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

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.

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The internal alternative NADH dehydrogenase of Neurospora crassa mitochondria

Biochemical Journal ◽

10.1042/bj20021374 ◽

2003 ◽

Vol 371 (3) ◽

pp. 1005-1011 ◽

Cited By ~ 26

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.

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Leishmania type II dehydrogenase is essential for parasite viability irrespective of the presence of an active complex I

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2103803118 ◽

2021 ◽

Vol 118 (42) ◽

pp. e2103803118

Author(s):

Margarida Duarte ◽

Cleide Ferreira ◽

Gurleen Kaur Khandpur ◽

Tamara Flohr ◽

Jannik Zimmermann ◽

...

Keyword(s):

Complex I ◽

Nadh Dehydrogenase ◽

Leishmania Major ◽

Proton Translocation ◽

Protozoan Parasite ◽

Type I ◽

Type Ii ◽

Active Complex ◽

Mitochondrial Inner Membrane ◽

Candidate Target

Type II NADH dehydrogenases (NDH2) are monotopic enzymes present in the external or internal face of the mitochondrial inner membrane that contribute to NADH/NAD+ balance by conveying electrons from NADH to ubiquinone without coupled proton translocation. Herein, we characterize the product of a gene present in all species of the human protozoan parasite Leishmania as a bona fide, matrix-oriented, type II NADH dehydrogenase. Within mitochondria, this respiratory activity concurs with that of type I NADH dehydrogenase (complex I) in some Leishmania species but not others. To query the significance of NDH2 in parasite physiology, we attempted its genetic disruption in two parasite species, exhibiting a silent (Leishmania infantum, Li) and a fully operational (Leishmania major, Lm) complex I. Strikingly, this analysis revealed that NDH2 abrogation is not tolerated by Leishmania, not even by complex I–expressing Lm species. Conversely, complex I is dispensable in both species, provided that NDH2 is sufficiently expressed. That a type II dehydrogenase is essential even in the presence of an active complex I places Leishmania NADH metabolism into an entirely unique perspective and suggests unexplored functions for NDH2 that span beyond its complex I–overlapping activities. Notably, by showing that the essential character of NDH2 extends to the disease-causing stage of Leishmania, we genetically validate NDH2—an enzyme without a counterpart in mammals—as a candidate target for leishmanicidal drugs.

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Phylogenomic Analysis and Predicted Physiological Role of the Proton-Translocating NADH:Quinone Oxidoreductase (Complex I) Across Bacteria

mBio ◽

10.1128/mbio.00389-15 ◽

2015 ◽

Vol 6 (2) ◽

Cited By ~ 28

Author(s):

Melanie A. Spero ◽

Frank O. Aylward ◽

Cameron R. Currie ◽

Timothy J. Donohue

Keyword(s):

Respiratory Chain ◽

Complex I ◽

Bacterial Genomes ◽

Bacterial Physiology ◽

Nadh:Quinone Oxidoreductase ◽

Different Types ◽

Respiratory Chains ◽

Bacterial Groups ◽

Bacterial Complex

ABSTRACTThe proton-translocating NADH:quinone oxidoreductase (complex I) is a multisubunit integral membrane enzyme found in the respiratory chains of both bacteria and eukaryotic organelles. Although much research has focused on the enzyme's central role in the mitochondrial respiratory chain, comparatively little is known about its role in the diverse energetic lifestyles of different bacteria. Here, we used a phylogenomic approach to better understand the distribution of complex I across bacteria, the evolution of this enzyme, and its potential roles in shaping the physiology of different bacterial groups. By surveying 970 representative bacterial genomes, we predict complex I to be present in ~50% of bacteria. While this includes bacteria with a wide range of energetic schemes, the presence of complex I is associated with specific lifestyles, including aerobic respiration and specific types of phototrophy (bacteria with only a type II reaction center). A phylogeny of bacterial complex I revealed five main clades of enzymes whose evolution is largely congruent with the evolution of the bacterial groups that encode complex I. A notable exception includes the gammaproteobacteria, whose members encode one of two distantly related complex I enzymes predicted to participate in different types of respiratory chains (aerobic versus anaerobic). Comparative genomic analyses suggest a broad role for complex I in reoxidizing NADH produced from various catabolic reactions, including the tricarboxylic acid (TCA) cycle and fatty acid beta-oxidation. Together, these findings suggest diverse roles for complex I across bacteria and highlight the importance of this enzyme in shaping diverse physiologies across the bacterial domain.IMPORTANCELiving systems use conserved energy currencies, including a proton motive force (PMF), NADH, and ATP. The respiratory chain enzyme, complex I, connects these energy currencies by using NADH produced during nutrient breakdown to generate a PMF, which is subsequently used for ATP synthesis. Our goal is to better understand the role of complex I in bacteria, whose energetic diversity allows us to view its function in a range of biological contexts. We analyzed sequenced bacterial genomes to predict the presence, evolution, and function of complex I in bacteria. We identified five main classes of bacterial complex I and predict that different classes participate in different types of respiratory chains (aerobic and anaerobic). We also predict that complex I helps maintain a cellular redox state by reoxidizing NADH produced from central metabolism. Our findings suggest diverse roles for complex I in bacterial physiology, highlighting the need for future laboratory-based studies.

