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

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

Frontiers in Chemistry ◽

10.3389/fchem.2021.672969 ◽

2021 ◽

Vol 9 ◽

Author(s):

Franziska Nuber ◽

Luca Mérono ◽

Sabrina Oppermann ◽

Johannes Schimpf ◽

Daniel Wohlwend ◽

...

Keyword(s):

Electron Transfer ◽

Complex I ◽

Proton Translocation ◽

Difference Spectrum ◽

Nadh:Ubiquinone Oxidoreductase ◽

Protonmotive Force ◽

Respiratory Complex ◽

Quinone Reduction ◽

Vis Spectroscopy ◽

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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Mammalian Respiratory Complex I Through the Lens of Cryo-EM

Annual Review of Biophysics ◽

10.1146/annurev-biophys-052118-115704 ◽

2019 ◽

Vol 48 (1) ◽

pp. 165-184 ◽

Cited By ~ 21

Author(s):

Ahmed-Noor A. Agip ◽

James N. Blaza ◽

Justin G. Fedor ◽

Judy Hirst

Keyword(s):

Complex I ◽

Catalytic Mechanism ◽

Structural Data ◽

Proton Pumping ◽

Nadh:Ubiquinone Oxidoreductase ◽

Respiratory Complex ◽

Data Collection Strategy ◽

Structural Work ◽

Membrane Bound ◽

Respiratory Complex I

Single-particle electron cryomicroscopy (cryo-EM) has led to a revolution in structural work on mammalian respiratory complex I. Complex I (mitochondrial NADH:ubiquinone oxidoreductase), a membrane-bound redox-driven proton pump, is one of the largest and most complicated enzymes in the mammalian cell. Rapid progress, following the first 5-Å resolution data on bovine complex I in 2014, has led to a model for mouse complex I at 3.3-Å resolution that contains 96% of the 8,518 residues and to the identification of different particle classes, some of which are assigned to biochemically defined states. Factors that helped improve resolution, including improvements to biochemistry, cryo-EM grid preparation, data collection strategy, and image processing, are discussed. Together with recent structural data from an ancient relative, membrane-bound hydrogenase, cryo-EM on mammalian complex I has provided new insights into the proton-pumping machinery and a foundation for understanding the enzyme's catalytic mechanism.

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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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Structure of Escherichia coli respiratory complex I reconstituted into lipid nanodiscs reveals an uncoupled conformation

10.1101/2021.04.09.439197 ◽

2021 ◽

Author(s):

Rouslan G. Efremov ◽

Piotr Kolata

Keyword(s):

Escherichia Coli ◽

Complex I ◽

Proton Translocation ◽

Structural Data ◽

Coupling Mechanism ◽

Membrane Domains ◽

Dynamic Features ◽

Respiratory Complex ◽

Respiratory Complex I ◽

Lipid Nanodiscs

Respiratory complex I is a multi-subunit membrane protein complex that reversibly couples NADH oxidation and ubiquinone reduction with proton translocation against trans-membrane potential. Complex I from Escherichia coli is among the best functionally characterized complexes, but its structure remains unknown, hindering further mechanistic studies to understand the enzyme coupling mechanism. Here we describe the single particle cryoelectron microscopy (cryo-EM) structure of the entire catalytically active E. coli complex I reconstituted into lipid nanodiscs. The structure of this mesophilic bacterial complex I displays highly dynamic connection between the peripheral and membrane domains. The peripheral domain assembly is stabilized by unique terminal extensions and an insertion loop. The membrane domain structure reveals novel dynamic features. Unusual conformation of the conserved interface between the cytoplasmic and membrane domains suggests an uncoupled conformation of the complex. Based on these structural data we suggest a new simple and testable coupling mechanism for the molecular machine.

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Structure of Escherichia coli respiratory complex I reconstituted into lipid nanodiscs reveals an uncoupled conformation

eLife ◽

10.7554/elife.68710 ◽

2021 ◽

Vol 10 ◽

Author(s):

Rouslan G Efremov ◽

Piotr Kolata

Keyword(s):

Escherichia Coli ◽

Complex I ◽

Proton Translocation ◽

Structural Data ◽

Coupling Mechanism ◽

Membrane Domains ◽

Dynamic Features ◽

Respiratory Complex ◽

Respiratory Complex I ◽

Lipid Nanodiscs

Respiratory complex I is a multi-subunit membrane protein complex that reversibly couples NADH oxidation and ubiquinone reduction with proton translocation against trans-membrane potential. Complex I from Escherichia coli is among the best functionally characterized complexes, but its structure remains unknown, hindering further mechanistic studies to understand the enzyme coupling mechanism. Here we describe the single particle cryo-electron microscopy (cryo-EM) structure of the entire catalytically active E. coli complex I reconstituted into lipid nanodiscs. The structure of this mesophilic bacterial complex I displays highly dynamic connection between the peripheral and membrane domains. The peripheral domain assembly is stabilized by unique terminal extensions and an insertion loop. The membrane domain structure reveals novel dynamic features. Unusual conformation of the conserved interface between the peripheral and membrane domains suggests an uncoupled conformation of the complex. Considering constraints imposed by the structural data we suggest a new simple hypothetical coupling mechanism for the molecular machine.

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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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