[FeFe] hydrogenases catalyze reversible hydrogen evolution at rates as high as 10,000 turnovers per second. This exceptional catalytic ability is attractive for the use of hydrogenases in renewable energy applications and biohydrogen production. In this presentation, we will discuss our investigation of unique representatives of this class of enzymes that uncovers the range of reactivities accessible to this class of enzymes. An [FeFe] hydrogenase from Clostridium beijerinckii (CbHydA1) does not degrade in the presence of O2 presents a long-awaited breakthrough in the field of enzymatic hydrogen catalysis because it presents an unprecedented opportunity to implement this very efficient enzyme into sustainable systems. The EPR, FTIR, and electrochemical data obtained provide crucial details necessary to understand the mechanism of O2 tolerance. Using this information, we developed a nanoconstruct combining a cyanobacterial photosystem I and CbHydA1 to demonstrate the plausibility of generating H2 photosynthetically in an aerobic environment. Furthermore, we discuss a recent discovery of an unusual natural chimera of a rubrerthrin and an [FeFe] hydrogenase in Clostridium perfringens (CperHydR), acting as an H2-dependent H2O2 reductase. This natural construct appears to withstand a high excess of H2O2, consistent with a postulated role of this enzyme in the oxidative stress response, likely constituting an additional virulent factor for this foodborne pathogen. The observed near-complete bias of this enzyme towards H2 oxidation presents an unprecedented case that urges further investigation into the structure-function relationships dictating the catalytic directionality in this family of enzymes.
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