Alternative NADH Research Articles

Identification of new inhibitors for alternative NADH dehydrogenase (NDH-II)

In bacterial membranes and plant, fungus and protist mitochondria, NADH dehydrogenase (NDH-II) serves as an alternative NADH : quinone reductase, a non-proton-pumping single-subunit enzyme bound to the membrane surface. Because NDH-II is absent in mammalian mitochondria, it is a promising target for new antibiotics. However, inhibitors for NDH-II are rare and unspecific. Taking advantage of the simple organization of the respiratory chain in Gluconobacter oxydans, we carried out screening of natural compounds and identified scopafungin and gramicidin S as inhibitors for G. oxydans NDH-II. Further, we examined their effects on Mycobacterium smegmatis and Plasmodium yoelii NDH-II as model pathogen enzymes.

Mitochondrial Dehydrogenases in the Aerobic Respiratory Chain of the Rodent Malaria Parasite Plasmodium yoelii yoelii

In the intraerythrocytic stages of malaria parasites, mitochondria lack obvious cristae and are assumed to derive energy through glycolysis. For understanding of parasite energy metabolism in mammalian hosts, we isolated rodent malaria mitochondria from Plasmodium yoelii yoelii grown in mice. As potential targets for antiplasmodial agents, we characterized two respiratory dehydrogenases, succinate:ubiquinone reductase (complex II) and alternative NADH dehydrogenase (NDH-II), which is absent in mammalian mitochondria. We found that P. y. yoelii complex II was a four-subunit enzyme and that kinetic properties were similar to those of mammalian enzymes, indicating that the Plasmodium complex II is favourable in catalysing the forward reaction of tricarboxylic acid cycle. Notably, Plasmodium complex II showed IC(50) value for atpenin A5 three-order of magnitudes higher than those of mammalian enzymes. Divergence of protist membrane anchor subunits from eukaryotic orthologs likely affects the inhibitor resistance. Kinetic properties and sensitivity to 2-heptyl-4-hydroxyquinoline-N-oxide and aurachin C of NADH: ubiquinone reductase activity of Plasmodium NDH-II were similar to those of plant and fungus enzymes but it can oxidize NADPH and deamino-NADH. Our findings are consistent with the notion that rodent malaria mitochondria are fully capable of oxidative phosphorylation and that these mitochondrial enzymes are potential targets for new antiplasmodials.

In Yarrowia lipolytica mitochondria, the alternative NADH dehydrogenase interacts specifically with the cytochrome complexes of the classic respiratory pathway

In Yarrowia lipolytica, mitochondria contain a branched respiratory chain constituted by the classic complexes I, II, III and IV, plus an alternative external NADH dehydrogenase (NDH2e) and an alternative oxidase (AOX). The alternative enzymes are peripheral, single-subunit oxido-reductases that do not pump protons. Thus, the oxidation of NADH via NDH2e-ubiquinone-AOX would not contribute to the proton-motive force. The futile oxidation of NADH may be prevented if either NDH2e or AOX bind to the classic complexes, channelling electrons. By oxymetry, it was observed that the electrons from complex I reached both cytochrome oxidase and AOX. In contrast, NDH2e-derived electrons were specifically channelled/directed to the cytochrome complexes. In addition, the presence of respiratory supercomplexes plus the interaction of NDH2e with these complexes was evaluated using blue native PAGE, clear native PAGE, in-gel activities, immunoblotting, mass spectrometry, and N-terminal sequencing. NDH2e (but not the redirected matrix NDH2i from a mutant strain, Δnubm) was detected in association with the cytochromic pathway; this interaction seems to be strong, as it was not disrupted by laurylmaltoside. The association of NDH2e to complex IV was also suggested when both enzymes coeluted from an ion exchange chromatography column. In Y. lipolytica mitochondria the cytochrome complexes probably associate into supercomplexes; those were assigned as follows: I–III 2, I–IV, I–III 2–IV 4, III 2–IV, III 2–IV 2, IV 2 and V 2. The molecular masses of all the complexes and putative supercomplexes detected in Y. lipolytica were estimated by comparison with the bovine mitochondrial complexes. To our knowledge, this is the first evidence of supercomplex formation in Y. lipolytica mitochondria and also, the first description of a specific association between an alternative NADH dehydrogenase and the classic cytochrome pathway.

