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.


RESULTS
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 beta-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 alpha-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.
Ng
Gordon
G
Department of Molecular, Cellular and Developmental Biology, Sinsheimer Laboratories, University of California Santa Cruz, Santa Cruz, California, United States of America.
Tom
Curtis G S
CG
Park
Angela S
AS
Zenad
Lounis
L
Ludwig
Robert A
RA
D020599:Azorhizobium caulinodans / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D017124:Conserved Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005966:Glucuronidase / Q000378:metabolism; D006859:Hydrogen / Q000378:metabolism*; D006864:Hydrogenase / Q000145:classification / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009586:Nitrogen Fixation / Q000235:genetics / Q000502:physiology*; D009876:Operon / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D006859:Hydrogen; D006864:Hydrogenase; C455599:hydrogenase 4, E coli; D005966:Glucuronidase