A Tale of Two Gases: Isotope Effects Associated with the Enzymatic Production of H2 and N2O

Tuesday, 16 December 2014
Hui Yang1, Hasand Gandhi1, Helen W Kreuzer2, James Moran2, Eric A Hill2, Ashley McQuarters3, Nicolai Lehnert3, Nathaniel E Ostrom1 and Eric L. Hegg1, (1)Michigan State Univ, East Lansing, MI, United States, (2)Pacific Northwest National Laboratory, Richland, WA, United States, (3)University of Michigan Ann Arbor, Ann Arbor, MI, United States
Stable isotopes can provide considerable insight into enzymatic mechanisms and fluxes in various biological processes. In our studies, we used stable isotopes to characterize both enzyme-catalyzed H2 and N2O production. H2 is a potential alternative clean energy source and also a key metabolite in many microbial communities. Biological H2 production is generally catalyzed by hydrogenases, enzymes that combine protons and electrons to produce H2 under anaerobic conditions. In our study, H isotopes and fractionation factors (α) were used to characterize two types of hydrogenases: [FeFe]- and [NiFe]-hydrogenases. Due to differences in the active site, the α associated with H2 production for [FeFe]- and [NiFe]-hydrogenases separated into two distinct clusters (αFeFe > αNiFe). The calculated kinetic isotope effects indicate that hydrogenase-catalyzed H2 production has a preference for light isotopes, consistent with the relative bond strengths of O-H and H-H bonds. Interestingly, the isotope effects associated with H2 consumption and H2-H2O exchange reactions were also characterized, but in this case no specific difference was observed between the different enzymes.

N2O is a potent greenhouse gas with a global warming potential 300 times that of CO2, and the concentration of N2O is currently increasing at a rate of ~0.25% per year. Thus far, bacterial and fungal denitrification processes have been identified as two of the major sources of biologically generated N2O. In this study, we measured the δ15N, δ18O, δ15Nα (central N atom in N2O), and δ15Nβ (terminal N atom in N2O) of N2O generated by purified fungal P450 nitric oxide reductase (P450nor) from Histoplasma capsulatum. We observed normal isotope effects for δ18O and δ15Nα, and inverse isotope effects for bulk δ15N (the average of Nα and Nβ) and δ15Nβ. The observed isotope effects have been used in conjunction with DFT calculations to provide important insight into the mechanism of P450nor. Similar experiments were performed with bacterial nitric oxide reductase from Paracoccus denitrificans (cNOR). In this case both Nα and Nβ exhibited inverse isotope effects, while O had a normal isotope effect. Together, these data highlight the utility in using stable isotopes as both tracers and mechanistic probes when studying metabolic processes.