Mitochondrial function in the yeast form of the pathogenic fungus Paracoccidioides brasiliensis

Differences between the respiratory chain of the fungus Paracoccidioides brasiliensis and its mammalian host are reported. Respiration, membrane potential, and oxidative phosphorylation in mitochondria from P. brasiliensis spheroplasts were evaluated in situ, and the presence of a complete (Complex I-V) functional respiratory chain was demonstrated. In succinate-energized mitochondria, ADP induced a transition from resting to phosphorylating respiration. The presence of an alternative NADH-ubiquinone oxidoreductase was indicated by: (i) the ability to oxidize exogenous NADH and (ii) the lack of sensitivity to rotenone and presence of sensitivity to flavone. Malate/NAD(+)-supported respiration suggested the presence of either a mitochondrial pyridine transporter or a glyoxylate pathway contributing to NADH and/or succinate production. Partial sensitivity of NADH/succinate-supported respiration to antimycin A and cyanide, as well as sensitivity to benzohydroxamic acids, suggested the presence of an alternative oxidase in the yeast form of the fungus. An increase in activity and gene expression of the alternative NADH dehydrogenase throughout the yeast's exponential growth phase was observed. This increase was coupled with a decrease in Complex I activity and gene expression of its subunit 6. These results support the existence of alternative respiratory chain pathways in addition to Complex I, as well as the utilization of NADH-linked substrates by P. brasiliensis. These specific components of the respiratory chain could be useful for further research and development of pharmacological agents against the fungus.

The iron–sulphur protein Ind1 is required for effective complex I assembly

NADH:ubiquinone oxidoreductase (complex I) of the mitochondrial inner membrane is a multi-subunit protein complex containing eight iron-sulphur (Fe-S) clusters. Little is known about the assembly of complex I and its Fe-S clusters. Here, we report the identification of a mitochondrial protein with a nucleotide-binding domain, named Ind1, that is required specifically for the effective assembly of complex I. Deletion of the IND1 open reading frame in the yeast Yarrowia lipolytica carrying an internal alternative NADH dehydrogenase resulted in slower growth and strongly decreased complex I activity, whereas the activities of other mitochondrial Fe-S enzymes, including aconitase and succinate dehydrogenase, were not affected. Two-dimensional gel electrophoresis, in vitro activity tests and electron paramagnetic resonance signals of Fe-S clusters showed that only a minor fraction (approximately 20%) of complex I was assembled in the ind1 deletion mutant. Using in vivo and in vitro approaches, we found that Ind1 can bind a [4Fe-4S] cluster that was readily transferred to an acceptor Fe-S protein. Our data suggest that Ind1 facilitates the assembly of Fe-S cofactors and subunits of complex I.

Protection by the NDI1 Gene against Neurodegeneration in a Rotenone Rat Model of Parkinson's Disease

It is widely recognized that mitochondrial dysfunction, most notably defects in the NADH-quinone oxidoreductase (complex I), is closely related to the etiology of sporadic Parkinson's disease (PD). In fact, rotenone, a complex I inhibitor, has been used for establishing PD models both in vitro and in vivo. A rat model with chronic rotenone exposure seems to reproduce pathophysiological conditions of PD more closely than acute mouse models as manifested by neuronal cell death in the substantia nigra and Lewy body-like cytosolic aggregations. Using the rotenone rat model, we investigated the protective effects of alternative NADH dehydrogenase (Ndi1) which we previously demonstrated to act as a replacement for complex I both in vitro and in vivo. A single, unilateral injection of recombinant adeno-associated virus carrying the NDI1 gene into the vicinity of the substantia nigra resulted in expression of the Ndi1 protein in the entire substantia nigra of that side. It was clear that the introduction of the Ndi1 protein in the substantia nigra rendered resistance to the deleterious effects caused by rotenone exposure as assessed by the levels of tyrosine hydroxylase and dopamine. The presence of the Ndi1 protein also prevented cell death and oxidative damage to DNA in dopaminergic neurons observed in rotenone-treated rats. Unilateral protection also led to uni-directional rotation of the rotenone-exposed rats in the behavioral test. The present study shows, for the first time, the powerful neuroprotective effect offered by the Ndi1 enzyme in a rotenone rat model of PD.

