Hu Z, Jollie D, Burgess B K, Stephens P J, Münck E
Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213-3890.
Biochemistry. 1994 Dec 6;33(48):14475-85. doi: 10.1021/bi00252a014.
Azotobacter vinelandii ferredoxin I (FdI) is a small protein that contains one Fe4S4 cluster and one Fe3S4 cluster. Previous studies of FdI have shown that the redox potential of the Fe3S4 cluster and the MCD and CD spectra of the reduced Fe3S4 cluster are pH-dependent. Using Mössbauer and EPR spectroscopy, we have studied FdI in different oxidation states and at different pH values. Here, we report the spin Hamiltonian parameters of the oxidized (S = 1/2) Fe3S4 cluster at pH 7.4 and the reduced (S = 2) Fe3S4 cluster at pH 6.0 and 8.5. The pH dependence observed by MCD is also evident in the Mössbauer spectra which show a change of the magnetic hyperfine tensor for one Fe site of the valence-delocalized pair. The Fe4S4 cluster is ligated by cysteines 20, 39, 42, and 45, but not by the adjacent cysteine 24. Treatment of FdI with 3 equiv of ferricyanide alters the Fe4S4 cluster, yielding a new species, [Fe4S4]'. The S = 1/2 EPR signal of [Fe4S4]' has previously been attributed to the formation of a cysteine disulfide radical from Cys24 and cluster sulfide. Here we show that the EPR signal is broadened by 57Fe, indicating that the electronic spin is significantly coupled to the cluster iron. Consistent with this, substantial magnetic hyperfine interactions are observed by Mössbauer spectroscopy. In addition, the average isomer shift of the four Fe sites is smaller for [Fe4S4]' than for [Fe4S4]2+, indicating that the oxidation is iron-based to at least some extent. Incubation of FdI with excess ferricyanide destroys the Fe4S4 cluster but leaves the Fe3S4 cluster intact. Our studies of (3Fe)FdI show that the S = 1/2 spin of the Fe3S4 cluster interacts with another paramagnet, presumably a radical generated at the site left vacant by the removal of the Fe4S4 cluster.
维涅兰德固氮菌铁氧化还原蛋白I（FdI）是一种小蛋白，包含一个Fe4S4簇和一个Fe3S4簇。先前对FdI的研究表明，Fe3S4簇的氧化还原电位以及还原态Fe3S4簇的磁圆二色性（MCD）和圆二色性（CD）光谱均依赖于pH值。我们使用穆斯堡尔光谱和电子顺磁共振（EPR）光谱，研究了处于不同氧化态以及不同pH值下的FdI。在此，我们报告了pH 7.4时氧化态（S = 1/2）的Fe3S4簇以及pH 6.0和8.5时还原态（S = 2）的Fe3S4簇的自旋哈密顿参数。MCD观察到的pH依赖性在穆斯堡尔光谱中也很明显，该光谱显示了价态离域对中一个铁位点的磁超精细张量的变化。Fe4S4簇由半胱氨酸20、39、42和45配位，但不与相邻的半胱氨酸24配位。用3当量的铁氰化物处理FdI会改变Fe4S4簇，产生一个新物种[Fe4S4]'。[Fe4S4]'的S = 1/2 EPR信号先前被归因于由Cys24和簇硫化物形成的半胱氨酸二硫自由基。在此我们表明，该EPR信号被57Fe加宽，这表明电子自旋与簇铁有显著耦合。与此一致的是，穆斯堡尔光谱观察到了大量的磁超精细相互作用。此外，[Fe4S4]'的四个铁位点的平均同质异能位移比[Fe4S4]2+的小，这表明氧化至少在一定程度上是基于铁的。将FdI与过量铁氰化物一起孵育会破坏Fe4S4簇，但使Fe3S4簇保持完整。我们对（3Fe）FdI的研究表明，Fe3S4簇的S = 1/2自旋与另一个顺磁体相互作用，推测是在去除Fe4S4簇后留下的空位处产生的一个自由基。
