National Academy of Sciences, Proceedings of the National Academy of Sciences, 44(111), p. 15735-15740, 2014
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In mitochondria, four respiratory-chain complexes drive oxidative phosphorylation by sustaining a proton-motive force across the inner membrane that is used to synthesize ATP. The question of how the densely packed proteins of the inner membrane are organized to optimize structure and function has returned to prominence with the characterization of respiratory-chain supercomplexes. Supercomplexes are increasingly accepted structural entities, but their functional and catalytic advantages are disputed. Notably, substrate "channeling" between the enzymes in supercomplexes has been proposed to confer a kinetic advantage, relative to the rate provided by a freely accessible, common substrate pool. Here, we focus on the mitochondrial ubiquinone/ubiquinol pool. We formulate and test three conceptually simple predictions of the behavior of the mammalian respiratory chain that depend on whether channeling in supercomplexes is kinetically important, and on whether the ubiquinone pool is partitioned between pathways. Our spectroscopic and kinetic experiments demonstrate how the metabolic pathways for NADH and succinate oxidation communicate and catalyze via a single, universally accessible ubiquinone/ubiquinol pool that is not partitioned or channeled. We reevaluate the major piece of contrary evidence from flux control analysis and find that the conclusion of substrate channeling arises from the particular behavior of a single inhibitor; we explain why different inhibitors behave differently and show that a robust flux control analysis provides no evidence for channeling. Finally, we discuss how the formation of respiratory-chain supercomplexes may confer alternative advantages on energy-converting membranes.