Lambda Red-Mediated Mutagenesis and Efficient Large Scale Affinity Purification of the Escherichia coli NADH:Ubiquinone Oxidoreductase (Complex I)

The proton-pumping NADH:ubiquinone oxidoreductase, the respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. The Escherichia coli complex I consists of 13 different subunits named NuoA-N (from NADH:ubiquinone oxidoreductase), that are coded by the genes of the nuo-operon. Genetic manipulation of the operon is difficult due to its enormous size. The enzymatic activity of variants is obscured by an alternative NADH dehydrogenase, and purification of the variants is hampered by their instability. To overcome these problems the entire E. coli nuo-operon was cloned and placed under control of the l-arabinose inducible promoter ParaBAD. The exposed N-terminus of subunit NuoF was chosen for engineering the complex with a hexahistidine-tag by lambda-Red-mediated recombineering. Overproduction of the complex from this construct in a strain which is devoid of any membrane-bound NADH dehydrogenase led to the assembly of a catalytically active complex causing the entire NADH oxidase activity of the cytoplasmic membranes. After solubilization with dodecyl maltoside the engineered complex binds to a Ni2+-iminodiacetic acid matrix allowing the purification of approximately 11 mg of complex I from 25 g of cells. The preparation is pure and monodisperse and comprises all known subunits and cofactors. It contains more lipids than earlier preparations due to the gentle and fast purification procedure. After reconstitution in proteoliposomes it couples the electron transfer with proton translocation in an inhibitor sensitive manner, thus meeting all prerequisites for structural and functional studies.

Expression of Ndi1p, an alternative NADH:ubiquinone oxidoreductase, increases mitochondrial membrane potential in a C. elegans model of mitochondrial disease

The NADH:ubiquinone oxidoreductase or complex I of the mitochondrial respiratory chain is an intricate enzyme with a vital role in energy metabolism. Mutations affecting complex I can affect at least three processes; they can impair the oxidation of NADH, reduce the enzyme's ability to pump protons for the generation of a mitochondrial membrane potential and increase the production of damaging reactive oxygen species. We have previously developed a nematode model of complex I-associated mitochondrial dysfunction that features hallmark characteristics of mitochondrial disease, such as lactic acidosis and decreased respiration. We have expressed the Saccharomyces cerevisiae NDI1 gene, which encodes a single subunit NADH dehydrogenase, in a strain of Caenorhabditis elegans with an impaired complex I. Expression of Ndi1p produces marked improvements in animal fitness and reproduction, increases respiration rates and restores mitochondrial membrane potential to wild type levels. Ndi1p functionally integrates into the nematode respiratory chain and mitigates the deleterious effects of a complex I deficit. However, we have also shown that Ndi1p cannot substitute for the absence of complex I. Nevertheless, the yeast Ndi1p should be considered as a candidate for gene therapy in human diseases involving complex I.

Growth inhibition of Toxoplasma gondii and Plasmodium falciparum by nanomolar concentrations of 1-hydroxy-2-dodecyl-4(1H)quinolone, a high-affinity inhibitor of alternative (type II) NADH dehydrogenases.

Both apicomplexan parasites Toxoplasma gondii and Plasmodium falciparum lack type I NADH dehydrogenases (complex I) but instead carry alternative (type II) NADH dehydrogenases, which are absent in mammalian cells and are thus considered promising antimicrobial drug targets. The quinolone-like compound 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ) was recently described as a high-affinity inhibitor of fungal alternative NADH dehydrogenases in enzymatic assays, probably by interfering with the ubiquinol binding site of the enzyme. We describe here that HDQ effectively inhibits the replication rates of P. falciparum and T. gondii in tissue culture. The 50% inhibitory concentration (IC50) of HDQ for T. gondii was determined to be 2.4+/-0.3 nM with a growth assay based on vacuole sizes and 3.7+/-1.4 nM with a growth assay based on beta-galactosidase activity. Quantification of the P. falciparum replication rate using a fluorometric assay revealed an IC50 of 14.0+/-1.9 nM. An important feature of the HDQ structure is the length of the alkyl side chain at position 2. Derivatives with alkyl side chains of C6, C8, C12 (HDQ), and C14 all displayed excellent anti-T. gondii activity, while a C5 derivative completely failed to inhibit parasite replication. A combined treatment of T. gondii-infected cells with HDQ and the antimalarial agent atovaquone, which blocks the ubiquinol oxidation site of cytochrome b in complex III, resulted in synergism, with a calculated fractional inhibitory concentration of 0.16 nM. Interference of the mitochondrial ubiquinone/ubiquinol cycle at two different locations thus appears to be a highly effective strategy for inhibiting parasite replication. HDQ and its derivatives, particularly in combination with atovaquone, represent promising compounds with a high potential for antimalarial and antitoxoplasmal therapy.

