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

Biological fixation of dinitrogen (N), 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 N fixation through its N isotope effect (ε), measured as the difference between the N/N ratios of diazotroph net new fixed N and N substrate. The value of ε underpins N cycle reconstructions and differs between diazotrophs using molybdenum-containing and molybdenum-free nitrogenases. By examining ε for strains with natural and mutated nitrogenases, we determined if ε reflects enzyme-scale isotope effects and, thus, N use efficiency. Distinct and relatively stable ε 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 ε as a proxy for isoform type among extant nitrogenases. Structural mutation of active site N access altered molybdenum-nitrogenase ε (3.0-6.8‰ for α-70VI mutant). Structure-function and isotopic modeling results indicated cellular N reduction is rate-limited by N diffusion inside nitrogenase due to highly efficient catalysis by the active site cofactor, exemplifying ε as a tool to probe N 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 ε values.