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Correlating kinetic and structural data on ubiquinone binding and reduction by respiratory complex I

  1. Judy Hirsta,1
  1. aMedical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge, CB2 0XY, United Kingdom;
  2. bDepartment of Chemistry, Technical University of Munich, D-85747 Garching, Germany
  1. Edited by Douglas C. Rees, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, and approved October 16, 2017 (received for review August 9, 2017)


Respiratory complex I, a redox-coupled proton pumping enzyme, is central to aerobic metabolism in mammalian mitochondria and implicated in many neuromuscular disorders. One of its substrates, ubiquinone-10, binds in an unusually long and narrow channel, which is at the intersection of the enzyme’s electron and proton transfer modules and a hotspot for disease-causing mutations. Here, we use a minimal, self-assembled respiratory chain to study complex I catalyzing with ubiquinones of different isoprenoid chain lengths. We show that the channel enhances the affinity of long-chain quinones, assists in their transfer along the channel, and organizes them for product release. Finally, we discuss how efficient binding and dissociation processes may help to link redox catalysis to proton pumping for energy conversion.


Respiratory complex I (NADH:ubiquinone oxidoreductase), one of the largest membrane-bound enzymes in mammalian cells, powers ATP synthesis by using the energy from electron transfer from NADH to ubiquinone-10 to drive protons across the energy-transducing mitochondrial inner membrane. Ubiquinone-10 is extremely hydrophobic, but in complex I the binding site for its redox-active quinone headgroup is ~20 ? above the membrane surface. Structural data suggest it accesses the site by a narrow channel, long enough to accommodate almost all of its ~50-? isoprenoid chain. However, how ubiquinone/ubiquinol exchange occurs on catalytically relevant timescales, and whether binding/dissociation events are involved in coupling electron transfer to proton translocation, are unknown. Here, we use proteoliposomes containing complex I, together with a quinol oxidase, to determine the kinetics of complex I catalysis with ubiquinones of varying isoprenoid chain length, from 1 to 10 units. We interpret our results using structural data, which show the hydrophobic channel is interrupted by a highly charged region at isoprenoids 4–7. We demonstrate that ubiquinol-10 dissociation is not rate determining and deduce that ubiquinone-10 has both the highest binding affinity and the fastest binding rate. We propose that the charged region and chain directionality assist product dissociation, and that isoprenoid stepping ensures short transit times. These properties of the channel do not benefit the exhange of short-chain quinones, for which product dissociation may become rate limiting. Thus, we discuss how the long channel does not hinder catalysis under physiological conditions and the possible roles of ubiquinone/ubiquinol binding/dissociation in energy conversion.


  • ?1To whom correspondence should be addressed. Email: jh{at}mrc-mbu.cam.ac.uk.

Published under the PNAS license.

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