[Fe-Fe] hydrogenases are enzymes that perform a reversible heterolytic splitting of hydrogen into two protons and two electrons. The major roadblock in the utilization of these very efficient catalysts in renewable energy devices is the irreversible damage by oxygen and reactive oxygen species. The exact mechanism of the oxygen-triggered inhibition is not known. However, it has been shown that in the presence of oxygen the active 6Fe metallocenter (H-cluster) is irreversibly degraded, perhaps via a generation of a reactive oxygen species that react with components of the H-cluster. In this work, we show that an [Fe-Fe] hydrogenase from <i>Clostridium </i><i>beijerinckii</i> shows staggering tolerance to oxygen in atmospheric concentrations. Using activity assays and Fourier Transform infrared (FTIR) spectrometry we illustrate that the H-cluster forms a stable adduct with oxygen that can be readily reactivated in the presence of hydrogen. Using protein film voltametry (PFV), IR spectroelectromemistry and EPR spectroscopy we provide a detailed description of the oxygen-reacted state of the active center. Guided by bioinformatics analysis of the [Fe-Fe] hydrogense family, we performed amino acid substitutions and subsequent biophysical characterization of the variants. The results of this study allow us to delineate the role of protein surrounding of the H-cluster and of the auxiliary [4Fe-4S] clusters in facilitating oxygen tolerance and catalytic bias observed in our PFV experiments.

Understanding oxygen tolerant [Fe-Fe] hydrogenase

2019 Spring National Meeting

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