Isotopic fractionation associated with [NiFe]- and [FeFe]-hydrogenases

RATIONALE: Hydrogenases catalyze the reversible formation of H-2 from electrons and protons with high efficiency. Understanding the relationships between H-2 production, H-2 uptake, and H-2-H2O exchange can provide insight into the metabolism of microbial communities in which H-2 is an essential component in energy cycling. METHODS: We used stable Hisotopes (H-1 and H-2) to probe the isotope effects associated with three [FeFe]-hydrogenases and three [NiFe]-hydrogenases. RESULTS: All six hydrogenases displayed fractionation factors for H-2 formation that were significantly less than 1, producing H-2 that was severely depleted in H-2 relative to the substrate, water. Consistent with differences in their active site structure, the fractionation factors for each class appear to cluster, with the three [NiFe]-hydrogenases (alpha = 0.27-0.40) generally having smaller values than the three [FeFe]-hydrogenases (alpha = 0.41-0.55). We also obtained isotopic fractionation factors associated with H-2 uptake and H-2-H2O exchange under conditions similar to those utilized for H-2 production, providing a more complete picture of the reactions catalyzed by hydrogenases. CONCLUSIONS: The fractionation factors determined in our studies can be used as signatures for different hydrogenases to probe their activity under different growth conditions and to ascertain which hydrogenases are most responsible for H-2 production and/or uptake in complex microbial communities. Copyright (c) 2015 John Wiley & Sons, Ltd.