Kisgeropoulos Effie C, Ratzloff Michael W, Stroeva-Dahl Ekaterina M, Hasan Sarah, Varghese Febin, Artz Jacob H, Peters John W, Mulder David W, King Paul W
Biosciences Center, National Renewable Energy Laboratory, Golden, Colorado 80401, United States.
Institute of Biological Chemistry, Washington State University, Pullman, Washington 99163, United States.
Biochemistry. 2025 Jun 3;64(11):2455-2466. doi: 10.1021/acs.biochem.5c00066. Epub 2025 May 13.
[FeFe]-Hydrogenases are structurally diverse enzymes that catalyze reversible H activation at a catalytic cofactor or H-cluster. The H-cluster is a [4Fe-4S] cubane linked by a cysteine thiolate to a diiron subsite containing unique CO, CN, and dithiomethylamine ligands. The established H-cluster resting state of [4Fe-4S]-[Fe-Fe], or H, functions in H binding and oxidation, or by proton-coupled reduction initiates H evolution. In contrast, in [FeFe]-hydrogenase III (CpIII) the resting state of the H-cluster is fully oxidized, [4Fe-4S]-[Fe-Fe], or H. To begin to understand if H has a role in the mechanism of CpIII, we determined the spectroscopic and redox properties of CpIII H-cluster states under catalytic conditions. CpIII poised in H and either equilibrated under 1 atm of H or reduced with sodium dithionite, resulted in a mixture of reduced states including H ( = -407 mV), H-like [4Fe-4S]-[Fe-Fe] ( = -418 mV), H [4Fe-4S]-[Fe-Fe], and H [4Fe-4S]-[Fe-Fe] ( = -455-480 mV). Under H the population of the H-like state was >20-fold higher than H, implicating a role in CpIII catalysis. Unlike other enzymes, there was no spectral evidence of fully reduced states, such as H ([4Fe-4S]-[Fe-Fe]) or H ([4Fe-4S]-[FeFe]-H). Thus, while the H-cluster states of CpIII encompass most of the catalytic intermediates, it is either unable to form H and H, or these states are highly destabilized in CpIII. Thus, these results demonstrate that catalytic intermediates of reduced CpIII differ from the typical intermediates of other catalytic [FeFe]-hydrogenases and may explain the catalytic preference for H production.
[铁铁]-氢化酶是结构多样的酶，可在催化辅因子或H-簇处催化可逆的H活化。H-簇是一个[4Fe-4S]立方烷，通过半胱氨酸硫醇盐与含有独特的CO、CN和二硫代甲胺配体的双铁亚位点相连。已确定的[4Fe-4S]-[Fe-Fe]或H的H-簇静止状态在H结合和氧化中起作用，或通过质子偶联还原引发H的释放。相比之下，在[铁铁]-氢化酶III（CpIII）中，H-簇的静止状态是完全氧化的，即[4Fe-4S]-[Fe-Fe]或H。为了开始了解H在CpIII的机制中是否起作用，我们测定了催化条件下CpIII H-簇状态的光谱和氧化还原性质。处于H状态且在1个大气压的H下平衡或用连二亚硫酸钠还原的CpIII，产生了包括H（E° = -407 mV）、类H的[4Fe-4S]-[Fe-Fe]（E° = -418 mV）、H[4Fe-4S]-[Fe-Fe]和H[4Fe-4S]-[Fe-Fe]（E° = -455 - 480 mV）在内的还原态混合物。在H2存在下，类H状态的丰度比H高20倍以上，这表明其在CpIII催化中起作用。与其他酶不同，没有光谱证据表明存在完全还原态，如H4（[4Fe-4S]-[Fe-Fe]）或H2（[4Fe-4S]-[FeFe]-H2）。因此，虽然CpIII的H-簇状态包含了大部分催化中间体，但它要么无法形成H4和H2，要么这些状态在CpIII中高度不稳定。因此，这些结果表明，还原态CpIII的催化中间体与其他催化性[铁铁]-氢化酶的典型中间体不同，这可能解释了其对H2产生的催化偏好。
