Wrathall Solomon L D, Procacci Barbara, Horch Marius, Saxton Emily, Furlan Chris, Walton Julia, Rippers Yvonne, Blaza James N, Greetham Gregory M, Towrie Michael, Parker Anthony W, Lynam Jason, Parkin Alison, Hunt Neil T
Department of Chemistry and York Biomedical Research Institute, University of York, York, YO10 5DD, UK.
Freie Universität Berlin, Department of Physics, Ultrafast Dynamics in Catalysis, Arnimallee 14, 14195 Berlin, Germany.
Phys Chem Chem Phys. 2022 Oct 19;24(40):24767-24783. doi: 10.1039/d2cp04188j.
Ultrafast two-dimensional infrared (2D-IR) spectroscopy of Hyd-1 (Hyd-1) reveals the structural and dynamic influence of the protein scaffold on the Fe(CO)(CN) unit of the active site. Measurements on as-isolated Hyd-1 probed a mixture of active site states including two, which we assign to Ni-S, that have not been previously observed in the enzyme. Explicit assignment of carbonyl (CO) and cyanide (CN) stretching bands to each state is enabled by 2D-IR. Energies of vibrational levels up to and including two-quantum vibrationally excited states of the CO and CN modes have been determined along with the associated vibrational relaxation dynamics. The carbonyl stretching mode potential is well described by a Morse function and couples weakly to the cyanide stretching vibrations. In contrast, the two CN stretching modes exhibit extremely strong coupling, leading to the observation of formally forbidden vibrational transitions in the 2D-IR spectra. We show that the vibrational relaxation times and structural dynamics of the CO and CN ligand stretching modes of the enzyme active site differ markedly from those of a model compound K[CpFe(CO)(CN)] in aqueous solution and conclude that the protein scaffold creates a unique biomolecular environment for the NiFe site that cannot be represented by analogy to simple models of solvation.
对Hyd-1进行的超快二维红外（2D-IR）光谱研究揭示了蛋白质支架对活性位点的Fe(CO)(CN)单元的结构和动力学影响。对原样分离的Hyd-1进行的测量探测了活性位点状态的混合物，其中包括两种我们归为Ni-S的状态，这两种状态此前在该酶中未曾观察到。二维红外光谱能够明确将羰基（CO）和氰基（CN）伸缩带分配到每种状态。已经确定了包括CO和CN模式的双量子振动激发态在内的振动能级的能量以及相关的振动弛豫动力学。羰基伸缩模式的势能可以用莫尔斯函数很好地描述，并且与氰基伸缩振动的耦合较弱。相比之下，两种CN伸缩模式表现出极强的耦合，导致在二维红外光谱中观察到形式上禁止的振动跃迁。我们表明，酶活性位点的CO和CN配体伸缩模式的振动弛豫时间和结构动力学与水溶液中的模型化合物K[CpFe(CO)(CN)]有显著差异，并得出结论，蛋白质支架为NiFe位点创造了一个独特的生物分子环境，不能通过类比简单的溶剂化模型来表示。
