A single external enzyme confers alternative NADH:ubiquinone oxidoreductase activity in Yarrowia lipolytica

1999 ◽  
Vol 112 (14) ◽  
pp. 2347-2354 ◽  
Author(s):  
S.J. Kerscher ◽  
J.G. Okun ◽  
U. Brandt
Keyword(s):  
Yarrowia Lipolytica ◽  
Complex I ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Inner Mitochondrial Membrane ◽  
Gene Encoding ◽  
Oxidoreductase Activity ◽  
Mitochondrial Inner Membrane ◽  
Energy Conserving ◽  
And Function ◽  
Complex I Activity

NADH:ubiquinone oxidoreductases catalyse the first step within the diverse pathways of mitochondrial NADH oxidation. In addition to the energy-conserving form commonly called complex I, fungi and plants contain much simpler alternative NADH:ubiquinone oxido-reductases that catalyze the same reaction but do not translocate protons across the inner mitochondrial membrane. Little is known about the distribution and function of these enzymes. We have identified YLNDH2 as the only gene encoding an alternative NADH:ubiquinone oxidoreductase (NDH2) in the obligate aerobic yeast Yarrowia lipolytica. Cells carrying a deletion of YLNDH2 were fully viable; full inhibition by piericidin A indicated that complex I activity was the sole NADH:ubiquinone oxidoreductase activity left in the deletion strains. Studies with intact mitochondria revealed that NDH2 in Y. lipolytica is oriented towards the external face of the mitochondrial inner membrane. This is in contrast to the situation seen in Saccharomyces cerevisiae, Neurospora crassa and in green plants, where internal alternative NADH:ubiquinone oxidoreductases have been reported. Phylogenetic analysis of known NADH:ubiquinone oxidoreductases suggests that during evolution conversion of an ancestral external alternative NADH:ubiquinone oxidoreductase to an internal enzyme may have paved the way for the loss of complex I in fermenting yeasts like S. cerevisiae.

External alternative NADH:ubiquinone oxidoreductase redirected to the internal face of the mitochondrial inner membrane rescues complex I deficiency in Yarrowia lipolytica

2001 ◽  
Vol 114 (21) ◽  
pp. 3915-3921 ◽  
Author(s):  
Stefan J. Kerscher ◽  
Andrea Eschemann ◽  
Pamela M. Okun ◽  
Ulrich Brandt
Keyword(s):  
Yarrowia Lipolytica ◽  
Complex I ◽  
Functional Expression ◽  
Proton Pumping ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
The Matrix ◽  
Cytosolic Side ◽  
Respiratory Membrane

Alternative NADH:ubiquinone oxidoreductases are single subunit enzymes capable of transferring electrons from NADH to ubiquinone without contributing to the proton gradient across the respiratory membrane. The obligately aerobic yeast Yarrowia lipolytica has only one such enzyme, encoded by the NDH2 gene and located on the external face of the mitochondrial inner membrane. In sharp contrast to ndh2 deletions, deficiencies in nuclear genes for central subunits of proton pumping NADH:ubiquinone oxidoreductases (complex I) are lethal. We have redirected NDH2 to the internal face of the mitochondrial inner membrane by N-terminally attaching the mitochondrial targeting sequence of NUAM, the largest subunit of complex I. Lethality of complex I mutations was rescued by the internal, but not the external version of alternative NADH:ubiquinone oxidoreductase. Internal NDH2 also permitted growth in the presence of complex I inhibitors such as 2-decyl-4-quinazolinyl amine (DQA). Functional expression of NDH2 on both sides of the mitochondrial inner membrane indicates that alternative NADH:ubiquinone oxidoreductase requires no additional components for catalytic activity. Our findings also demonstrate that shuttle mechanisms for the transfer of redox equivalents from the matrix to the cytosolic side of the mitochondrial inner membrane are insufficient in Y. lipolytica.

scholarly journals Structural organization of mitochondrial human complex I: role of the ND4 and ND5 mitochondria-encoded subunits and interaction with prohibitin

