Nitrogen stable isotope fractionation by biological nitrogen fixation reveals cellular nitrogenase is diffusion limited.
Journal Article
Overview
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‰ and 5.8-6.6‰, 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 (3.0-6.8‰ 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 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.
