A Class of Its Own: Function-Discovery of HydY, A Novel Class of [FeFe]-Hydrogenases
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Hydrogen has received widespread attention as a potential energy carrier due to its high energy content and clean combustion product H2O. [FeFe]-H2ases exhibit the highest H2 production rates utilizing a complex iron sulfur cofactor, called the H-cluster that requires three biosynthetic maturation proteins. Mutagenesis studies of conserved residues surrounding the H-cluster led to variants with either decreased overall activity or minimal H-cluster incorporation. These studies underscore the importance of the protein matrix in tuning active site chemistry. Here, we investigated a new class of enzymes consisting of N-terminal [FeFe]-H2ase fused to a C-terminal rubrerythrin domain (named HydY). Using protein film electrochemistry and colorimetric assays, HydY was found to function differently than standard [FeFe]-H2ases: it exhibits strong product inhibition for H+ reduction, a low KM for H2 oxidation, bias toward H2 oxidation and significant overpotential. We hypothesized that the altered reactivity for HydY was due to hydrogen bonds from conserved residue substitutions vital for H-cluster coordination. Consistent with this hypothesis, HydY variants result in enzymes with catalytic properties more similar to traditional [FeFe]-H2ases. In addition, the C-terminal domain (CTD) of HydY efficiently reduces H2O2 to H2O. Electronic absorbance, EPR and Mӧssbauer spectroscopic studies of the CTD are consistent with a rubrerythrin diiron active site with flanking mononuclear iron sites. A 1.77 Å crystal structure of the CTD reveals a domain swapped dimer in which ligands for a modified di-iron rubrerythrin active site are provided by residues across the dimer interface. Further, our results indicate that electrons generated by the oxidation of H2 are transferred to the CTD, presumably for H2O2 reduction. This is the first example of H2-dependent peroxidase. We hypothesize that evolution ‘tuned’ HydY to favor H2 oxidation and that HydY has a protective role in anaerobic bacteria that allows survival upon transient oxygen exposure. Additional bioinformatics indicate that HydY belongs to a broader class of [FeFe]-H2ases that also use H2 as a reductant for various substrates. Overall, these studies identify determinants for controlling active site chemistry of [FeFe]-H2ases that may lead to improved design of biomimetic compounds with implications in energy production.
Martin Jr., Darrell Wayne (2016). A Class of Its Own: Function-Discovery of HydY, A Novel Class of [FeFe]-Hydrogenases. Doctoral dissertation, Texas A&M University. Available electronically from