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Five decades of research on mitochondrial NADH-quinone oxidoreductase (complex I)

Biological Chemistry ◽

10.1515/hsz-2018-0164 ◽

2018 ◽

Vol 399 (11) ◽

pp. 1249-1264 ◽

Cited By ~ 16

Author(s):

Tomoko Ohnishi ◽

S. Tsuyoshi Ohnishi ◽

John C. Salerno

Keyword(s):

Electron Transfer ◽

Respiratory Chain ◽

Complex I ◽

Proton Translocation ◽

Atp Synthesis ◽

Mitochondrial Respiratory Chain ◽

Proton Pumping ◽

Entry Site ◽

Quinone Oxidoreductase ◽

Terminal Electron Acceptor

AbstractNADH-quinone oxidoreductase (complex I) is the largest and most complicated enzyme complex of the mitochondrial respiratory chain. It is the entry site into the respiratory chain for most of the reducing equivalents generated during metabolism, coupling electron transfer from NADH to quinone to proton translocation, which in turn drives ATP synthesis. Dysfunction of complex I is associated with neurodegenerative diseases such as Parkinson’s and Alzheimer’s, and it is proposed to be involved in aging. Complex I has one non-covalently bound FMN, eight to 10 iron-sulfur clusters, and protein-associated quinone molecules as electron transport components. Electron paramagnetic resonance (EPR) has previously been the most informative technique, especially in membranein situanalysis. The structure of complex 1 has now been resolved from a number of species, but the mechanisms by which electron transfer is coupled to transmembrane proton pumping remains unresolved. Ubiquinone-10, the terminal electron acceptor of complex I, is detectable by EPR in its one electron reduced, semiquinone (SQ) state. In the aerobic steady state of respiration the semi-ubiquinone anion has been observed and studied in detail. Two distinct protein-associated fast and slow relaxing, SQ signals have been resolved which were designated SQNfand SQNs. This review covers a five decade personal journey through the field leading to a focus on the unresolved questions of the role of the SQ radicals and their possible part in proton pumping.

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Redox-induced activation of the proton pump in the respiratory complex I

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1503761112 ◽

2015 ◽

Vol 112 (37) ◽

pp. 11571-11576 ◽

Cited By ~ 66

Author(s):

Vivek Sharma ◽

Galina Belevich ◽

Ana P. Gamiz-Hernandez ◽

Tomasz Róg ◽

Ilpo Vattulainen ◽

...

Keyword(s):

Proton Pump ◽

Conformational Changes ◽

Complex I ◽

Large Scale ◽

Reductase Activity ◽

Proton Pumping ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Respiratory Chains ◽

Dynamics Simulations

Complex I functions as a redox-linked proton pump in the respiratory chains of mitochondria and bacteria, driven by the reduction of quinone (Q) by NADH. Remarkably, the distance between the Q reduction site and the most distant proton channels extends nearly 200 Å. To elucidate the molecular origin of this long-range coupling, we apply a combination of large-scale molecular simulations and a site-directed mutagenesis experiment of a key residue. In hybrid quantum mechanics/molecular mechanics simulations, we observe that reduction of Q is coupled to its local protonation by the His-38/Asp-139 ion pair and Tyr-87 of subunit Nqo4. Atomistic classical molecular dynamics simulations further suggest that formation of quinol (QH2) triggers rapid dissociation of the anionic Asp-139 toward the membrane domain that couples to conformational changes in a network of conserved charged residues. Site-directed mutagenesis data confirm the importance of Asp-139; upon mutation to asparagine the Q reductase activity is inhibited by 75%. The current results, together with earlier biochemical data, suggest that the proton pumping in complex I is activated by a unique combination of electrostatic and conformational transitions.