2004 ◽  
Vol 383 (3) ◽  
pp. 491-499 ◽  
Author(s):  
Ingrid BOURGES ◽  
Claire RAMUS ◽  
Bénédicte MOUSSON de CAMARET ◽  
Réjane BEUGNOT ◽  
Claire REMACLE ◽  
...  
Keyword(s):  
Complex I ◽  
Nadh Dehydrogenase ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Membrane Association ◽  
Oxidoreductase Activity ◽  
Mitochondrial Inner Membrane ◽  
The Matrix ◽  
Complex I Assembly ◽  
Human Complex

Mitochondria-encoded ND (NADH dehydrogenase) subunits, as components of the hydrophobic part of complex I, are essential for NADH:ubiquinone oxidoreductase activity. Mutations or lack of expression of these subunits have significant pathogenic consequences in humans. However, the way these events affect complex I assembly is poorly documented. To understand the effects of particular mutations in ND subunits on complex I assembly, we studied four human cell lines: ND4 non-expressing cells, ND5 non-expressing cells, and rho° cells that do not express any ND subunits, in comparison with normal complex I control cells. In control cells, all the seven analysed nuclear-encoded complex I subunits were found to be attached to the mitochondrial inner membrane, except for the 24 kDa subunit, which was nearly equally partitioned between the membranes and the matrix. Absence of a single ND subunit, or even all the seven ND subunits, caused no major changes in the nuclear-encoded complex I subunit content of mitochondria. However, in cells lacking ND4 or ND5, very low amounts of 24 kDa subunit were found associated with the membranes, whereas most of the other nuclear-encoded subunits remained attached. In contrast, membrane association of most of the nuclear subunits was significantly reduced in the absence of all seven ND proteins. Immunopurification detected several subcomplexes. One of these, containing the 23, 30 and 49 kDa subunits, also contained prohibitin. This is the first description of prohibitin interaction with complex I subunits and suggests that this protein might play a role in the assembly or degradation of mitochondrial complex I.

Mutants of Chlamydomonas reinhardtii Deficient in Mitochondrial Complex I: Characterization of Two Mutations Affecting the nd1 Coding Sequence

Genetics ◽  
2001 ◽  
Vol 158 (3) ◽  
pp. 1051-1060
Author(s):  
Claire Remacle ◽  
Denis Baurain ◽  
Pierre Cardol ◽  
René F Matagne
Keyword(s):  
Mitochondrial Genome ◽  
Chlamydomonas Reinhardtii ◽  
Complex I ◽  
Slow Growth ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Sequencing Analysis ◽  
Mitochondrial Complex ◽  
Genetical Analysis ◽  
Complex I Activity

Abstract The mitochondrial rotenone-sensitive NADH:ubiquinone oxidoreductase (complex I) comprises more than 30 subunits, the majority of which are encoded by the nucleus. In Chlamydomonas reinhardtii, only five components of complex I are coded for by mitochondrial genes. Three mutants deprived of complex I activity and displaying slow growth in the dark were isolated after mutagenic treatment with acriflavine. A genetical analysis demonstrated that two mutations (dum20 and dum25) affect the mitochondrial genome whereas the third mutation (dn26) is of nuclear origin. Recombinational analyses showed that dum20 and dum25 are closely linked on the genetic map of the mitochondrial genome and could affect the nd1 gene. A sequencing analysis confirmed this conclusion: dum20 is a deletion of one T at codon 243 of nd1; dum25 corresponds to a 6-bp deletion that eliminates two amino acids located in a very conserved hydrophilic segment of the protein.

