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

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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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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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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Accessory Subunits of the Matrix Arm of Mitochondrial Complex I with a Focus on Subunit NDUFS4 and Its Role in Complex I Function and Assembly

Life ◽

10.3390/life11050455 ◽

2021 ◽

Vol 11 (5) ◽

pp. 455

Author(s):

Flora Kahlhöfer ◽

Max Gansen ◽

Volker Zickermann

Keyword(s):

Complex I ◽

Hot Spot ◽

Proton Translocation ◽

Chain Complex ◽

Nadh:Ubiquinone Oxidoreductase ◽

Inner Mitochondrial Membrane ◽

Accessory Subunits ◽

The Matrix ◽

Assembly Factor ◽

Accessory Subunit

NADH:ubiquinone-oxidoreductase (complex I) is the largest membrane protein complex of the respiratory chain. Complex I couples electron transfer to vectorial proton translocation across the inner mitochondrial membrane. The L shaped structure of complex I is divided into a membrane arm and a matrix arm. Fourteen central subunits are conserved throughout species, while some 30 accessory subunits are typically found in eukaryotes. Complex I dysfunction is associated with mutations in the nuclear and mitochondrial genome, resulting in a broad spectrum of neuromuscular and neurodegenerative diseases. Accessory subunit NDUFS4 in the matrix arm is a hot spot for mutations causing Leigh or Leigh-like syndrome. In this review, we focus on accessory subunits of the matrix arm and discuss recent reports on the function of accessory subunit NDUFS4 and its interplay with NDUFS6, NDUFA12, and assembly factor NDUFAF2 in complex I assembly.

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

Biochemical Society Transactions ◽

10.1042/bst0360971 ◽

2008 ◽

Vol 36 (5) ◽

pp. 971-975 ◽

Cited By ~ 16

Author(s):

Thomas Pohl ◽

Daniel Schneider ◽

Ruth Hielscher ◽

Stefan Stolpe ◽

Katerina Dörner ◽

...

Keyword(s):

Electron Microscopy ◽

Escherichia Coli ◽

Conformational Changes ◽

Complex I ◽

Proton Translocation ◽

Attenuated Total Reflection ◽

Nadh:Ubiquinone Oxidoreductase ◽

Respiratory Complex I ◽

Part Structure ◽

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 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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The respiratory complexes I from the mitochondria of two Pichia species

Biochemical Journal ◽

10.1042/bj20090492 ◽

2009 ◽

Vol 422 (1) ◽

pp. 151-159 ◽

Cited By ~ 19

Author(s):

Hannah R. Bridges ◽

Ljuban Grgic ◽

Michael E. Harbour ◽

Judy Hirst

Keyword(s):

Complex I ◽

Catalytic Mechanism ◽

Specific Activity ◽

Proton Translocation ◽

High Specific Activity ◽

Nadh Oxidation ◽

Model Systems ◽

Nadh:Ubiquinone Oxidoreductase ◽

Protonmotive Force ◽

Wide Range

NADH:ubiquinone oxidoreductase (complex I) is an entry point for electrons into the respiratory chain in many eukaryotes. It couples NADH oxidation and ubiquinone reduction to proton translocation across the mitochondrial inner membrane. Because complex I deficiencies occur in a wide range of neuromuscular diseases, including Parkinson's disease, there is a clear need for model eukaryotic systems to facilitate structural, functional and mutational studies. In the present study, we describe the purification and characterization of the complexes I from two yeast species, Pichia pastoris and Pichia angusta. They are obligate aerobes which grow to very high cell densities on simple medium, as yeast-like, spheroidal cells. Both Pichia enzymes catalyse inhibitor-sensitive NADH:ubiquinone oxidoreduction, display EPR spectra which match closely to those from other eukaryotic complexes I, and show patterns characteristic of complex I in SDS/PAGE analysis. Mass spectrometry was used to identify several canonical complex I subunits. Purified P. pastoris complex I has a particularly high specific activity, and incorporating it into liposomes demonstrates that NADH oxidation is coupled to the generation of a protonmotive force. Interestingly, the rate of NADH-induced superoxide production by the Pichia enzymes is more than twice as high as that of the Bos taurus enzyme. Our results both resolve previous disagreement about whether Pichia species encode complex I, furthering understanding of the evolution of complex I within dikarya, and they provide two new, robust and highly active model systems for study of the structure and catalytic mechanism of eukaryotic complexes I.

