scholarly journals Human Ind1, an Iron-Sulfur Cluster Assembly Factor for Respiratory Complex I

2009 ◽  
Vol 29 (22) ◽  
pp. 6059-6073 ◽  
Author(s):  
Alex D. Sheftel ◽  
Oliver Stehling ◽  
Antonio J. Pierik ◽  
Daili J. A. Netz ◽  
Stefan Kerscher ◽  
...  
Keyword(s):  
Complex I ◽  
Mitochondrial Diseases ◽  
Mitochondrial Morphology ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Activity Levels ◽  
Cluster Assembly ◽  
Respiratory Complex ◽  
Iron Sulfur ◽  
Assembly Factor ◽  
Respiratory Complex I

ABSTRACT Respiratory complex I (NADH:ubiquinone oxidoreductase) is a large mitochondrial inner membrane enzyme consisting of 45 subunits and 8 iron-sulfur (Fe/S) clusters. While complex I dysfunction is the most common reason for mitochondrial diseases, the assembly of complex I and its Fe/S cofactors remains elusive. Here, we identify the human mitochondrial P-loop NTPase, designated huInd1, that is critically required for the assembly of complex I. huInd1 can bind an Fe/S cluster via a conserved CXXC motif in a labile fashion. Knockdown of huInd1 in HeLa cells by RNA interference technology led to strong decreases in complex I protein and activity levels, remodeling of respiratory supercomplexes, and alteration of mitochondrial morphology. In addition, huInd1 depletion resulted in massive decreases in several subunits (NDUFS1, NDUFV1, NDUFS3, and NDUFA13) of the peripheral arm of complex I, with the concomitant appearance of a 450-kDa subcomplex representing part of the membrane arm. By a novel radiolabeling technique, the amount of iron associated with complex I was also shown to reflect the dependence of this enzyme on huInd1 for assembly. Together, these data identify huInd1 as a new assembly factor for human respiratory complex I with a possible role in the delivery of one or more Fe/S clusters to complex I subunits.

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 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.

scholarly journals The multitude of iron–sulfur clusters in respiratory complex I

2016 ◽  
Vol 1857 (8) ◽  
pp. 1068-1072 ◽  
Author(s):  
Emmanuel Gnandt ◽  
Katerina Dörner ◽  
Marc F.J. Strampraad ◽  
Simon de Vries ◽  
Thorsten Friedrich
Keyword(s):  
Complex I ◽  
Iron Sulfur Clusters ◽  
Respiratory Complex ◽  
Iron Sulfur ◽  
Respiratory Complex I

Mammalian Respiratory Complex I Through the Lens of Cryo-EM

2019 ◽  
Vol 48 (1) ◽  
pp. 165-184 ◽  
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.

Fluorescent signals associated with respiratory Complex I revealed conformational changes in the catalytic site

2019 ◽  
Vol 366 (12) ◽  
Author(s):  
Marina Verkhovskaya ◽  
Nikolai Belevich
Keyword(s):  
Conformational Changes ◽  
Complex I ◽  
Catalytic Site ◽  
Flavin Mononucleotide ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Type I ◽  
Fluorescent Signal ◽  
Maximum Emission ◽  
Respiratory Complex ◽  
Respiratory Complex I

ABSTRACT Fluorescent signals associated with Complex I (NADH:ubiquinone oxidoreductase type I) upon its reduction by NADH without added acceptors and upon NADH:ubiquinone oxidoreduction were studied. Two Complex I-associated redox-dependent signals were observed: with maximum emission at 400 nm (λex = 320 nm) and 526 nm (λex = 450 nm). The 400 nm signal derived from ubiquinol accumulated in Complex I/DDM (n-dodecyl β-D-maltopyranoside) micelles. The 526 nm redox signal unexpectedly derives mainly from FMN (flavin mononucleotide), whose fluorescence in oxidized protein is fully quenched, but arises transiently upon reduction of Complex I by NADH. The paradoxical flare-up of FMN fluorescence is discussed in terms of conformational changes in the catalytic site upon NADH binding. The difficulties in revealing semiquinone fluorescent signal are considered.

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