Nucleotide-induced conformational changes in the Escherichia coli NADH:ubiquinone oxidoreductase (complex I)

The energy-converting NADH:ubiquinone oxidoreductase, also known as respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Electron microscopy revealed the two-part structure of the complex consisting of a peripheral and a membrane arm. The peripheral arm contains all known cofactors and the NADH-binding site, whereas the membrane arm has to be involved in proton translocation. Owing to this, a conformation-linked mechanism for redox-driven proton translocation is discussed. By means of electron microscopy, we show that both arms of the Escherichia coli complex I are widened after the addition of NADH but not of NADPH. NADH-induced conformational changes were also detected in solution: ATR-FTIR (attenuated total reflection Fourier-transform infrared) of the soluble NADH dehydrogenase fragment of the complex indicates protein re-arrangements induced by the addition of NADH. EPR spectroscopy of surface mutants of the complex containing a covalently bound spin label at distinct positions demonstrates NADH-dependent conformational changes in both arms of the complex.

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Ion translocation by the Escherichia coli NADH:ubiquinone oxidoreductase (complex I)

Biochemical Society Transactions ◽

10.1042/bst0330836 ◽

2005 ◽

Vol 33 (4) ◽

pp. 836-839 ◽

Cited By ~ 8

Author(s):

T. Friedrich ◽

S. Stolpe ◽

D. Schneider ◽

B. Barquera ◽

P. Hellwig

Keyword(s):

Escherichia Coli ◽

Enzyme Activity ◽

Fourier Transform ◽

Complex I ◽

Nadh:Ubiquinone Oxidoreductase ◽

E Coli ◽

Ion Translocation ◽

Activity Generation ◽

Respiratory Complex I ◽

Energy Converting

The energy-converting NADH:ubiquinone oxidoreductase, also known as respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of ions across the membrane. It was assumed that the complex exclusively works as a proton pump. Recently, it has been proposed that complex I from Klebsiella pneumoniae and Escherichia coli work as Na+ pumps. We have used an E. coli complex I preparation to determine the type of ion(s) translocated by means of enzyme activity, generation of a membrane potential and redox-induced Fourier-transform infrared spectroscopy. We did not find any indications for Na+ translocation by the E. coli complex I.

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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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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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Constraining the Lateral Helix of Respiratory Complex I by Cross-linking Does Not Impair Enzyme Activity or Proton Translocation

Journal of Biological Chemistry ◽

10.1074/jbc.m115.660381 ◽

2015 ◽

Vol 290 (34) ◽

pp. 20761-20773 ◽

Cited By ~ 22

Author(s):

Shaotong Zhu ◽

Steven B. Vik

Keyword(s):

Conformational Changes ◽

Complex I ◽

Nadh Oxidase ◽

Membrane Surface ◽

Proton Translocation ◽

Membrane Vesicles ◽

Peripheral Region ◽

Significant Loss ◽

Cysteine Residues ◽

Nadh:Ubiquinone Oxidoreductase

Complex I (NADH:ubiquinone oxidoreductase) is a multisubunit, membrane-bound enzyme of the respiratory chain. The energy from NADH oxidation in the peripheral region of the enzyme is used to drive proton translocation across the membrane. One of the integral membrane subunits, nuoL in Escherichia coli, has an unusual lateral helix of ∼75 residues that lies parallel to the membrane surface and has been proposed to play a mechanical role as a piston during proton translocation (Efremov, R. G., Baradaran, R., and Sazanov, L. A. (2010) Nature 465, 441–445). To test this hypothesis we have introduced 11 pairs of cysteine residues into Complex I; in each pair one is in the lateral helix, and the other is in a nearby region of subunit N, M, or L. The double mutants were treated with Cu2+ ions or with bi-functional methanethiosulfonate reagents to catalyze cross-link formation in membrane vesicles. The yields of cross-linked products were typically 50–90%, as judged by immunoblotting, but in no case did the activity of Complex I decrease by >10–20%, as indicated by deamino-NADH oxidase activity or rates of proton translocation. In contrast, several pairs of cysteine residues introduced at other interfaces of N:M and M:L subunits led to significant loss of activity, in particular, in the region of residue Glu-144 of subunit M. The results do not support the hypothesis that the lateral helix of subunit L functions like a piston, but rather, they suggest that conformational changes might be transmitted more directly through the functional residues of the proton translocation apparatus.