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Chemo-mechanical Coupling in the Transport Cycle of a Type II ABC Transporter

10.1101/471920 ◽

2018 ◽

Author(s):

Koichi Tamura ◽

Hiroshi Sugimoto ◽

Yoshitsugu Shiro ◽

Yuji Sugita

Keyword(s):

Atpase Activity ◽

Abc Transporter ◽

Conformational Changes ◽

Abc Transporters ◽

Md Simulations ◽

Coupling Mechanism ◽

Transmembrane Helices ◽

Mechanical Coupling ◽

Binding Domains ◽

Wide Range

AbstractAT P -binding cassette (ABC) transporters are integral membrane proteins that translocate a wide range of substrates across biological membranes, harnessing free energy from the binding and hydrolysis of ATP. To understand the mechanism of the inward- to outward-facing transition that could be achieved by tight regulation of ATPase activity through extensive conformational changes of the protein, we applied template-based iterative all-atom molecular dynamics (MD) simulation to the heme ABC transporter BhuUV-T. The simulations, together with biased MDs, predict two new conformations of the protein, namely, occluded (Occ) and outward-facing (OF) conformations. The comparison between the inward-facing crystal structure and the predicted two structures shows atomic details of the gating motions at the transmembrane helices and dimerization of the nucleotide-binding domains (NBDs). The MD simulations further reveal a novel role of the ABC signature motifs (LSGG[Q/E]) at the NBDs in decelerating ATPase activity in the Occ form through sporadic flipping of the side chains of the LSGG[Q/E] catalytic serine residues. The orientational changes are coupled to loose NBD dimerization in the Occ state, whereas they are blocked in the OF form where the NBDs are tightly dimerized. The chemo-mechanical coupling mechanism may apply to other types of ABC transporters having the conserved LSGG[Q/E] signature motifs.

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Paracoccus denitrificans: a genetically tractable model system for studying respiratory complex I

Scientific Reports ◽

10.1038/s41598-021-89575-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Owen D. Jarman ◽

Olivier Biner ◽

John J. Wright ◽

Judy Hirst

Keyword(s):

Complex I ◽

Paracoccus Denitrificans ◽

Model System ◽

Proton Pumping ◽

Model Systems ◽

Metabolic Enzyme ◽

Nadh:Ubiquinone Oxidoreductase ◽

Mitochondrial Complex I ◽

Mitochondrial Complex ◽

Inner Mitochondrial Membrane

AbstractMitochondrial complex I (NADH:ubiquinone oxidoreductase) is a crucial metabolic enzyme that couples the free energy released from NADH oxidation and ubiquinone reduction to the translocation of four protons across the inner mitochondrial membrane, creating the proton motive force for ATP synthesis. The mechanism by which the energy is captured, and the mechanism and pathways of proton pumping, remain elusive despite recent advances in structural knowledge. Progress has been limited by a lack of model systems able to combine functional and structural analyses with targeted mutagenic interrogation throughout the entire complex. Here, we develop and present the α-proteobacterium Paracoccus denitrificans as a suitable bacterial model system for mitochondrial complex I. First, we develop a robust purification protocol to isolate highly active complex I by introducing a His6-tag on the Nqo5 subunit. Then, we optimize the reconstitution of the enzyme into liposomes, demonstrating its proton pumping activity. Finally, we develop a strain of P. denitrificans that is amenable to complex I mutagenesis and create a catalytically inactive variant of the enzyme. Our model provides new opportunities to disentangle the mechanism of complex I by combining mutagenesis in every subunit with established interrogative biophysical measurements on both the soluble and membrane bound enzymes.

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