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

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.

scholarly journals Structure of the deactive state of mammalian respiratory complex I

2017 ◽  
Author(s):  
James N. Blaza ◽  
Kutti R. Vinothkumar ◽  
Judy Hirst
Keyword(s):  
Complex I ◽  
Bos Taurus ◽  
Atp Synthesis ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Structural Elements ◽  
Highly Active ◽  
Mammalian Mitochondria ◽  
Energy Conserving ◽  
Respiratory Complex I ◽  
Active Preparation

AbstractComplex I (NADH:ubiquinone oxidoreductase) is central to energy metabolism in mammalian mitochondria. It couples NADH oxidation by ubiquinone to proton transport across the energy-conserving inner membrane, catalyzing respiration and driving ATP synthesis. In the absence of substrates, ‘active’ complex I gradually enters a pronounced resting or ‘deactive’ state. The active-deactive transition occurs during ischemia and is crucial for controlling how respiration recovers upon reperfusion. Here, we set a highly-active preparation of Bos taurus complex I into the biochemically-defined deactive state, and used single-particle electron cryomicroscopy to determine its structure to 4.1 Å resolution. The deactive state arises when critical structural elements that form the ubiquinone-binding site become disordered, and we propose reactivation is induced when substrate binding templates their reordering. Our structure both rationalizes biochemical data on the deactive state, and offers new insights into its physiological and cellular roles.

scholarly journals Loss of m-AAA protease in mitochondria causes complex I deficiency and increased sensitivity to oxidative stress in hereditary spastic paraplegia

2003 ◽  
Vol 163 (4) ◽  
pp. 777-787 ◽  
Author(s):  
Luigia Atorino ◽  
Laura Silvestri ◽  
Mirko Koppen ◽  
Laura Cassina ◽  
Andrea Ballabio ◽  
...  
Keyword(s):  
Hereditary Spastic Paraplegia ◽  
Complex I ◽  
Oxidant Stress ◽  
Cellular Level ◽  
Spastic Paraplegia ◽  
Functional Conservation ◽  
Homologous Protein ◽  
Mitochondrial Inner Membrane ◽  
Increased Sensitivity ◽  
Complex I Activity

Mmutations in paraplegin, a putative mitochondrial metallopeptidase of the AAA family, cause an autosomal recessive form of hereditary spastic paraplegia (HSP). Here, we analyze the function of paraplegin at the cellular level and characterize the phenotypic defects of HSP patients' cells lacking this protein. We demonstrate that paraplegin coassembles with a homologous protein, AFG3L2, in the mitochondrial inner membrane. These two proteins form a high molecular mass complex, which we show to be aberrant in HSP fibroblasts. The loss of this complex causes a reduced complex I activity in mitochondria and an increased sensitivity to oxidant stress, which can both be rescued by exogenous expression of wild-type paraplegin. Furthermore, complementation studies in yeast demonstrate functional conservation of the human paraplegin–AFG3L2 complex with the yeast m-AAA protease and assign proteolytic activity to this structure. These results shed new light on the molecular pathogenesis of HSP and functionally link AFG3L2 to this neurodegenerative disease.

Mitochondrial network complexity and pathological decrease in complex I activity are tightly correlated in isolated human complex I deficiency

2005 ◽  
Vol 289 (4) ◽  
pp. C881-C890 ◽  
Author(s):  
Werner J. H. Koopman ◽  
Henk-Jan Visch ◽  
Sjoerd Verkaart ◽  
Lambertus W. P. J. van den Heuvel ◽  
Jan A. M. Smeitink ◽  
...  
Keyword(s):  
Form Factor ◽  
Aspect Ratio ◽  
Complex I ◽  
Mitochondrial Morphology ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Mitochondrial Network ◽  
Mitochondrial Structure ◽  
Complex I Deficiency ◽  
Complex I Activity ◽  
Human Complex

