H‑cluster Intermediates and Catalytic Properties of Clostridium pasteurianum [FeFe]-Hydrogenase III

[FeFe]-Hydrogenases are structurally diverse enzymes that catalyze reversible H2 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]2+-[FeII-FeI], or Hox, functions in H2 binding and oxidation, or by proton-coupled reduction initiates H2 evolution. In contrast, in Clostridium pasteurianum [FeFe]-hydrogenase III (CpIII) the resting state of the H-cluster is fully oxidized, [4Fe-4S]2+-[FeII-FeII], or Hox+1. To begin to understand if Hox+1 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 Hox+1 and either equilibrated under 1 atm of H2 or reduced with sodium dithionite, resulted in a mixture of reduced states including Hox (Em8 = −407 mV), Htrans-like [4Fe-4S]+-[FeII-FeII] (Em8 = −418 mV), Hred [4Fe-4S]+-[FeII-FeI], and HredH+ [4Fe-4S]2+-[FeI-FeI] (Em8 = −455–480 mV). Under H2 the population of the Htrans-like state was >20-fold higher than Hox, implicating a role in CpIII catalysis. Unlike other enzymes, there was no spectral evidence of fully reduced states, such as HsredH+ ([4Fe-4S]+-[FeI-FeI]) or Hhyd ([4Fe-4S]+-[FeII‑FeII]-H–). Thus, while the H-cluster states of CpIII encompass most of the catalytic intermediates, it is either unable to form HsredH+ and Hhyd, 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 H2 production.[FeFe]-氢化酶（[FeFe]-Hydrogenases）是一类结构多样的酶，可在催化辅因子（catalytic cofactor）——即H簇（H-cluster）——处催化可逆的氢气活化反应。H簇是一种通过半胱氨酸硫醇盐（cysteine thiolate）连接至双铁亚位点（diiron subsite）的[4Fe-4S]立方烷（[4Fe-4S] cubane），该双铁亚位点含有独特的CO、CN⁻与二硫代甲胺配体（ligands）。已确定的H簇静息态（resting state）为[4Fe-4S]²⁺-[FeII-FeI]，即Hox态（Hox），其功能在于结合氢气并催化其氧化，或是通过质子偶联还原（proton-coupled reduction）启动氢气生成过程。与之形成对比的是，巴氏梭菌（Clostridium pasteurianum）来源的[FeFe]-氢化酶III（CpIII）中，H簇的静息态为完全氧化形式[4Fe-4S]²⁺-[FeII-FeII]，即Hox+1态。为探明Hox+1态是否在CpIII的催化机制中发挥功能，我们测定了催化条件下CpIII的H簇状态的光谱学与氧化还原特性。将处于Hox+1态的CpIII在1标准大气压氢气下平衡，或是用连二亚硫酸钠（sodium dithionite）还原后，可得到一系列还原态混合物，包括Hox态（Em8 = -407 mV）、类Htrans态（Htrans-like）[4Fe-4S]⁺-[FeII-FeII]（Em8 = -418 mV）、Hred态（Hred）[4Fe-4S]⁺-[FeII-FeI]以及HredH+态（HredH+）[4Fe-4S]²⁺-[FeI-FeI]（Em8 = -455~480 mV）。在氢气氛围下，类Htrans态的占比是Hox态的20倍以上，这提示其在CpIII的催化过程中扮演重要角色。与其他[FeFe]-氢化酶不同，本研究未观测到完全还原态的光谱学证据，例如HsredH+态（HsredH+）([4Fe-4S]⁺-[FeI-FeI])或是Hhyd态（Hhyd）([4Fe-4S]⁺-[FeII-FeII]-H⁻)。综上，尽管CpIII的H簇状态涵盖了绝大多数催化中间体，但它要么无法形成HsredH+与Hhyd态，要么这两种状态在CpIII中极不稳定。本研究结果表明，还原态CpIII的催化中间体与其他典型催化型[FeFe]-氢化酶的中间体存在显著差异，这或许可以解释CpIII偏好催化氢气生成的分子机制。