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Mitochondrial type II NADH dehydrogenase of Plasmodium falciparum is dispensable and not the functional target of putative NDH2 quinolone inhibitors

10.1101/436881 ◽

2018 ◽

Author(s):

Hangjun Ke ◽

Suresh M. Ganesan ◽

Swati Dass ◽

Joanne M. Morrisey ◽

Sovitj Pou ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Antimalarial Drug ◽

Drug Target ◽

Nadh Dehydrogenase ◽

Antimalarial Activity ◽

Reductase Activity ◽

Proton Pumping ◽

Type Ii ◽

Long Time ◽

Specific Inhibitors

AbstractThe battle against malaria has been substantially impeded by the recurrence of drug resistance in Plasmodium falciparum, the deadliest human malaria parasite. To counter the problem, novel antimalarial drugs are urgently needed, especially those that target unique pathways of the parasite, since they are less likely to have side effects. The mitochondrial type II NADH dehydrogenase of P. falciparum, PfNDH2 (PF3D7_0915000), has been considered a good prospective antimalarial drug target for over a decade, since malaria parasites lack the conventional multi-subunit NADH dehydrogenase, or Complex I, present in the mammalian mitochondrial electron transport chain (mtETC). Instead, Plasmodium parasites contain a single subunit NDH2, which lacks proton pumping activity and is absent in humans. A significant amount of effort has been expended to develop PfNDH2 specific inhibitors, yet the essentiality of PfNDH2 has not been convincingly verified. Herein, we knocked out PfNDH2 in P. falciparum via a CRISPR/Cas9 mediated approach. Deletion of PfNDH2 does not alter the parasite’s susceptibility to multiple mtETC inhibitors, including atovaquone and ELQ-300. We also show that the antimalarial activity of the fungal NDH2 inhibitor HDQ and its new derivative CK-2-68 is due to inhibition of the parasite cytochrome bc1 complex rather than PfNDH2. These compounds directly inhibit the ubiquinol-cytochrome c reductase activity of the malarial bc1 complex. Our results call into question the validity of PfNDH2 as an antimalarial drug target.ImportanceFor a long time, PfNDH2 has been considered an attractive antimalarial drug target. However, the conclusion that PfNDH2 is essential was based on preliminary and incomplete data. Here we generate a PfNDH2 KO (knockout) parasite in the blood stages of Plasmodium falciparum, showing that the gene is not essential. We also show that previously reported PfNDH2-specific inhibitors kill the parasites primarily via targeting the cytochrome bc1 complex, not PfNDH2. Overall, we provide genetic and biochemical data that help to resolve a long-debated issue in the field regarding the potential of PfNDH2 as an antimalarial drug target.

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A yeast-based drug discovery platform to identify Plasmodium falciparum type II NADH dehydrogenase inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02470-20 ◽

2021 ◽

Author(s):

Yu Cao ◽

Chen Sun ◽

Han Wen ◽

Mengfei Wang ◽

Pan Zhu ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Complex I ◽

Nadh Dehydrogenase ◽

Specific Inhibitor ◽

Type I ◽

Type Ii ◽

Protein Activity ◽

Asexual Blood Stage

Conventional methods utilizing in vitro protein activity assay or in vivo parasite survival to screen for malaria inhibitors suffer from high experimental background and/or inconvenience. Here we introduce a yeast-based system to facilitate chemical screen for specific protein or pathway inhibitors. The platform comprises several isogeneic Pichia strains that only differ in the target of interest, so that a compound which inhibits one strain but not the other is implicated in working specifically against the target. We used Plasmodium falciparum NDH2(PfNDH2), a type II NADH dehydrogenase, as a proof of principle to show how well this works. Three isogenic Pichia strains harboring respectively exogeneously introduced PfNDH2, its own complex I (a type I NADH dehydrogenase), and PfNDH2 with its own complex I were constructed. In a pilot screen of more than2000 compounds, we identified a highly specific inhibitor that acts on PfNDH2. This compound poorly inhibit the parasites at the asexual blood stage, however, is highly effective in repressing oocyst maturation in the mosquito stage. Our results demonstrate that the yeast cell based screen platform is feasible, efficient, economical and with very low background noise. Similar strategies could be extended to the functional screen for interacting molecules of other targets.

