Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
J Am Chem Soc. 2013 May 29;135(21):7992-8000. doi: 10.1021/ja402150q. Epub 2013 May 14.
Metal centers in metalloproteins involve multiple metal-ligand bonds. The release of metal ions from metalloproteins can have significant biological consequences, so understanding of the mechanisms by which metal ion dissociates has broad implications. By definition, the release of metal ions from metalloproteins involves the disruption of multiple metal-ligand bonds, and this process is often accompanied by unfolding of the protein. Detailed pathways for metal ion release from metalloproteins have been difficult to elucidate by classical ensemble techniques. Here, we combine single molecule force spectroscopy and protein engineering techniques to investigate the mechanical dissociation mechanism of iron from the active site of the simplest iron-sulfur protein, rubredoxin, at the single molecule level. Our results reveal that the mechanical rupture of this simplest iron center is stochastic and follows multiple, complex pathways that include concurrent rupture of multiple ferric-thiolate bonds as well as sequential rupture of ferric-thiolate bonds that lead to the formation of intermediate species. Our results uncover the surprising complexity of the rupture process of the seemingly simple iron center in rubredoxin and provide the first unambiguous experimental evidence concerning the detailed mechanism of mechanical disruption of a metal center in its native protein environment in aqueous solution. This study opens up a new avenue to investigating the rupture mechanism of metal centers in metalloproteins with unprecedented resolution by using single molecule force spectroscopy techniques.
金属蛋白中的金属中心涉及多个金属配体键。金属蛋白中金属离子的释放可能会产生重大的生物学后果，因此，理解金属离子解离的机制具有广泛的意义。根据定义，金属蛋白中金属离子的释放涉及到多个金属配体键的破坏，这个过程通常伴随着蛋白质的展开。通过经典的整体技术来阐明金属离子从金属蛋白中释放的详细途径一直具有挑战性。在这里，我们结合单分子力谱技术和蛋白质工程技术，在单分子水平上研究了最简单的铁硫蛋白——细胞色素 c 中的铁从活性部位机械解离的机制。我们的结果表明，这种最简单的铁中心的机械断裂是随机的，并遵循多种复杂的途径，包括同时断裂多个铁-硫键以及铁-硫键的顺序断裂，导致中间产物的形成。我们的结果揭示了细胞色素 c 中看似简单的铁中心的断裂过程的惊人复杂性，并为在水溶液中原位蛋白质环境中机械破坏金属中心的详细机制提供了第一个明确的实验证据。这项研究通过使用单分子力谱技术，为研究金属蛋白中金属中心的断裂机制开辟了一条新的途径，具有前所未有的分辨率。
