Nitrogen stable isotope fractionation by biological nitrogen fixation reveals cellular nitrogenase is diffusion limited

Biological fixation of dinitrogen (N₂), the primary natural source of new bioavailable nitrogen (N) on Earth, is catalyzed by the enzyme nitrogenase through a complex mechanism at its active site metal cofactor. How this reaction functions in cellular environments, including its rate-limiting step, and how enzyme structure affects functioning remain unclear. Here, we investigated cellular N₂ fixation through its N isotope effect (¹⁵ε_fix), measured as the difference between the ¹⁵N/¹⁴N ratios of diazotroph net new fixed N and N₂ substrate. The value of ¹⁵ε_fix underpins N cycle reconstructions and differs between diazotrophs using molybdenum-containing and molybdenum-free nitrogenases. By examining ¹⁵ε_fix for Azotobacter vinelandii strains with natural and mutated nitrogenases, we determined if ¹⁵ε_fix reflects enzyme-scale isotope effects and, thus, N₂ use efficiency. Distinct and relatively stable ¹⁵ε_fix values for wild-type molybdenum- and vanadium-nitrogenase isoforms (2.5‰ and 5.8–6.6‰, respectively), despite changing cellular growth rate and electron availability, support ¹⁵ε_fix as a proxy for isoform type among extant nitrogenases. Structural mutation of active site N₂ access altered molybdenum-nitrogenase ¹⁵ε_fix (3.0–6.8‰ for α-70VI mutant). Structure-function and isotopic modeling results indicated cellular N₂ reduction is rate-limited by N₂ diffusion inside nitrogenase due to highly efficient catalysis by the active site cofactor, exemplifying ¹⁵ε_fix as a tool to probe N₂ fixation mechanisms. Diffusion-constrained reactions could reflect structural tradeoffs that protect the oxygen-sensitive cofactor from oxygen inactivation. This suggests that nitrogenase function is optimized for modern oxygenated environments and that pre-Great Oxidative Event nitrogenases were less diffusion-limited and potentially exhibited larger ¹⁵ε_fix values.