Abstract

[NiFe]-hydrogenases are gas-processing metalloenzymes that catalyze the conversion of dihydrogen (H₂) to protons and electrons in a broad range of microorganisms. Within the framework of green chemistry, the molecular proceedings of biological hydrogen turnover inspired the design of novel catalytic compounds for H₂ generation. The bidirectional “O₂-sensitive” [NiFe]-hydrogenase from Escherichia coli HYD-2 has recently been crystallized; however, a systematic infrared characterization in the presence of natural reactants is not available yet. In this study, we analyze HYD-2 from E. coli by in situ attenuated total reflection Fourier-transform infrared spectroscopy (ATR FTIR) under quantitative gas control. We provide an experimental assignment of all catalytically relevant redox intermediates alongside the O₂- and CO-inhibited cofactor species. Furthermore, the reactivity and mutual competition between H₂, O₂, and CO was probed in real time, which lays the foundation for a comparison with other enzymes, e.g., “O₂-tolerant” [NiFe]-hydrogenases. Surprisingly, only Ni-B was observed in the presence of O₂ with no indications for the “unready” Ni-A state. The presented work proves the capabilities of in situ ATR FTIR spectroscopy as an efficient and powerful technique for the analysis of biological macromolecules and enzymatic small molecule catalysis. View Full-Text