Mycobacterium smegmatis is a heterotrophic, obligately aerobic soil dwelling bacterium that requires organic carbon for growth. Due to fierce competition for resources in soil, the bacterium often experiences deprivation of both oxygen and organic carbon sources. To cope with this, M. smegmatis has evolved to persist during hypoxia and carbon starvation through metabolic flexibility, upregulating enzymes that enable it to utilise electron donors and acceptors other than organic carbon and molecular oxygen. One class of enzyme that M. smegmatis upregulates during organic carbon limitation is hydrogenases. M. smegmatis possesses three hydrogenases that mediate distinct but related functions. One of these is the uptake hydrogenase Huc, a high affinity enzyme that is oxygen tolerant and capable of oxidising atmospheric hydrogen. Huc provides electrons from H2 to the cytochrome bcc-aa3 oxidase supercomplex, a terminal electron acceptor of the respiratory chain, via the quinone pool, which results in generation of the proton motive force. The molecular details of how the electrons produced by Huc are transferred to the respiratory chain have not been determined. Additionally, while it has been shown that M. smegmatis is capable of oxidising atmospheric H2 under ambient atmospheric conditions, it has not been determined whether purified hydrogenases are capable of oxidising atmospheric H2 or if they are oxygen tolerant in isolation. In this work we purified Huc by chromosomal strep tagging it’s small subunit in an M. smegmatis strain that overexpresses the enzyme. Purified Huc was visualised by cryo-electron microscopy, where high resolution electron density maps of Huc were obtained. Further, we performed gas chromatography and hydrogen electrode experiments demonstrating that purified Huc is a high affinity hydrogenase capable of oxidising atmospheric hydrogen in isolation from the respiratory chain. These experiments also demonstrated that Huc is completely insensitive to inhibition by to oxygen, confirming cell-based data. This work represented the first molecular characterisation of a member of a novel family of hydrogenases capable of scavenging H2 at atmospheric concentrations.