Complex I (NADH:ubiquinone oxidoreductase) is the largest multisubunit assembly of the oxidative phosphorylation system, and its malfunction is associated with a wide variety of clinical syndromes ranging from highly progressive, often early lethal, encephalopathies to neurodegenerative disorders in adult life. The changes in mitochondrial structure and function that are at the basis of the clinical symptoms are poorly understood. Video-rate confocal microscopy of cells pulse-loaded with mitochondria-specific rhodamine 123 followed by automated analysis of form factor (combined measure of length and degree of branching), aspect ratio (measure of length), and number of revealed marked differences between primary cultures of skin fibroblasts from 13 patients with an isolated complex I deficiency. These differences were independent of the affected subunit, but plotting of the activity of complex I, normalized to that of complex IV, against the ratio of either form factor or aspect ratio to number revealed a linear relationship. Relatively small reductions in activity appeared to be associated with an increase in form factor and never with a decrease in number, whereas relatively large reductions occurred in association with a decrease in form factor and/or an increase in number. These results demonstrate that complex I activity and mitochondrial structure are tightly coupled in human isolated complex I deficiency. To further prove the relationship between aberrations in mitochondrial morphology and pathological condition, fibroblasts from two patients with a different mutation but a highly fragmented mitochondrial phenotype were fused. Full restoration of the mitochondrial network demonstrated that this change in mitochondrial morphology was indeed associated with human complex I deficiency.

scholarly journals A ferredoxin bridge connects the two arms of plant mitochondrial complex I

2020 ◽  
Author(s):  
Niklas Klusch ◽  
Jennifer Senkler ◽  
Özkan Yildiz ◽  
Werner Kühlbrandt ◽  
Hans-Peter Braun
Keyword(s):  
Arabidopsis Thaliana ◽  
Complex I ◽  
Mitochondrial Membrane ◽  
Mitochondrial Complex I ◽  
Mitochondrial Complex ◽  
Inner Mitochondrial Membrane ◽  
Main Site ◽  
The Matrix ◽  
Quinol Binding ◽  
Complex I Activity

SUMMARYMitochondrial complex I is the main site for electron transfer to the respiratory chain and generates much of the proton gradient across the inner mitochondrial membrane. It is composed of two arms, which form a conserved L-shape. We report the structures of the intact, 47-subunit mitochondrial complex I from Arabidopsis thaliana and from the green alga Polytomella sp. at 3.2 and 3.3 Å resolution. In both, a heterotrimeric γ-carbonic anhydrase domain is attached to the membrane arm on the matrix side. Two states are resolved in A. thaliana complex I, with different angles between the two arms and different conformations of the ND1 loop near the quinol binding site. The angle appears to depend on a bridge domain, which links the peripheral arm to the membrane arm and includes an unusual ferredoxin. We suggest that the bridge domain regulates complex I activity.One sentence summaryThe activity of complex I depends on the angel between its two arms, which, in plants, is adjusted by a protein bridge that includes an unusual ferredoxin.The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) are: Hans-Peter Braun ([email protected]) and Werner Kühlbrandt ([email protected]).

scholarly journals Structure of inhibitor-bound mammalian complex I

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Hannah R. Bridges ◽  
Justin G. Fedor ◽  
James N. Blaza ◽  
Andrea Di Luca ◽  
Alexander Jussupow ◽  
...  
Keyword(s):  
Complex I ◽  
Energy Coupling ◽  
Competitive Inhibitor ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Mouse Heart ◽  
Coupling Mechanism ◽  
Substrate Reduction ◽  
Mitochondrial Inner Membrane ◽  
Respiratory Complex I ◽  
Ubiquinone Binding

Abstract Respiratory complex I (NADH:ubiquinone oxidoreductase) captures the free energy from oxidising NADH and reducing ubiquinone to drive protons across the mitochondrial inner membrane and power oxidative phosphorylation. Recent cryo-EM analyses have produced near-complete models of the mammalian complex, but leave the molecular principles of its long-range energy coupling mechanism open to debate. Here, we describe the 3.0-Å resolution cryo-EM structure of complex I from mouse heart mitochondria with a substrate-like inhibitor, piericidin A, bound in the ubiquinone-binding active site. We combine our structural analyses with both functional and computational studies to demonstrate competitive inhibitor binding poses and provide evidence that two inhibitor molecules bind end-to-end in the long substrate binding channel. Our findings reveal information about the mechanisms of inhibition and substrate reduction that are central for understanding the principles of energy transduction in mammalian complex I.

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