Achieving a unified understanding of the mechanism of a multicenter redox enzyme such as [NiFe] hydrogenase is complicated by difficulties in reconciling information obtained by using different techniques and on samples in different physical forms. Measurements of the activity of the enzyme, and of factors which perturb activity, are generally carried out using biochemical assays in solution or with electrode-immobilized enzymes using protein film electrochemistry (PFE). Conversely, spectroscopy aimed at reporting on features of the metalloclusters in the enzyme, such as electron paramagnetic resonance (EPR) or X-ray absorption spectroscopy (XAS), is often conducted on frozen samples and is thus difficult to relate to catalytically relevant states as information about turnover and activity has been lost. To complicate matters further, most of our knowledge of the atomic-level structure of metalloenzymes comes from X-ray diffraction studies in the solid, crystalline state, which are again difficult to link to turnover conditions. Taking [NiFe] hydrogenases as our case study, we show here how it is possible to apply infrared (IR) spectroscopic sampling approaches to unite direct spectroscopic study with catalytic turnover. Using a method we have named protein film IR electrochemistry (PFIRE), we reveal the steady-state distribution of intermediates during catalysis and identify catalytic “bottlenecks” introduced by site-directed mutagenesis. We also show that it is possible to study dynamic transitions between active site states of enzymes in single crystals, uniting solid state and solution spectroscopic information. In all of these cases, the spectroscopic data complement and enhance interpretation of purely activity-based measurements by providing direct chemical insight that is otherwise hidden. The [NiFe] hydrogenases possess a bimetallic [NiFe] active site, coordinated by CO and CN^– ligands, linked to the protein via bridging and terminal cysteine sulfur ligands, as well as an electron relay chain of iron sulfur clusters. Infrared spectroscopy is ideal for probing hydrogenases because the CO and CN^– ligands are strong IR absorbers, but the suite of IR-based approaches we describe here will be equally valuable in studying substrate- or intermediate-bound states of other metalloenzymes where key mechanistic questions remain open, such as nitrogenase, formate dehydrogenase, or carbon monoxide dehydrogenase. We therefore hope that this Account will encourage future studies which unify information from different techniques across bioinorganic chemistry.

中文翻译：

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[NiFe]氢化酶的统一活性，结构和光谱：结合技术以澄清机理的理解。

由于难以协调使用不同技术和不同物理形式的样品获得的信息，因此难以对多中心氧化还原酶（例如[NiFe]氢化酶）的机理达成统一的理解。通常使用溶液中的生化测定法或使用蛋白膜电化学（PFE）的电极固定化酶对酶的活性和干扰活性的因素进行测量。相反，旨在报告酶中金属团簇特征的光谱，例如电子顺磁共振（EPR）或X射线吸收光谱（XAS），通常是在冷冻样品上进行的，因此很难与催化相关状态关联有关营业额和活动的信息已丢失。为了进一步使事情复杂化，我们对金属酶的原子级结构的大多数了解来自处于固态，结晶状态的X射线衍射研究，这又很难与营业额条件联系起来。以[NiFe]氢化酶为例，我们在这里展示了如何应用红外（IR）光谱采样方法将直接光谱研究与催化转化率相结合。使用一种称为蛋白质膜IR电化学（PFIRE）的方法，我们揭示了催化过程中中间体的稳态分布，并鉴定了定点诱变引入的催化“瓶颈”。我们还表明，有可能研究单晶中酶的活性位点状态之间的动态过渡，并结合固态和溶液光谱信息。在所有这些情况下，光谱数据可提供直接的化学见解，从而补充和增强对纯粹基于活动的测量的解释。[NiFe]氢化酶具有双金属[NiFe]活性位点，由CO和CN协调^–通过桥接和末端半胱氨酸硫配体与蛋白质连接的配体，以及铁硫簇的电子中继链。红外光谱是理想的，因为CO和CN探测氢化^-配体是强有力的IR吸收剂，但基于IR的套件方法，我们在这里描述将在研究其他金属酶，其中关键机理问题的底物或中间束缚态同样宝贵保持开放状态，例如固氮酶，甲酸脱氢酶或一氧化碳脱氢酶。因此，我们希望该帐户能够鼓励未来的研究，这些研究将跨生物无机化学的不同技术的信息统一起来。