Ye Hong, Garifullina Gulnara F, Abdel-Ghany Salah E, Zhang Lihong, Pilon-Smits Elizabeth A H, Pilon Marinus
Department of Biology, Colorado State University, Anatomy/Zoology Building, Fort Collins, CO 80523, USA.
Planta. 2005 Feb;220(4):602-8. doi: 10.1007/s00425-004-1388-1. Epub 2004 Oct 8.
Plastids are known to be able to synthesize their own iron-sulfur clusters, but the biochemical machinery responsible for this process is not known. In this study it is investigated whether CpNifS, the chloroplastic NifS-like cysteine desulfurase of Arabidopsis thaliana (L.) Heynh. is responsible for the release of sulfur from cysteine for the biogenesis of iron-sulfur (Fe-S) clusters in chloroplasts. Using an in vitro reconstitution assay it was found that purified CpNifS was sufficient for Fe-S cluster formation in ferredoxin in the presence of cysteine and a ferrous iron salt. Antibody-depletion experiments using stromal extract showed that CpNifS is also essential for the Fe-S cluster formation activity of chloroplast stroma. The activity of CpNifS in the stroma was 50- to 80-fold higher than that of purified CpNifS on a per-protein basis, indicating that other stromal factors cooperate in Fe-S cluster formation. When stromal extract was separated on a gel-filtration column, most of the CpNifS eluted as a dimer of 86 kDa, but a minor fraction of the stromal CpNifS eluted at a molecular weight of approx. 600 kDa, suggesting the presence of a multi-protein complex. The possible nature of the interacting proteins is discussed.
已知质体能够合成自身的铁硫簇，但负责这一过程的生化机制尚不清楚。在本研究中，对拟南芥（Arabidopsis thaliana (L.) Heynh.）叶绿体中类NifS半胱氨酸脱硫酶CpNifS是否负责从半胱氨酸中释放硫以用于叶绿体中铁硫（Fe-S）簇的生物合成进行了研究。通过体外重组试验发现，在存在半胱氨酸和亚铁盐的情况下，纯化的CpNifS足以在铁氧还蛋白中形成Fe-S簇。使用基质提取物进行的抗体去除实验表明，CpNifS对于叶绿体基质的Fe-S簇形成活性也是必不可少的。以每蛋白计，基质中CpNifS的活性比纯化的CpNifS高50至80倍，这表明其他基质因子在Fe-S簇形成过程中协同作用。当基质提取物在凝胶过滤柱上分离时，大部分CpNifS以86 kDa的二聚体形式洗脱，但一小部分基质CpNifS以约600 kDa的分子量洗脱，这表明存在一种多蛋白复合物。文中讨论了相互作用蛋白的可能性质。
