Krebs Cycle Metabolon: Structural Evidence of Substrate Channeling Revealed by Cross‐Linking and Mass Spectrometry

Abstract It has been hypothesized that the high metabolic flux in the mitochondria is due to the self‐assembly of enzyme supercomplexes (called metabolons) that channel substrates from one enzyme to another, but there has been no experimental confirmation of this structure or the channeling. A structural investigation of enzyme organization within the Krebs cycle metabolon was accomplished by in vivo cross‐linking and mass spectrometry. Eight Krebs cycle enzyme components were isolated upon chemical fixation, and interfacial residues between mitochondrial malate dehydrogenase, citrate synthase, and aconitase were identified. Using constraint protein docking, a low‐resolution structure for the three‐enzyme complex was achieved, as well as the two‐fold symmetric octamer. Surface analysis showed formation of electrostatic channeling upon protein–protein association, which is the first structural evidence of substrate channeling in the Krebs cycle metabolon.