Exploring Structure and Function of Redox Intermediates in [NiFe]-Hydrogenases by an Advanced Experimental Approach for Solvated, Lyophilized and Crystallized Metalloenzymes

To study metalloenzymes in detail, we developed a new experimental setup allowing the controlled preparation of catalytic intermediates for characterization by various spectroscopic techniques. The in situ monitoring of redox transitions by infrared spectroscopy in enzyme lyophilizate, crystals, and solution during gas exchange in a wide temperature range can be accomplished as well. Two O-2-tolerant [NiFe]-hydrogenases were investigated as model systems. First, we utilized our platform to prepare highly concentrated hydrogenase lyophilizate in a paramagnetic state harboring a bridging hydride. This procedure proved beneficial for Fe-57 nuclear resonance vibrational spectroscopy and revealed, in combination with density functional theory calculations, the vibrational fingerprint of this catalytic intermediate. The same in situ IR setup, combined with resonance Raman spectroscopy, provided detailed insights into the redox chemistry of enzyme crystals, underlining the general necessity to complement X-ray crystallographic data with spectroscopic analyses.

Automated summary

A new experimental setup was developed to study metalloenzymes in detail, allowing the controlled preparation and characterization of catalytic intermediates using various spectroscopic techniques. By investigating two O-2-tolerant [NiFe]-hydrogenases as model systems, detailed insights into the catalytic mechanisms and vibrational fingerprints were revealed. Additionally, the study highlighted the importance of complementing X-ray crystallographic data with spectroscopic analyses.