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Respiratory Chain Analysis of Zymomonas mobilis Mutants Producing High Levels of Ethanol

Applied and Environmental Microbiology ◽

10.1128/aem.00733-12 ◽

2012 ◽

Vol 78 (16) ◽

pp. 5622-5629 ◽

Cited By ~ 27

Author(s):

Takeshi Hayashi ◽

Tsuyoshi Kato ◽

Kensuke Furukawa

Keyword(s):

Ethanol Production ◽

Dehydrogenase Activity ◽

Respiratory Chain ◽

Zymomonas Mobilis ◽

Nadh Dehydrogenase ◽

Nucleotide Sequence Analysis ◽

Wild Type ◽

Aerobic Growth ◽

Content Type ◽

Respiratory Chains

ABSTRACTWe previously isolated respiratory-deficient mutant (RDM) strains ofZymomonas mobilis, which exhibited greater growth and enhanced ethanol production under aerobic conditions. These RDM strains also acquired thermotolerance. Morphologically, the cells of all RDM strains were shorter compared to the wild-type strain. We investigated the respiratory chains of these RDM strains and found that some RDM strains lost NADH dehydrogenase activity, whereas others exhibited reduced cytochromebd-type ubiquinol oxidase or ubiquinol peroxidase activities. Complementation experiments restored the wild-type phenotype. Some RDM strains seem to have certain mutations other than the corresponding respiratory chain components. RDM strains with deficient NADH dehydrogenase activity displayed the greatest amount of aerobic growth, enhanced ethanol production, and thermotolerance. Nucleotide sequence analysis revealed that all NADH dehydrogenase-deficient strains were mutated within thendhgene, which includes insertion, deletion, or frameshift. These results suggested that the loss of NADH dehydrogenase activity permits the acquisition of higher aerobic growth, enhanced ethanol production, and thermotolerance in this industrially important strain.

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The respiratory-chain NADH dehydrogenase (complex I) of mitochondria

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1991.tb15945.x ◽

1991 ◽

Vol 197 (3) ◽

pp. 563-576 ◽

Cited By ~ 335

Author(s):

Hanns WEISS ◽

Thorsten FRIEDRICH ◽

Gotz HOFHAUS ◽

Dagmar PREIS

Keyword(s):

Respiratory Chain ◽

Complex I ◽

Nadh Dehydrogenase ◽

Dehydrogenase Complex

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Novel mitochondrial complex I-inhibiting peptides restrain NADH dehydrogenase activity

Scientific Reports ◽

10.1038/s41598-019-50114-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Yao-Peng Xue ◽

Mou-Chieh Kao ◽

Chung-Yu Lan

Keyword(s):

Antimicrobial Peptides ◽

Complex I ◽

Fungal Pathogens ◽

Nadh Dehydrogenase ◽

Mitochondrial Complex I ◽

Mitochondrial Complex ◽

Human Fungal Pathogen ◽

New Class ◽

Transport Chain ◽

Nadh Dehydrogenases

Abstract The emergence of drug-resistant fungal pathogens is becoming increasingly serious due to overuse of antifungals. Antimicrobial peptides have potent activity against a broad spectrum of pathogens, including fungi, and are considered a potential new class of antifungals. In this study, we examined the activities of the newly designed peptides P-113Du and P-113Tri, together with their parental peptide P-113, against the human fungal pathogen Candida albicans. The results showed that these peptides inhibit mitochondrial complex I, specifically NADH dehydrogenase, of the electron transport chain. Moreover, P-113Du and P-113Tri also block alternative NADH dehydrogenases. Currently, most inhibitors of the mitochondrial complex I are small molecules or artificially-designed antibodies. Here, we demonstrated novel functions of antimicrobial peptides in inhibiting the mitochondrial complex I of C. albicans, providing insight in the development of new antifungal agents.

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