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

[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. Infrared (IR) absorption spectroscopy represents a well-established and versatile technique that can identify and characterize all active and inactive states of the H-cluster by means of structurally sensitive and spectrally isolated CO and CN stretching vibrations. However, the amount of information that can be extracted from these linear experiments is inherently limited. Here we introduce experimental and computational two-dimensional (2D-)IR spectroscopy for the characterization of [FeFe] hydrogenases. Utilizing the H_inact state of the H-cluster as a model system, we demonstrate that this nonlinear technique yields direct information about the nature and interactions of the CO and CN stretching vibrations. These insights allow, for the first time, to quantitatively describe the character of these widely used reporter vibrations, their spatial localization, and the way they change upon structural variation of the H-cluster. The strength of this approach is demonstrated by correctly identifying the proposed structure of the H_inact state, in solution and at ambient temperature. In conclusion, the introduced combination of experimental and computational 2D-IR spectroscopy represents a powerful approach for studying [FeFe] hydrogenases and other complex organometallic targets.