Alteration of the a-70 "gatekeeping" residue of the Mo-nitrogenase active site increases cellular scale nitrogen isotope fractionation during biological nitrogen fixation

Biological nitrogen fixation (BNF) by the metalloenzyme nitrogenase is the main source of biologically available, fixed nitrogen (N) to terrestrial and marine ecosystems. The N stable isotope composition of biomass (¹⁵N/¹⁴N, typically expressed as δ¹⁵N) is a powerful tool for reconstructing N cycling, but its interpretation depends on understanding the isotope signatures of nitrogen fixation. The cellular scale expression of N stable isotope fractionation for biological nitrogen fixation (¹⁵ɛ_fix= δ¹⁵N_{dissolved N2} - δ¹⁵N_biomass) is generally small ( 0 to 2‰ for BNF based on molybdenum nitrogenase), but can reach higher values (7 to 8‰) when alternative vanadium and iron-only nitrogenase isoforms are employed. Variations in the active site between these different nitrogenases suggest that active site structure constrains fractionation. Here, we explore how the active site environment of Mo-nitrogenase influences ¹⁵ɛ_fixusing strains of the model diazotroph, Azotobacter vinelandii,which have alterations in a gatekeeping residue (α-70 valine) that modulates N₂access to the enzyme active site. In batch cultures, substitution of the α-70 valine residue with the smaller amino acid, alanine, or with the larger amino acid, isoleucine, led to larger fractionations relative to wild type values (¹⁵ɛ_fix-Mo-small= 3.4‰ and ¹⁵ɛ_fix-Mo-large=5.5‰ versus ¹⁵ɛ_{fix-Mo WT} = 0-2‰) as well as slower growth. These results demonstrate that a single amino acid change at a critical active site position can alter the isotope signature of BNF. Ongoing investigations using chemostats, measurements of dissolved nitrogen, enzyme abundance, and N₂ fixation rates will be used to decipher how the biochemistry and physiology of BNF, along with environmental parameters, determinethe δ¹⁵N of nitrogen fixer biomass.