scholarly journals The internal alternative NADH dehydrogenase of Neurospora crassa mitochondria

2003 ◽  
Vol 371 (3) ◽  
pp. 1005-1011 ◽  
Author(s):  
Margarida DUARTE ◽  
Markus PETERS ◽  
Ulrich SCHULTE ◽  
Arnaldo VIDEIRA
Keyword(s):  
Neurospora Crassa ◽  
Complex I ◽  
Nadh Dehydrogenase ◽  
Point Mutations ◽  
Proton Pumping ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Prosthetic Group ◽  
Reading Frame ◽  
Mature Enzyme ◽  
Nadh Dehydrogenases

An open reading frame homologous with genes of non-proton-pumping NADH dehydrogenases was identified in the genome of Neurospora crassa. The 57 kDa NADH:ubiquinone oxidoreductase acts as internal (alternative) respiratory NADH dehydrogenase (NDI1) in the fungal mitochondria. The precursor polypeptide includes a pre-sequence of 31 amino acids, and the mature enzyme comprises one FAD molecule as a prosthetic group. It catalyses specifically the oxidation of NADH. Western blot analysis of fungal mitochondria fractionated with digitonin indicated that the protein is located at the inner face of the inner membrane of the organelle (internal enzyme). The corresponding gene was inactivated by the generation of repeat-induced point mutations. The respiratory activity of mitochondria from the resulting null-mutant ndi1 is almost fully inhibited by rotenone, an inhibitor of the proton-pumping complex I, when matrix-generated NADH is used as substrate. Although no effects of the NDI1 defect on vegetative growth and sexual differentiation were observed, the germination of both sexual and asexual ndi1 mutant spores is significantly delayed. Crosses between the ndi1 mutant strain and complex I-deficient mutants yielded no viable double mutants. Our data indicate: (i) that NDI1 represents the sole internal alternative NADH dehydrogenase of Neurospora mitochondria; (ii) that NDI1 and complex I are functionally complementary to each other; and (iii) that NDI1 is specially needed during spore germination.

scholarly journals Physiological relevance, localization and substrate specificity of the alternative (type II) mitochondrial NADH dehydrogenases of Ogataea parapolymorpha

2021 ◽  
Author(s):  
Hannes Juergens ◽  
Álvaro Mielgo-Gómez ◽  
Albert Godoy-Hernández ◽  
Jolanda ter Horst ◽  
Janine M. Nijenhuis ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Respiratory Chain ◽  
Complex I ◽  
Nadh Dehydrogenase ◽  
Physiological Role ◽  
Proton Pumping ◽  
Type Ii ◽  
Respiratory Chains ◽  
Alternative Type ◽  
Nadh Dehydrogenases

AbstractMitochondria from Ogataea parapolymorpha harbor a branched electron-transport chain containing a proton-pumping Complex I NADH dehydrogenase and three alternative (type II) NADH dehydrogenases (NDH2s). To investigate the physiological role, localization and substrate specificity of these enzymes, growth of various NADH dehydrogenase mutants was quantitatively characterized in shake-flask and chemostat cultures, followed by oxygen-uptake experiments with isolated mitochondria. Furthermore, NAD(P)H:quinone oxidoreduction of the three NDH2s were individually assessed. Our findings show that the O. parapolymorpha respiratory chain contains an internal NADH-accepting NDH2 (Ndh2-1/OpNdi1), at least one external NAD(P)H-accepting enzyme and likely additional mechanisms for respiration-linked oxidation of cytosolic NADH. Metabolic regulation appears to prevent competition between OpNdi1 and Complex I for mitochondrial NADH. With the exception of OpNdi1, the respiratory chain of O. parapolymorpha exhibits metabolic redundancy and tolerates deletion of multiple NADH-dehydrogenase genes without compromising fully respiratory metabolism.ImportanceTo achieve high productivity and yields in microbial bioprocesses, efficient use of the energy substrate is essential. Organisms with branched respiratory chains can respire via the energy-efficient proton-pumping Complex I, or make use of alternative NADH dehydrogenases (NDH2s). The yeast Ogataea parapolymorpha contains three uncharacterized, putative NDH2s which were investigated in this work. We show that O. parapolymorpha contains at least one ‘internal’ NDH2, which provides an alternative to Complex I for mitochondrial NADH oxidation, albeit at a lower efficiency. The use of this NDH2 appeared to be limited to carbon excess conditions and the O. parapolymorpha respiratory chain tolerated multiple deletions without compromising respiratory metabolism, highlighting opportunities for metabolic (redox) engineering. By providing a more comprehensive understanding of the physiological role of NDH2s, including insights into their metabolic capacity, orientation and substrate specificity this study also extends our fundamental understanding of respiration in organisms with branched respiratory chains.