Substrate‐inducible versions of internal alternative NADH : ubiquinone oxidoreductase from Yarrowia lipolytica

In standard laboratory strains of the obligate aerobic yeast Yarrowia lipolytica, respiratory chain complex I (proton-translocating NADH : ubiquinone oxidoreductase) is an essential enzyme, since alternative NADH dehydrogenase activity is located exclusively at the external face of the mitochondrial inner membrane. Deletions and other loss-of-function mutations in genes for nuclear coded subunits of complex I can be obtained only when an internal version of the latter enzyme, termed NDH2i, is introduced. In contrast to recent findings with Neurospora crassa, external alternative NADH dehydrogenase activity is dispensable in complex I deletion strains of Y. lipolytica. We used regulable promoters to create strains which express internal alternative NADH dehydrogenase in a substrate-dependent manner. The ability to switch between complex I-dependent and -independent mode of growth simply by changing the carbon source is an important prerequisite for screens for both loss-of-function and inhibitor resistance mutation. The isocitrate lyase promoter (pICL1), in combination with a NDH2i allele that results in reduced expression and activity, was most promising. In the presence of complex I inhibitors, this construct allowed growth on acetate, but not on glucose minimal media. A somewhat higher background was observed with the acyl-CoA oxidase 2 (pPOX2) promoter on glucose minimal media.

Agrobacterium tumefaciens type II NADH dehydrogenase

Type II NADH dehydrogenases (NDH-2) are monomeric enzymes that catalyse quinone reduction and allow electrons to enter the respiratory chain in different organisms including higher plant mitochondria, bacteria and yeasts. In this study, an Agrobacterium tumefaciens gene encoding a putative alternative NADH dehydrogenase (AtuNDH-2) was isolated and expressed in Escherichia coli as a (His)6-tagged protein. The purified 46 kDa protein contains FAD as a prosthetic group and oxidizes both NADH and NADPH with similar Vmax values, but with a much higher affinity for NADH than for NADPH. AtuNDH-2 complements the growth (on a minimal medium) of an E. coli mutant strain deficient in both NDH-1 and NDH-2, and is shown to supply electrons to the respiratory chain when incubated with bacterial membranes prepared from this mutant. By measuring photosystem II chlorophyll fluorescence on thylakoid membranes prepared from the green alga Chlamydomonas reinhardtii, we show that AtuNDH-2 is able to stimulate NADH-dependent reduction of the plastoquinone pool. We discuss the possibility of using heterologous expression of NDH-2 enzymes to improve nonphotochemical reduction of plastoquinones and H2 production in C. reinhardtii.

Functional characterization and target validation of alternative complex I of Plasmodium falciparum mitochondria.

This study reports on the first characterization of the alternative NADH:dehydrogenase (also known as alternative complex I or type II NADH:dehydrogenase) of the human malaria parasite Plasmodium falciparum, known as PfNDH2. PfNDH2 was shown to actively oxidize NADH in the presence of quinone electron acceptors CoQ(1) and decylubiquinone with an apparent K(m) for NADH of approximately 17 and 5 muM, respectively. The inhibitory profile of PfNDH2 revealed that the enzyme activity was insensitive to rotenone, consistent with recent genomic data indicating the absence of the canonical NADH:dehydrogenase enzyme. PfNDH2 activity was sensitive to diphenylene iodonium chloride and diphenyl iodonium chloride, known inhibitors of alternative NADH:dehydrogenases. Spatiotemporal confocal imaging of parasite mitochondria revealed that loss of PfNDH2 function provoked a collapse of mitochondrial transmembrane potential (Psi(m)), leading to parasite death. As with other alternative NADH:dehydrogenases, PfNDH2 lacks transmembrane domains in its protein structure, and therefore, it is proposed that this enzyme is not directly involved in mitochondrial transmembrane proton pumping. Rather, the enzyme provides reducing equivalents for downstream proton-pumping enzyme complexes. As inhibition of PfNDH2 leads to a depolarization of mitochondrial Psi(m), this enzyme is likely to be a critical component of the electron transport chain (ETC). This notion is further supported by proof-of-concept experiments revealing that targeting the ETC's Q-cycle by inhibition of both PfNDH2 and the bc(1) complex is highly synergistic. The potential of targeting PfNDH2 as a chemotherapeutic strategy for drug development is discussed.

