The mitochondrial-encoded subunits of respiratory complex I (NADH:ubiquinone oxidoreductase): identifying residues important in mechanism and disease

2011 ◽  
Vol 39 (3) ◽  
pp. 799-806 ◽  
Author(s):  
Hannah R. Bridges ◽  
James A. Birrell ◽  
Judy Hirst
Keyword(s):  
Complex I ◽  
Structural Model ◽  
Proton Translocation ◽  
Site Directed Mutagenesis ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Intramolecular Electron Transfer ◽  
Hydrophobic Domain ◽  
Bacterial Cytoplasm ◽  
Conserved Core ◽  
Hydrophilic Domain

Complex I (NADH:ubiquinone oxidoreductase) is crucial to respiration in many aerobic organisms. The hydrophilic domain of complex I, containing nine or more redox cofactors, and comprising seven conserved core subunits, protrudes into the mitochondrial matrix or bacterial cytoplasm. The α-helical membrane-bound hydrophobic domain contains a further seven core subunits that are mitochondrial-encoded in eukaryotes and named the ND subunits (ND1–ND6 and ND4L). Complex I couples the oxidation of NADH in the hydrophilic domain to ubiquinone reduction and proton translocation in the hydrophobic domain. Although the mechanisms of NADH oxidation and intramolecular electron transfer are increasingly well understood, the mechanisms of ubiquinone reduction and proton translocation remain only poorly defined. Recently, an α-helical model of the hydrophobic domain of bacterial complex I [Efremov, Baradaran and Sazanov (2010) Nature 465, 441–447] revealed how the 63 transmembrane helices of the seven core subunits are arranged, and thus laid a foundation for the interpretation of functional data and the formulation of mechanistic proposals. In the present paper, we aim to correlate information from sequence analyses, site-directed mutagenesis studies and mutations that have been linked to human diseases, with information from the recent structural model. Thus we aim to identify and discuss residues in the ND subunits of mammalian complex I which are important in catalysis and for maintaining the enzyme's structural and functional integrity.

scholarly journals High-resolution structure and dynamics of mitochondrial complex I – insights into the proton pumping mechanism

2021 ◽  
Author(s):  
Kristian Parey ◽  
Jonathan Lasham ◽  
Deryck J. Mills ◽  
Amina Djurabekova ◽  
Outi Haapanen ◽  
...  
Keyword(s):  
High Resolution ◽  
Complex I ◽  
Large Scale ◽  
Catalytic Mechanism ◽  
Proton Translocation ◽  
Bound Water ◽  
Structural Data ◽  
Proton Pumping ◽  
Site Directed Mutagenesis ◽  
Nadh:Ubiquinone Oxidoreductase

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is a 1 MDa membrane protein complex with a central role in energy metabolism. Redox-driven proton translocation by complex I contributes substantially to the proton motive force that drives ATP synthase. Several structures of complex I from bacteria and mitochondria have been determined but its catalytic mechanism has remained controversial. We here present the cryo-EM structure of complex I from Yarrowia lipolytica at 2.1 Å resolution, which reveals the positions of more than 1600 protein-bound water molecules, of which ~100 are located in putative proton translocation pathways. Another structure of the same complex under steady-state activity conditions at 3.4 Å resolution indicates conformational transitions that we associate with proton injection into the central hydrophilic axis. By combining high-resolution structural data with site-directed mutagenesis and large-scale molecular dynamics simulations, we define details of the proton translocation pathways, and offer new insights into the redox-coupled proton pumping mechanism of complex I.

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

2017 ◽  
Vol 114 (48) ◽  
pp. 12737-12742 ◽  
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.

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

2015 ◽  
Vol 290 (34) ◽  
pp. 20761-20773 ◽  
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.

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

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.

A Possible Role for Iron-Sulfur Cluster N2 in Proton Translocation by the NADH:Ubiquinone Oxidoreductase (Complex I)

2005 ◽  
Vol 10 (2-4) ◽  
pp. 208-222 ◽  
Author(s):  
Dirk Flemming ◽  
Stefan Stolpe ◽  
Daniel Schneider ◽  
Petra Hellwig ◽  
Thorsten Friedrich
Keyword(s):  
Complex I ◽  
Proton Translocation ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Iron Sulfur

scholarly journals 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 ◽  
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.

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

2013 ◽  
Vol 41 (5) ◽  
pp. 1265-1271 ◽  
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.

scholarly journals 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?

2006 ◽  
Vol 400 (3) ◽  
pp. 541-550 ◽  
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.

Sign in / Sign up

Export Citation Format

Share Document