Investigating Metalloprotein Mechanism Using ‘Physiological’ X-ray Absorption Spectroscopy

Development of sustainable hydrogen-based fuel systems requires both efficient ways to produce hydrogen and extract energy from it. In nature, this reaction is performed efficiently by hydrogenase enzymes. Studying the mechanism of hydrogenase enzymes is difficult due to their exceptionally high turnover frequencies. As a result, new techniques are required to probe their catalytic cycle.

X-ray Absorption Spectroscopy is a promising technique, due to the structural and electronic information obtainable, but requirements to utilize cryogenic samples to avoid photodamage hinder its applicability. To avoid this, a specially designed cell was developed to allow room-temperature X-ray spectroscopy to be performed on hydrogenase samples under electrochemical control. This cell was successfully trialed using HERFD-XAS measurements of both [NiFe] and [FeFe] hydrogenases and demonstrated promise in mitigating photodamage via novel electrochemical protective methods. Preliminary work using model nickel complexes also demonstrated the feasibility of accessing VtC transitions during X-ray Emission Spectroscopy, allowing access to new probes of the active site.

The feasibility of Time-Resolved Multiple Probe spectroscopy, an ultrafast infrared spectroscopy method, was also tested in order to investigate sub-turnover kinetics of [NiFe] hydrogenases. This demonstrated the existence of intermediates with lifetimes below that accessible by more conventional methods.

This work provides multiple avenues to investigate the mechanism of [NiFe] hydrogenases.