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 through metabolic flexibility, upregulating enzymes, such as the hydrogenase Huc, that enable it to utilise electron donors and acceptors other than organic carbon and molecular oxygen. Huc provides electrons from H2 to the cytochrome bcc-aa3 oxidase supercomplex 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. In this work, we purified Huc by chromosomal strep tagging in an M. smegmatis strain that overexpresses the enzyme. Purified Huc was visualised by cryo-electron microscopy, where we obtained electron density maps with a maximum resolution of 1.52 Å. Huc was revealed to be an octamer of the large and small subunits, with an additional novel membrane-associated central stalk, critical for transporting menaquinone directly from the membrane to the enzyme. 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. Through these experiments, molecular dynamics simulations and mutagenesis we demonstrate that Huc is completely insensitive to inhibition by oxygen. This is a key feature of Huc that could be exploited for industrial purposes. This work represents the first molecular characterisation of a member of a novel family of hydrogenases capable of scavenging H2 at atmospheric concentrations.