Respiratory Membrane endo-Hydrogenase Activity in the Microaerophile Azorhizobium caulinodans Is Bidirectional

BACKGROUND: The microaerophilic bacterium Azorhizobium caulinodans, when fixing N(2) both in pure cultures held at 20 µM dissolved O(2) tension and as endosymbiont of Sesbania rostrata legume nodules, employs a novel, respiratory-membrane endo-hydrogenase to oxidize and recycle endogenous H(2) produced by soluble Mo-dinitrogenase activity at the expense of O(2). METHODS AND FINDINGS: From a bioinformatic analysis, this endo-hydrogenase is a core (6 subunit) version of (14 subunit) NADH:ubiquinone oxidoreductase (respiratory complex I). In pure A. caulinodans liquid cultures, when O(2) levels are lowered to <1 µM dissolved O(2) tension (true microaerobic physiology), in vivo endo-hydrogenase activity reverses and continuously evolves H(2) at high rates. In essence, H(+) ions then supplement scarce O(2) as respiratory-membrane electron acceptor. Paradoxically, from thermodynamic considerations, such hydrogenic respiratory-membrane electron transfer need largely uncouple oxidative phosphorylation, required for growth of non-phototrophic aerobic bacteria, A. caulinodans included. CONCLUSIONS: A. caulinodans in vivo endo-hydrogenase catalytic activity is bidirectional. To our knowledge, this study is the first demonstration of hydrogenic respiratory-membrane electron transfer among aerobic (non-fermentative) bacteria. When compared with O(2) tolerant hydrogenases in other organisms, A. caulinodans in vivo endo-hydrogenase mediated H(2) production rates (50,000 pmol 10(9)·cells(-1) min(-1)) are at least one-thousandfold higher. Conceivably, A. caulinodans respiratory-membrane hydrogenesis might initiate H(2) crossfeeding among spatially organized bacterial populations whose individual cells adopt distinct metabolic states in response to variant O(2) availability. Such organized, physiologically heterogeneous cell populations might benefit from augmented energy transduction and growth rates of the populations, considered as a whole

Structure-function rendering of L-type Hyq <i>endo</i>-hydrogenase by analogy and homology to respiratory complex I.

<p>Inferred membrane ubiquinone (Q) or ubiquinol (QH₂) binding at the interface of HyqC, HyqG and Hyq I requires partial (14Å) extraction from the respiratory membrane hydrophobic phase; yellow rods represent linked transmembrane and transverse α-helices <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036744#pone.0036744-Efremov1" target="_blank">[14]</a>. Any HyqG catalytic site remains speculative; <i>in vivo</i> activity is in principle fully reversible (see Discussion).</p

H₂ evolution by <i>A. caulinodans</i> diazotrophic cultures.

†<p>pmol 10⁹·cells⁻¹ min⁻¹ (typical, single experiment);</p>‡<p>multiple experiments.</p

<i>A. caulinodans</i> Nuo (NADH:quinone oxidoreductase) and Hyq (<i>endo</i>-hydrogenase) structural homologs.

†<p>5′-end of <i>hyqG</i> encodes residues 1–156;</p>‡<p>3′-end of <i>hyqG</i> encodes residues 157–504;</p>††<p>CLUSTAL 2.1 pairwise alignments.</p

<i>Azorhizobium caulinodans</i> strains.

<p><i>Azorhizobium caulinodans</i> strains.</p