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 Large-scale chromatographic purification of F1F0-ATPase and complex I from bovine heart mitochondria

1996 ◽  
Vol 318 (1) ◽  
pp. 343-349 ◽  
Author(s):  
Susan K BUCHANAN ◽  
John E. WALKER
Keyword(s):  
Complex I ◽  
Large Scale ◽  
Gel Filtration ◽  
Atp Synthesis ◽  
Lipid Vesicles ◽  
Proton Pumping ◽  
Heart Mitochondria ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Bovine Heart ◽  
F1fo Atpase

A new chromatographic procedure has been developed for the isolation of F1Fo-ATPase and NADH:ubiquinone oxidoreductase (complex I) from a single batch of bovine heart mitochondria. The method employed dodecyl β-Δ-maltoside, a monodisperse, homogeneous detergent in which many respiratory complexes exhibit high activity, for solubilization and subsequent purification by ammonium sulphate fractionation and column chromatography. A combination of anion-exchange, gel-filtration, and dye-ligand affinity chromatography was used to purify both complexes to homogeneity. The F1Fo-ATPase preparation contains only the 16 known subunits of the enzyme. It has oligomycin-sensitive ATP hydrolysis activity and, as demonstrated elsewhere, when reconstituted into lipid vesicles it is capable of ATP-dependent proton pumping and of ATP synthesis driven by a proton gradient [Groth and Walker (1996) Biochem. J. 318, 351–357]. The complex I preparation contains all of the subunits identified in other preparations of the enzyme, and has rotenone-sensitive NADH:ubiquinone oxidoreductase and NADH:ferricyanide oxidoreductase activities. The procedure is rapid and reproducible, yielding 50–80 mg of purified F1Fo-ATPase and 20–40 mg of purified complex I from 1 g of mitochondrial membranes. Both preparations are devoid of phospholipids, and gel filtration and dynamic light scattering experiments indicate that they are monodisperse. Therefore, the preparations fulfil important prerequisites for structural analysis.

scholarly journals Roles of subunit NuoL in the proton pumping coupling mechanism of NADH:ubiquinone oxidoreductase (complex I) fromEscherichia coli

2016 ◽  
Vol 160 (4) ◽  
pp. 205-215 ◽  
Author(s):  
Madhavan Narayanan ◽  
Joseph A. Sakyiama ◽  
Mahmoud M. Elguindy ◽  
Eiko Nakamaru-Ogiso
Keyword(s):  
Complex I ◽  
Proton Pumping ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Coupling Mechanism ◽  
Fromescherichia Coli

scholarly journals The mitochondrial AAA protease FTSH3 regulates Complex I abundance by promoting its disassembly

2021 ◽  
Author(s):  
Aneta Ivanova ◽  
Abi S Ghifari ◽  
Oliver Berkowitz ◽  
James Whelan ◽  
Monika W Murcha
Keyword(s):  
Complex I ◽  
Nadh Dehydrogenase ◽  
Slow Growth ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Forward Genetic Screen ◽  
Slow Growth Rate ◽  
Partial Restoration ◽  
Screen Approach

Abstract ATP is generated in mitochondria by oxidative phosphorylation. Complex I (NADH:ubiquinone oxidoreductase or NADH dehydrogenase) is the first multisubunit protein complex of this pathway, oxidising NADH and transferring electrons to the ubiquinone pool. Typically Complex I mutants display a slow growth rate compared to wild-type plants. Here, using a forward genetic screen approach for restored growth of a Complex I mutant, we have identified the mitochondrial ATP dependent metalloprotease, Filamentous Temperature Sensitive H 3 (FTSH3), as a factor that is required for the disassembly of Complex I. An ethyl methanesulfonate-induced mutation in FTSH3, named rmb1 (restoration of mitochondrial biogenesis 1), restored Complex I abundance and plant growth. Complementation could be achieved with FTSH3 lacking proteolytic activity, suggesting the unfoldase function of FTSH3 has a role in Complex I disassembly. The introduction of the rmb1 to an additional, independent, and extensively characterised Complex I mutant, ndufs4, resulted in similar increases to Complex I abundance and a partial restoration of growth. These results show that disassembly or degradation of Complex I plays a role in determining its steady-state abundance and thus turnover may vary under different conditions.

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.

Challenges in elucidating structure and mechanism of proton pumping NADH:ubiquinone oxidoreductase (complex I)

2008 ◽  
Vol 40 (5) ◽  
pp. 475-483 ◽  
Author(s):  
Volker Zickermann ◽  
Stefan Dröse ◽  
Maja A. Tocilescu ◽  
Klaus Zwicker ◽  
Stefan Kerscher ◽  
...  
Keyword(s):  
Complex I ◽  
Proton Pumping ◽  
Nadh:Ubiquinone Oxidoreductase

scholarly journals Accessory NUMM (NDUFS6) subunit harbors a Zn-binding site and is essential for biogenesis of mitochondrial complex I

2015 ◽  
Vol 112 (18) ◽  
pp. 5685-5690 ◽  
Author(s):  
Katarzyna Kmita ◽  
Christophe Wirth ◽  
Judith Warnau ◽  
Sergio Guerrero-Castillo ◽  
Carola Hunte ◽  
...  
Keyword(s):  
Binding Site ◽  
Complex I ◽  
Proton Pumping ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
X Ray Crystallography ◽  
Late Step ◽  
Multistep Process ◽  
Assembly Factor ◽  
Accessory Subunit ◽  
Assembly Intermediate

Mitochondrial proton-pumping NADH:ubiquinone oxidoreductase (respiratory complex I) comprises more than 40 polypeptides and contains eight canonical FeS clusters. The integration of subunits and insertion of cofactors into the nascent complex is a complicated multistep process that is aided by assembly factors. We show that the accessory NUMM subunit of complex I (human NDUFS6) harbors a Zn-binding site and resolve its position by X-ray crystallography. Chromosomal deletion of the NUMM gene or mutation of Zn-binding residues blocked a late step of complex I assembly. An accumulating assembly intermediate lacked accessory subunit N7BM (NDUFA12), whereas a paralog of this subunit, the assembly factor N7BML (NDUFAF2), was found firmly bound instead. EPR spectroscopic analysis and metal content determination after chromatographic purification of the assembly intermediate showed that NUMM is required for insertion or stabilization of FeS cluster N4.

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