Schmidt Andrea, Kalms Jacqueline, Lorent Christian, Katz Sagie, Frielingsdorf Stefan, Evans Rhiannon M, Fritsch Johannes, Siebert Elisabeth, Teutloff Christian, Armstrong Fraser A, Zebger Ingo, Lenz Oliver, Scheerer Patrick
Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Medical Physics and Biophysics (CC2), Group Structural Biology of Cellular Signaling Charitéplatz 1 10117 Berlin Germany
Institut für Chemie, Biophysical Chemistry, Technische Universität Berlin Straße des 17. Juni 135 10623 Berlin Germany
Chem Sci. 2023 Sep 20;14(40):11105-11120. doi: 10.1039/d3sc03739h. eCollection 2023 Oct 18.
The membrane-bound [NiFe]-hydrogenase of is a rare example of a truly O-tolerant hydrogenase. It catalyzes the oxidation of H into 2e and 2H in the presence of high O concentrations. This characteristic trait is intimately linked to the unique Cys[4Fe-3S] cluster located in the proximal position to the catalytic center and coordinated by six cysteine residues. Two of these cysteines play an essential role in redox-dependent cluster plasticity, which bestows the cofactor with the capacity to mediate two redox transitions at physiological potentials. Here, we investigated the individual roles of the two additional cysteines by replacing them individually as well as simultaneously with glycine. The crystal structures of the corresponding MBH variants revealed the presence of Cys[4Fe-4S] or Cys[4Fe-4S] clusters of different architecture. The protein X-ray crystallography results were correlated with accompanying biochemical, spectroscopic and electrochemical data. The exchanges resulted in a diminished O tolerance of all MBH variants, which was attributed to the fact that the modified proximal clusters mediated only one redox transition. The previously proposed O protection mechanism that detoxifies O to HO using four protons and four electrons supplied by the cofactor infrastructure, is extended by our results, which suggest efficient shutdown of enzyme function by formation of a hydroxy ligand in the active site that protects the enzyme from O binding under electron-deficient conditions.
嗜热栖热菌的膜结合[NiFe]氢化酶是真正耐氧氢化酶的罕见例子。在高氧浓度存在下，它催化H氧化为2e和2H。这一特性与位于催化中心近端、由六个半胱氨酸残基配位的独特Cys[4Fe-3S]簇密切相关。其中两个半胱氨酸在依赖氧化还原的簇可塑性中起重要作用，赋予辅因子在生理电位下介导两个氧化还原转变的能力。在这里，我们通过将另外两个半胱氨酸分别或同时替换为甘氨酸来研究它们各自的作用。相应的MBH变体的晶体结构揭示了不同结构的Cys[4Fe-4S]或Cys[4Fe-4S]簇的存在。蛋白质X射线晶体学结果与伴随的生化、光谱和电化学数据相关联。这些替换导致所有MBH变体的耐氧性降低，这归因于修饰后的近端簇仅介导一个氧化还原转变这一事实。我们的结果扩展了先前提出的利用辅因子基础设施提供的四个质子和四个电子将O解毒为HO的氧保护机制，结果表明在电子缺乏条件下，通过在活性位点形成羟基配体来有效关闭酶功能，从而保护酶不与O结合。