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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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The coupling mechanism of mammalian respiratory complex I

Science ◽

10.1126/science.abc4209 ◽

2020 ◽

Vol 370 (6516) ◽

pp. eabc4209 ◽

Cited By ~ 6

Author(s):

Domen Kampjut ◽

Leonid A. Sazanov

Keyword(s):

Conformational Changes ◽

Complex I ◽

Electrostatic Interactions ◽

Proton Translocation ◽

Proton Pumping ◽

State Transitions ◽

Coupling Mechanism ◽

Cryo Electron Microscopy ◽

Open Conformation ◽

Respiratory Complex I

Mitochondrial complex I couples NADH:ubiquinone oxidoreduction to proton pumping by an unknown mechanism. Here, we present cryo–electron microscopy structures of ovine complex I in five different conditions, including turnover, at resolutions up to 2.3 to 2.5 angstroms. Resolved water molecules allowed us to experimentally define the proton translocation pathways. Quinone binds at three positions along the quinone cavity, as does the inhibitor rotenone that also binds within subunit ND4. Dramatic conformational changes around the quinone cavity couple the redox reaction to proton translocation during open-to-closed state transitions of the enzyme. In the induced deactive state, the open conformation is arrested by the ND6 subunit. We propose a detailed molecular coupling mechanism of complex I, which is an unexpected combination of conformational changes and electrostatic interactions.

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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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A long road towards the structure of respiratory complex I, a giant molecular proton pump

Biochemical Society Transactions ◽

10.1042/bst20130193 ◽

2013 ◽

Vol 41 (5) ◽

pp. 1265-1271 ◽

Cited By ~ 19

Author(s):

Leonid A. Sazanov ◽

Rozbeh Baradaran ◽

Rouslan G. Efremov ◽

John M. Berrisford ◽

Gurdeep Minhas

Keyword(s):

Conformational Changes ◽

Complex I ◽

Thermus Thermophilus ◽

Proton Translocation ◽

Nadh:Ubiquinone Oxidoreductase ◽

Coupling Mechanism ◽

Membrane Domains ◽

Transmembrane Helices ◽

Inner Mitochondrial Membrane ◽

Wide Range

Complex I (NADH:ubiquinone oxidoreductase) is central to cellular energy production, being the first and largest enzyme of the respiratory chain in mitochondria. It couples electron transfer from NADH to ubiquinone with proton translocation across the inner mitochondrial membrane and is involved in a wide range of human neurodegenerative disorders. Mammalian complex I is composed of 44 different subunits, whereas the ‘minimal’ bacterial version contains 14 highly conserved ‘core’ subunits. The L-shaped assembly consists of hydrophilic and membrane domains. We have determined all known atomic structures of complex I, starting from the hydrophilic domain of Thermus thermophilus enzyme (eight subunits, nine Fe–S clusters), followed by the membrane domains of the Escherichia coli (six subunits, 55 transmembrane helices) and T. thermophilus (seven subunits, 64 transmembrane helices) enzymes, and finally culminating in a recent crystal structure of the entire intact complex I from T. thermophilus (536 kDa, 16 subunits, nine Fe–S clusters, 64 transmembrane helices). The structure suggests an unusual and unique coupling mechanism via long-range conformational changes. Determination of the structure of the entire complex was possible only through this step-by-step approach, building on from smaller subcomplexes towards the entire assembly. Large membrane proteins are notoriously difficult to crystallize, and so various non-standard and sometimes counterintuitive approaches were employed in order to achieve crystal diffraction to high resolution and solve the structures. These steps, as well as the implications from the final structure, are discussed in the present review.

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