A Quinol Anion as Catalytic Intermediate Coupling Proton Translocation With Electron Transfer in E. coli Respiratory Complex I

Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, plays a major role in cellular energy metabolism. It couples NADH oxidation and quinone reduction with the translocation of protons across the membrane, thus contributing to the protonmotive force. Complex I has an overall L-shaped structure with a peripheral arm catalyzing electron transfer and a membrane arm engaged in proton translocation. Although both reactions are arranged spatially separated, they are tightly coupled by a mechanism that is not fully understood. Using redox-difference UV-vis spectroscopy, an unknown redox component was identified in Escherichia coli complex I as reported earlier. A comparison of its spectrum with those obtained for different quinone species indicates features of a quinol anion. The re-oxidation kinetics of the quinol anion intermediate is significantly slower in the D213GH variant that was previously shown to operate with disturbed quinone chemistry. Addition of the quinone-site inhibitor piericidin A led to strongly decreased absorption peaks in the difference spectrum. A hypothesis for a mechanism of proton-coupled electron transfer with the quinol anion as catalytically important intermediate in complex I is discussed.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1714074114 ◽

2017 ◽

Vol 114 (48) ◽

pp. 12737-12742 ◽

Cited By ~ 36

Author(s):

Justin G. Fedor ◽

Andrew J. Y. Jones ◽

Andrea Di Luca ◽

Ville R. I. Kaila ◽

Judy Hirst

Keyword(s):

Electron Transfer ◽

Complex I ◽

Mammalian Cells ◽

Membrane Surface ◽

Proton Translocation ◽

Structural Data ◽

Nadh:Ubiquinone Oxidoreductase ◽

Respiratory Complex ◽

Respiratory Complex I ◽

Ubiquinone 10

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 Å above the membrane surface. Structural data suggest it accesses the site by a narrow channel, long enough to accommodate almost all of its ∼50-Å 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.

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Biochemical consequences of two clinically relevant ND-gene mutations in Escherichia coli respiratory complex I

Scientific Reports ◽

10.1038/s41598-021-91631-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Franziska Nuber ◽

Johannes Schimpf ◽

Jean-Paul di Rago ◽

Déborah Tribouillard-Tanvier ◽

Vincent Procaccio ◽

...

Keyword(s):

Escherichia Coli ◽

Complex I ◽

Proton Translocation ◽

Gene Mutations ◽

Stable Complex ◽

Nadh:Ubiquinone Oxidoreductase ◽

Common Source ◽

Respiratory Complex ◽

Quinone Reduction ◽

Respiratory Complex I

AbstractNADH:ubiquinone oxidoreductase (respiratory complex I) plays a major role in energy metabolism by coupling electron transfer from NADH to quinone with proton translocation across the membrane. Complex I deficiencies were found to be the most common source of human mitochondrial dysfunction that manifest in a wide variety of neurodegenerative diseases. Seven subunits of human complex I are encoded by mitochondrial DNA (mtDNA) that carry an unexpectedly large number of mutations discovered in mitochondria from patients’ tissues. However, whether or how these genetic aberrations affect complex I at a molecular level is unknown. Here, we used Escherichia coli as a model system to biochemically characterize two mutations that were found in mtDNA of patients. The V253AMT-ND5 mutation completely disturbed the assembly of complex I, while the mutation D199GMT-ND1 led to the assembly of a stable complex capable to catalyze redox-driven proton translocation. However, the latter mutation perturbs quinone reduction leading to a diminished activity. D199MT-ND1 is part of a cluster of charged amino acid residues that are suggested to be important for efficient coupling of quinone reduction and proton translocation. A mechanism considering the role of D199MT-ND1 for energy conservation in complex I is discussed.

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The mechanism of coupling between electron transfer and proton translocation in respiratory complex I

Journal of Bioenergetics and Biomembranes ◽

10.1007/s10863-014-9554-z ◽

2014 ◽

Vol 46 (4) ◽

pp. 247-253 ◽

Cited By ~ 41

Author(s):

Leonid A. Sazanov

Keyword(s):

Electron Transfer ◽

Complex I ◽

Proton Translocation ◽

Respiratory Complex ◽

Respiratory Complex I

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Investigation of the mechanism of proton translocation by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria: does the enzyme operate by a Q-cycle mechanism?

Biochemical Journal ◽

10.1042/bj20060766 ◽

2006 ◽

Vol 400 (3) ◽

pp. 541-550 ◽

Cited By ~ 26

Author(s):

Steven Sherwood ◽

Judy Hirst

Keyword(s):

Complex I ◽

Active Sites ◽

Proton Translocation ◽

Energy Transduction ◽

Heart Mitochondria ◽

Nadh:Ubiquinone Oxidoreductase ◽

Protonmotive Force ◽

Bovine Heart ◽

Quinone Reduction ◽

Q Cycle

Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the membrane-bound electron transport chain in mitochondria. It conserves energy, from the reduction of ubiquinone by NADH, as a protonmotive force across the inner membrane, but the mechanism of energy transduction is not known. The structure of the hydrophilic arm of thermophilic complex I supports the idea that proton translocation is driven at (or close to) the point of quinone reduction, rather than at the point of NADH oxidation, with a chain of iron–sulfur clusters transferring electrons between the two active sites. Here, we describe experiments to determine whether complex I, isolated from bovine heart mitochondria, operates via a Q-cycle mechanism analogous to that observed in the cytochrome bc1 complex. No evidence for the ‘reductant-induced oxidation’ of ubiquinol could be detected; therefore no support for a Q-cycle mechanism was obtained. Unexpectedly, in the presence of NADH, complex I inhibited by either rotenone or piericidin A was found to catalyse the exchange of redox states between different quinone and quinol species, providing a possible route for future investigations into the mechanism of energy transduction.

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