浓度的Fe 2+、Zn 2+或Cd 2+的搅拌溶液中，则通过偶对A '（[3Fe-4S]+/0）的还原通道引发快速变化。在随后的几秒钟的循环过程中，波A '和C'同时消失，并被新的成对波D '（M）所取代。E度的值如下：D '（Fe），-393 +/-10 mV; D'（Zn），-492 +/-10 mV; D '（cd），-569 +/-10 mV。新的电对的位置密切对应的循环伏安图的Fd III在溶液相进行改造。通过EPR和MCD光谱表征，分别鉴定后一种物质为[4Fe-4S] 2 +/+、[Zn3Fe-4S] 2 +/+和[Cd3Fe-4S] 2 +/+。[Zn3Fe-4S] 2+和[Cd3Fe-4S] 2+与[3Fe-4S] 0等电子，基态S = 2，轴向负零场分裂.还原态[Zn_3Fe-4S]~+和[Cd_3Fe-4S]~+具有基态自旋S = 5/2，具有特征EPR谱。限制在电极表面的蛋白质分子的金属离子吸收的速率和平衡取决于金属离子的特性和浓度。由{M2 +}{3Fe-4S] 0}/{[M3Fe-4S] 2 +}给出的平衡解离常数K（d）的值如下：Fe，30 +/-15 μ M; Zn，1.6 +/-1.0 μ M; Cd，0.8 +/-0.5 μ M。由此建立了Cd~（2+）与Zn~（2+）>> Fe~（2+）的亲合力顺序。结果表明，与Fe相比，在蛋白质中[M3Fe-4S]簇的M亚位点处，对Zn（生物丰富的元素）的内在偏好是可行的。
Facile transformation of Fe-S clusters in proteins, as described by [3Fe-4S{0 + M2+ reversible [M3Fe-4S]2+ in which metal ion M enters the vacant subsite of a 3Fe cluster to complete a cubane-type structure, is identified and studied by a convenient and highly economical voltammetric procedure. The technique extends a recent discovery that ferredoxins coadsorb with aminocyclitols at a pyrolytic graphite ''edge- (PGE) electrode, giving a stable electroactive film, and is demonstrated by an investigation of cluster interconversions in Desulfovibrio africanus ferredoxin Ill (Fd 111). Cyclic voltammetric scanning (typically over the region 0 to -850 mV vs SHE) of a preformed film, transferred to an EGTA-containing buffer solution at pH 7, reveals well-defined voltammetric signals due to three redox couples. One of these (B') corresponds to the stable [4Fe-4S]2+/+ cluster; the other two (A' and C') are associated with the [3Fe-4S] cluster and assigned respectively as the normal 1+/0 couple and a chemically reversible two-electron process of as yet unestablished formulation. If the coated electrode is then transferred to stirred solutions devoid of EGTA but containing low concentrations of Fe2+, Zn2+, or Cd2+, reductive passage through couple A' ([3Fe-4S]+/0) initiates rapid changes. During subsequent cycles over the course of several seconds, waves A' and C' disappear simultaneously and are replaced by a new couple D'(M). Values of E-degrees' are as follows: D'(Fe), -393 +/- 10 mV; D'(Zn), -492 +/- 10 mV; D'(cd), -569 +/- 10 mV. The positions of the new couples correspond closely with cyclic voltammograms of Fd III undergoing transformations in the solution phase. Characterization by EPR and MCD spectroscopy identifies the latter species to be [4Fe-4S]2+/+, [Zn3Fe-4S]2+/+, and [Cd3Fe-4S]2+/+, respectively. The clusters [Zn3Fe-4S]2+ and [Cd3Fe-4S]2+ are shown to be isoelectronic with [3Fe-4S]0 and to have a ground electronic state S = 2 subject to a negative axial zero-field splitting. The reduced states [Zn3Fe-4S]+ and [Cd3Fe-4S]+ have a ground state spin S = 5/2 with characteristic EPR spectra. Rates and equilibria of metal ion uptake for protein molecules confined to the electrode surface depend upon the identity and concentration of the metal ion. Values of K(d), the equilibrium dissociation constant given by {M2+}{3Fe-4S]0}/{[M3Fe-4S]2+} are as follows: Fe, 30 +/- 15-mu-M; Zn, 1.6 +/- 1.0-mu-M; Cd, 0.8 +/- 0.5-mu-M. The affinity order Cd2+ greater-than-or-equal-to Zn2+ >> Fe2+ is thus established. The results demonstrate the feasibility of an intrinsic preference for Zn (a biologically abundant element) as compared to Fe, at the M subsite of [M3Fe-4S] clusters in proteins.