Abstract
Azotobacter vinelandii selectively utilizes three types of nitrogenase (molybdenum, vanadium, and iron only) to fix N(2), with their expression regulated by the presence or absence of different metal cofactors in its environment. Each alternative nitrogenase isoenzyme is predicted to have different electron flux requirements based on in vitro measurements, with the molybdenum nitrogenase requiring the lowest flux and the iron-only nitrogenase requiring the highest. Here, prior characterized strains, derepressed in nitrogenase synthesis and also deficient in uptake hydrogenase, were further modified to generate new mutants lacking the ability to produce poly-β-hydroxybutyrate (PHB). PHB is a storage polymer generated under oxygen-limiting conditions and can represent up to 70% of the cells' dry weight. The absence of such granules facilitated the study of relationships between catalytic biomass and product molar yields across different adaptive respiration conditions. The released hydrogen gas observed during growth, due to the inability of the mutants to recapture hydrogen, allowed for direct monitoring of in vivo nitrogenase activity for each isoenzyme. The data presented here show that increasing oxygen exposure limits equally the in vivo activities of all nitrogenase isoenzymes, while under comparative conditions, the Mo nitrogenase enzyme evolves more hydrogen per unit of biomass than the alternative isoenzymes.IMPORTANCEA. vinelandii has been a focus of intense research for over 100 years. It has been investigated for a variety of functions, including agricultural fertilization and hydrogen production. All of these endeavors are centered around A. vinelandii's ability to fix nitrogen aerobically using three nitrogenase isoenzymes. The majority of research up to this point has targeted in vitro measurements of the molybdenum nitrogenase, and robust data contrasting how oxygen impacts the in vivo activity of each nitrogenase isoenzyme are lacking. This article aims to provide in vivo nitrogenase activity data using a real-time evaluation of hydrogen gas released by derepressed nitrogenase mutants lacking an uptake hydrogenase and PHB accumulation.