Stress-induced co-expression of alternative respiratory chain components in Arabidopsis thaliana

Plant mitochondria contain non-phosphorylating bypasses of the respiratory chain, catalysed by the alternative oxidase (AOX) and alternative NADH dehydrogenases (NDH), as well as uncoupling (UCP) protein. Each of these components either circumvents or short-circuits proton translocation pathways, and each is encoded by a small gene family in Arabidopsis. Whole genome microarray experiments were performed with suspension cell cultures to examine the effects of various 3 h treatments designed to induce abiotic stress. The expression of over 60 genes encoding components of the classical, phosphorylating respiratory chain and tricarboxylic acid cycle remained largely constant when cells were subjected to a broad range of abiotic stresses, but expression of the alternative components responded differentially to the various treatments. In detailed time-course quantitative PCR analysis, specific members of both AOX and NDH gene families displayed coordinated responses to treatments. In particular, the co-expression of AOX1a and NDB2 observed under a number of treatments suggested co-regulation that may be directed by common sequence elements arranged hierarchically in the upstream promoter regions of these genes. A series of treatment sets were identified, representing the response of specific AOX and NDH genes to mitochondrial inhibition, plastid inhibition and abiotic stresses. These treatment sets emphasise the multiplicity of pathways affecting alternative electron transport components in plants.

HDQ (1-Hydroxy-2-dodecyl-4(1H)quinolone), a High Affinity Inhibitor for Mitochondrial Alternative NADH Dehydrogenase: EVIDENCE FOR A PING-PONG MECHANISM

Alternative NADH dehydrogenases (NADH:ubiquinone oxidoreductases) are single subunit respiratory chain enzymes found in plant and fungal mitochondria and in many bacteria. It is unclear how these peripheral membrane proteins interact with their hydrophobic substrate ubiquinone. Known inhibitors of alternative NADH dehydrogenases bind with rather low affinities. We have identified 1-hydroxy-2-dodecyl-4(1H)quinolone as a high affinity inhibitor of alternative NADH dehydrogenase from Yarrowia lipolytica. Using this compound, we have analyzed the bisubstrate and inhibition kinetics for NADH and decylubiquinone. We found that the kinetics of alternative NADH dehydrogenase follow a ping-pong mechanism. This suggests that NADH and the ubiquinone headgroup interact with the same binding pocket in an alternating fashion.

The nuclear genes encoding the internal ( KlNDI1) and external ( KlNDE1) alternative NAD(P)H:ubiquinone oxidoreductases of mitochondria from Kluyveromyces lactis

Cloning, sequence and functional analyses of the Kluyveromyces lactis genes KlNDI1 and KlNDE1 are reported. These genes encode for proteins with high homology to the mitochondrial internal (Ndi1p) and external (Nde1p) alternative NADH:ubiquinone oxidoreductases from Saccharomyces cerevisiae and complement the respective mutations. Analysis of KlNDI1 transcriptional regulation showed that expression of this gene is lower in 2% glucose than in 0.5% glucose or non-fermentable carbon sources. Beta-galactosidase activity values, shown by lacZ fusions of KlNDI1 promoter deletions, suggested that two Adr1p binding sites mediate this carbon source regulation of KlNDI1. The expression of the KlNDE1 gene in S. cerevisiae mutant strains and measurement of respiration with isolated mitochondria showed that the protein encoded by KlNDE1 oxidizes NADPH, this being an important difference with respect to the conventional yeast S. cerevisiae. Moreover, Northern blot experiments using a phosphoglucose isomerase mutant showed that KlNDE1 gene transcription increases with glucose metabolism through the pentose phosphate pathway.

