<p>Bacterial lipoquinones are two-proton and two-electron carriers that serve as electron shuttles between the intricate, modular networks of electron donor and acceptor proteins of electron transfer systems (ETS). These molecules have historically been used extensively for taxonomy purposes as different bacterial taxa have evolved distinct structural variations which are highly conserved. However, relatively little is known about how the structural variations seen in bacterial lipoquinones affect the functionality and/or the relative importance of the enzymes that utilize them as electron acceptors or donors. This variability combined with the modular nature of the enzymes and protein complexes involved in the bacterial ETS necessitates characterization in situ. To address these complex issues, it was determined that the mycobacterial lipoquinones comprise a single pool shared between enzymes rather than isolated pools dedicated to specific enzymes and/or complexes. Subsequently, a method that selectively depletes endogenous lipoquinones from <i>Mycobacterium smegmatis</i> membranes was implemented using the 405&#xa0;nm wavelength output from a violet-blue laser diode. Exposure of membranes to coherent violet-blue light resulted in photolysis of endogenous lipoquinone with concomitant loss of lipoquinone-dependent enzyme activities, which could be rescued by repletion with chemically defined synthetic lipoquinones. Irradiation did not significantly reduce enzyme activity in rescued relative to non-irradiated membranes, indicating that minimal damage to proteins was caused by exposure to the violet-blue light. These results allowed characterization of endogenous NADH dehydrogenases and malate:quinone oxidoreductase activities and inhibitor specificities. This work lays the foundation for future efforts to characterize the relationship between lipoquinone structure and ETS functionality in mycobacteria and other bacterial species.</p>