Cryo-EM of native membranes reveals an intimate connection between the Krebs cycle and respiration in mycobacteria
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Imaging of endogenous protein complexes in their native membranes can reveal protein-protein interactions that are lost upon detergent solubilization. To investigate interactions in the mycobacterial oxidative phosphorylation machinery, we prepared inverted membrane vesicles from Mycobacterium smegmatis and enriched for vesicles containing complexes of interest by affinity chromatography. Electron cryomicroscopy (cryo-EM) of these vesicles revealed that malate-quinone oxidoreductase (Mqo), an enzyme from the Krebs cycle, physically associates with the electron transport chain Complex III 2 IV 2 (CIII 2 CIV 2 ) supercomplex. Analysis of the Mqo:CIII 2 CIV 2 interaction shows that CIII 2 CIV 2 is necessary for malate-driven, but not NADH- driven, electron transport chain activity and oxygen consumption. Further, the association of Mqo with CIII 2 CIV 2 enables electron transfer from malate to CIII 2 CIV 2 with millisecond kinetics. Together, these findings indicate a connection between the Krebs cycle and respiration that directs electrons along a single branch of the mycobacterial electron transport chain.