Functional properties of the alternative NADH:ubiquinone oxidoreductase from E. coli through comparative 3-D modelling

The alternative NADH:ubiquinone oxidoreductase (NDH-2) from Escherichia coli is a membrane protein playing a prominent role in respiration by linking the reduction of NADH to the quinone pool. Remote sequence similarity reveals an evolutionary relation between alternative NADH:quinone oxidoreductases and the SCOP-family “FAD/NAD-linked reductases”. We have created a structural model for NDH-2 from E. coli through comparative modelling onto a template from this family. Combined analysis of our model and sequence conservation allowed us to include the cofactor FAD and the substrate NADH in atomic detail. Furthermore, we propose the most plausible orientation of NDH-2 relative to the membrane and specify a region of the protein potentially involved in ubiquinone binding.

In situ evidence of an alternative oxidase and an uncoupling protein in the respiratory chain of Aspergillus fumigatus

Aspergillus fumigatus is an unusual pathogen in immunocompetent individuals; its incidence has increased in the last decades in patients immunocompromised, like those with chronic granulomatosis disease and AIDS. The aim of this study was to identify differences between the respiratory chain of host and the fungus planning to use the later as a pharmacological target. We evaluated respiration, membrane potential and oxidative phosphorylation of mitochondria of the spheroplasts of A. fumigatus in situ, after permeabilization with digitonin. Firstly, a functional respiratory chain (complex I–V) was demonstrated: adenosine 5′-diphosphate (ADP) induced an oligomycin-sensitive transition from resting to phophorylating respiration in the presence of the oxidizable substrates malate, glutamate, α-ketoglutarate, pyruvate, dihydroorotate, succinate, N,N,N′ ,N′-tetramethyl- p-phenylenediamine (TMPD) and exogenous NADH. In addition, the ability of the fungus to oxidize exogenous NADH, as well as the insensitivity of its respiration to rotenone, in association with the sensitivity to flavone, indicate the presence of an alternative NADH–ubiquinone oxidoreductase; the partial sensitivity of respiration to antimycin A and cyanide, in association with the sensitivity to benzohydroxamic acid, indicates the presence of an alternative oxidase. The fatty acid-uncoupled respiration was partly reversed by bovine serum albumin (BSA) and guanosine 5′-triphosphate (GTP) and was insensitive to either carboxyatractyloside or ADP. These results, together with evidences obtained using antibodies raised against uncoupling protein (UCP) from potato, indicate in addition, the presence of an uncoupling protein in the respiratory chain of A. fumigatus.

The internal alternative NADH dehydrogenase of Neurospora crassa mitochondria.

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.

External alternative NADH dehydrogenase of Saccharomyces cerevisiae: a potential source of superoxide.

Three rotenone-insensitive NADH dehydrogenases are present in the mitochondria of yeast Saccharomyces cerevisiae, which lack complex I. To elucidate the functions of these enzymes, superoxide production was determined in yeast mitochondria. The low levels of hydrogen peroxide (0.10 to 0.18 nmol/min/mg) produced in mitochondria incubated with succinate, malate, or NADH were stimulated 9-fold by antimycin A. Myxothiazol and stigmatellin blocked completely hydrogen peroxide formation with succinate or malate, indicating that the cytochrome bc(1) complex is the source of superoxide; however, these inhibitors only inhibited 46% hydrogen peroxide formation with NADH as substrate. Diphenyliodonium inhibited hydrogen peroxide formation (with NADH as substrate) by 64%. Superoxide formation, determined by EPR and acetylated cytochrome c reduction in mitochondria was stimulated by antimycin A, and partially inhibited by myxothiazol and stigmatellin. Proteinase K digestion of mitoplasts reduced 95% NADH dehydrogenase activity with a similar inhibition of superoxide production. Mild detergent treatment of the proteinase-treated mitoplasts resulted in an increase in NADH dehydrogenase activity due to the oxidation of exogenous NADH by the internal NADH dehydrogenase; however, little increase in superoxide production was observed. These results suggest that the external NADH dehydrogenase is a potential source of superoxide in S. cerevisiae mitochondria.

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

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.