Chandramouli Kala, Johnson Michael K
Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602, USA
Biochemistry. 2006 Sep 19;45(37):11087-95. doi: 10.1021/bi061237w.
The role of the Azotobacter vinelandii HscA/HscB cochaperone system in ISC-mediated iron-sulfur cluster biogenesis has been investigated in vitro by using CD and EPR spectrometry to monitor the effect of HscA, HscB, MgATP, and MgADP on the time course of cluster transfer from [2Fe-2S]IscU to apo-Isc ferredoxin. CD spectra indicate that both HscB and HscA interact with [2Fe-2S]IscU and the rate of cluster transfer was stimulated more than 20-fold in the presence stoichiometric HscA and HscB and excess MgATP. No stimulation was observed in the absence of either HscB or MgATP, and cluster transfer was found to be an ATP-dependent reaction based on concomitant phosphate production and the enhanced rates of cluster transfer in the presence of KCl which is known to stimulate HscA ATPase activity. The results demonstrate a role of the ISC HscA/HscB cochaperone system in facilitating efficient [2Fe-2S] cluster transfer from the IscU scaffold protein to acceptor proteins and that [2Fe-2S] cluster transfer from IscU is an ATP-dependent process. The data are consistent with the proposed regulation of the HscA ATPase cycle by HscB and IscU [Silberg, J. J., Tapley, T. L., Hoff, K. G., and Vickery, L. E. (2004) J. Biol. Chem. 279, 53924-53931], and mechanistic proposals for coupling of the HscA ATPase cycle with cluster transfer from [2Fe-2S]IscU to apo-IscFdx are discussed.
通过使用圆二色光谱（CD）和电子顺磁共振光谱（EPR）来监测HscA、HscB、MgATP和MgADP对[2Fe-2S]铁硫簇从IscU转移至脱辅基Isc铁氧还蛋白的时间进程的影响，已在体外研究了棕色固氮菌HscA/HscB伴侣蛋白系统在铁硫簇（ISC）介导的铁硫簇生物合成中的作用。CD光谱表明，HscB和HscA均与[2Fe-2S]IscU相互作用，在化学计量的HscA和HscB以及过量MgATP存在的情况下，簇转移速率被刺激提高了20多倍。在没有HscB或MgATP的情况下未观察到刺激作用，并且基于伴随的磷酸盐产生以及在已知可刺激HscA ATP酶活性的KCl存在下簇转移速率的提高，发现簇转移是一个ATP依赖性反应。结果证明了ISC HscA/HscB伴侣蛋白系统在促进[2Fe-2S]簇从IscU支架蛋白高效转移至受体蛋白中的作用，并且从IscU进行的[2Fe-2S]簇转移是一个ATP依赖性过程。这些数据与HscB和IscU对HscA ATP酶循环的拟议调节一致[Silberg, J. J., Tapley, T. L., Hoff, K. G., and Vickery, L. E. (2004) J. Biol. Chem. 279, 53924 - 53931]，并且讨论了将HscA ATP酶循环与[2Fe-2S]簇从IscU转移至脱辅基IscFdx相偶联的机制提议。
