Abstract
Summary M ycobacterium smegmatis is an obligate aerobe that harbours three predicted [ NiFe ] hydrogenases, Hyd 1 ( MSMEG _2262–2263), Hyd 2 ( MSMEG _2720‐2719) and Hyd 3 ( MSMEG _3931‐3928). We show here that these three enzymes differ in their phylogeny, regulation and catalytic activity. Phylogenetic analysis revealed that Hyd 1 groups with hydrogenases that oxidize H 2 produced by metabolic processes, and Hyd 2 is homologous to a novel group of putative high‐affinity hydrogenases. Hyd 1 and Hyd 2 respond to carbon and oxygen limitation, and, in the case of Hyd 1, hydrogen supplementation. Hydrogen consumption measurements confirmed that both enzymes can oxidize hydrogen. In contrast, the phylogenetic analysis and activity measurements of Hyd 3 are consistent with the enzyme evolving hydrogen. Hyd 3 is controlled by DosR , a regulator that responds to hypoxic conditions. The strict dependence of hydrogen oxidation of Hyd 1 and Hyd 2 on oxygen suggests that the enzymes are oxygen tolerant and linked to the respiratory chain. This unique combination of hydrogenases allows M . smegmatis to oxidize hydrogen at high ( Hyd 1) and potentially tropospheric ( Hyd 2) concentrations, as well as recycle reduced equivalents by evolving hydrogen ( Hyd 3). The distribution of these hydrogenases throughout numerous soil and marine species of actinomycetes suggests that oxic hydrogen metabolism provides metabolic flexibility in environments with changing nutrient fluxes.