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

Abstract:

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 (α_FeFe > α_NiFe). 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, δ¹⁵N^α (central N atom in N₂O), and δ¹⁵N^β (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 δ¹⁵N^α, and inverse isotope effects for bulk δ¹⁵N (the average of N^α and N^β) and δ¹⁵N^β. 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 N^α and N^β 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.