@article{1371,
  author = {E. Han and S.H. Kopf and A. Maloney and X. Ai and D.M. Sigman and X. Zhang},
  title = {Nitrogen stable isotope fractionation by biological nitrogen fixation reveals cellular nitrogenase is diffusion limited},
  abstract = {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 (15εfix), measured as the difference between the 15N/14N ratios of diazotroph net new fixed N and N2 substrate. The value of 15εfix underpins N cycle reconstructions and differs between diazotrophs using molybdenum-containing and molybdenum-free nitrogenases. By examining 15εfix for Azotobacter vinelandii strains with natural and mutated nitrogenases, we determined if 15εfix reflects enzyme-scale isotope effects and, thus, N2 use efficiency. Distinct and relatively stable 15εfix values for wild-type molybdenum- and vanadium-nitrogenase isoforms (2.5{\textperthousand} and 5.8{\textendash}6.6{\textperthousand}, respectively), despite changing cellular growth rate and electron availability, support 15εfix as a proxy for isoform type among extant nitrogenases. Structural mutation of active site N2 access altered molybdenum-nitrogenase 15εfix (3.0{\textendash}6.8{\textperthousand} 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 15εfix 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 15εfix values.},
  year = {2025},
  journal = {PNAS Nexus},
  volume = {4},
  number = {3},
  pages = {pgaf061},
  month = {02},
  issn = {2752-6542},
  url = {https://doi.org/10.1093/pnasnexus/pgaf061},
  doi = {10.1093/pnasnexus/pgaf061},
}