A Novel Endo-Hydrogenase Activity Recycles Hydrogen Produced by Nitrogen Fixation

Background Nitrogen (N 2 ) fixation also yields hydrogen (H 2 ) at 1∶1 stoichiometric amounts. In aerobic diazotrophic (able to grow on N 2 as sole N-source) bacteria, orthodox respiratory hupSL -encoded hydrogenase activity, associated with the cell membrane but facing the periplasm ( exo -hydrogenase), has nevertheless been presumed responsible for recycling such endogenous hydrogen. Methods and Findings As shown here, for Azorhizobium caulinodans diazotrophic cultures open to the atmosphere, exo -hydrogenase activity is of no consequence to hydrogen recycling. In a bioinformatic analysis, a novel seven-gene A. caulinodans hyq cluster encoding an integral-membrane, group-4, Ni,Fe-hydrogenase with homology to respiratory complex I (NADH : quinone dehydrogenase) was identified. By analogy, Hyq hydrogenase is also integral to the cell membrane, but its active site faces the cytoplasm ( endo -hydrogenase). An A. caulinodans in-frame hyq operon deletion mutant, constructed by “crossover PCR”, showed markedly decreased growth rates in diazotrophic cultures; normal growth was restored with added ammonium—as expected of an H 2 -recycling mutant phenotype. Using A. caulinodans hyq merodiploid strains expressing β-glucuronidase as promoter-reporter, the hyq operon proved strongly and specifically induced in diazotrophic culture; as well, hyq operon induction required the NIFA transcriptional activator. Therefore, the hyq operon is constituent of the nif regulon. Conclusions Representative of aerobic N 2 -fixing and H 2 -recycling α-proteobacteria, A. caulinodans possesses two respiratory Ni,Fe-hydrogenases: HupSL exo -hydrogenase activity drives exogenous H 2 respiration, and Hyq endo -hydrogenase activity recycles endogenous H 2 , specifically that produced by N 2 fixation. To benefit human civilization, H 2 has generated considerable interest as potential renewable energy source as its makings are ubiquitous and its combustion yields no greenhouse gases. As such, the reversible, group-4 Ni,Fe-hydrogenases, such as the A. caulinodans Hyq endo -hydrogenase, offer promise as biocatalytic agents for H 2 production and/or consumption.