Two-dimensional infrared spectroscopy as a tool to reveal the vibrational and molecular structure of [FeFe] hydrogenases.

[FeFe] hydrogenases are Nature's most efficient catalysts for the cleavage and evolution of molecular hydrogen. Despite decades of research, key aspects of the catalytic cycle and the underlying geometrical and electronic properties of the active-site cofactor, called the H-cluster, are not fully understood. Spectroscopic techniques have played a central role in establishing the current state of knowledge on [FeFe] hydrogenases, and further advances in the field depend critically on novel techniques that yield so-far inaccessible insights into structural and mechanistic aspects.

Understanding the [NiFe] Hydrogenase Active Site Environment through Ultrafast Infrared and 2D-IR Spectroscopy of the Subsite Analogue K[CpFe(CO)(CN)] in Polar and Protic Solvents.

The [CpFe(CO)(CN)] unit is an excellent structural model for the Fe(CO)(CN) moiety of the active site found in [NiFe] hydrogenases. Ultrafast infrared (IR) pump-probe and 2D-IR spectroscopy have been used to study K[CpFe(CO)(CN)] () in a range of protic and polar solvents and as a dry film. Measurements of anharmonicity, intermode vibrational coupling strength, vibrational relaxation time, and solvation dynamics of the CO and CN stretching modes of in HO, DO, methanol, dimethyl sulfoxide, and acetonitrile reveal that H-bonding to the CN ligands plays an important role in defining the spectroscopic characteristics and relaxation dynamics of the Fe(CO)(CN) unit.

Reversible Glutamate Coordination to High-Valent Nickel Protects the Active Site of a [NiFe] Hydrogenase from Oxygen.

NAD-reducing [NiFe] hydrogenases are valuable biocatalysts for H-based energy conversion and the regeneration of nucleotide cofactors. While most hydrogenases are sensitive toward O and elevated temperatures, the soluble NAD-reducing [NiFe] hydrogenase from (SH) is O-tolerant and thermostable. Thus, it represents a promising candidate for biotechnological applications.

Understanding the structure and dynamics of hydrogenases by ultrafast and two-dimensional infrared spectroscopy.

Hydrogenases are valuable model enzymes for sustainable energy conversion approaches using H, but rational utilization of these base-metal biocatalysts requires a detailed understanding of the structure and dynamics of their complex active sites. The intrinsic CO and CN ligands of these metalloenzymes represent ideal chromophores for infrared (IR) spectroscopy, but structural and dynamic insight from conventional IR absorption experiments is limited. Here, we apply ultrafast and two-dimensional (2D) IR spectroscopic techniques, for the first time, to study hydrogenases in detail.