Composition and physiological function of the chloroplast NADH dehydrogenase‐like complex in Marchantia polymorpha

The chloroplast NADH dehydrogenase‐like (NDH) complex mediates cyclic electron transport and chloro‐respiration and consists of five sub‐omplexes, which in angiosperms further associate with photosystem I (PSI) to form a super‐complex. In Marchantia polymorpha, 11 plastid‐encoded subunits and all the nuclear‐encoded subunits of the A, B, membrane and ferredoxin‐binding sub‐complexes are conserved. However, it is unlikely that the genome of this liverwort encodes Lhca5 and Lhca6, both of which mediate NDH–PSI super‐complex formation. It is also unlikely that the subunits of the lumen sub‐complex, PnsL1–L4, are encoded by the genome. Consistent with this in silico prediction, the results of blue‐native gel electrophoresis showed that NDH subunits were detected in a protein complex with lower molecular mass in Marchantia than the NDH–PSI super‐complex in Arabidopsis. Using the plastid transformation technique, we knocked out the ndhB gene in Marchantia. Although the wild‐type genome copies were completely segregated out, the ΔndhB lines grew like the wild‐type photoautotrophically. A post‐illumination transient increase in chlorophyll fluorescence, which reflects NDH activity in vivo in angiosperms, was absent in the thalli of the ΔndhB lines. In ruptured chloroplasts, antimycin A‐insensitive, and ferredoxin‐dependent plastoquinone reduction was impaired, suggesting that chloroplast NDH mediates similar electron transport in Marchantia and Arabidopsis, despite its possible difference in structure. As in angiosperms, linear electron transport was not strongly affected in the ΔndhB lines. However, the plastoquinone pool was slightly more reduced at low light intensity, suggesting that chloroplast NDH functions in redox balancing of the inter system, especially under low light conditions.     

Oxidation of NADH by plant mitochondria: Kinetics and effects of calcium ions

The kinetics of NADH oxidation by the outer membrane electron transport system of intact beetroot (Beta vulgaris L.) mitochondria were investigated. Very different values for V_max and the K_m for NADH were obtained when either antimycin A-insensitive NADH-cytochrome c activity (V_max= 31 ± 2.5 nmol cytochrome c (mg protein)^?1 min^?1; K_m= 3.1 ± 0.8 μM) or antimycin A-insensitive NADH-ferricyanide activity (V_max= 1.7 ± 0.7 μmol ferricyanide (mg protein)^?1 min^?1; K_m= 83 ± 20 μM) were measured. As ferricyanide is believed to accept electrons closer to the NADH binding site than cytochrome c, it was concluded that 83 ± 20 μM NADH represented a more accurate estimate of the binding affinity of the outer membrane dehydrogenase for NADH. The low K_m determined with NADH-cytochrome c activity may be due to a limitation in electron flow through the components of the outer membrane electron transport chain. The K_m for NADH of the externally-facing inner membrane NADH dehydrogenase of pea leaf (Pisum sativum L. cv. Massey Gem) mitochondria was 26.7 ± 4.3 μM when oxygen was the electron acceptor. At an NADH concentration at which the inner membrane dehydrogenase should predominate, the Ca²⁺ chelator, ethyleneglycol-(β-aminoethylether)-N,N,-tetraacetic acid (EGTA), inhibited the oxidation of NADH through to oxygen and to the ubiquinone-10 analogues, duroquinone and ubiquinone-1, but had no effect on the antimycin A-insensitive ferricyanide reduction. It is concluded that the site of action of Ca²⁺ involves the interaction of the enzyme with ubiquinone and not with NADH.     

Identification of a Cytosolically Directed NADH Dehydrogenase in Mitochondria of Saccharomyces cerevisiae

The reoxidation of NADH generated in reactions within the mitochondrial matrix of Saccharomyces cerevisiae is catalyzed by an NADH dehydrogenase designated Ndi1p (C. A. M. Marres, S. de Vries, and L. A. Grivell, Eur. J. Biochem. 195:857–862, 1991). Gene disruption analysis was used to examine possible metabolic functions of two proteins encoded by open reading frames having significant primary sequence similarity to Ndi1p. Disruption of the gene designated NDH1 results in a threefold reduction in total mitochondrial NADH dehydrogenase activity in cells cultivated with glucose and in a fourfold reduction in the respiration of isolated mitochondria with NADH as the substrate. Thus, Ndh1p appears to be a mitochondrial dehydrogenase capable of using exogenous NADH. Disruption of a closely related gene designated NDH2 has no effect on these properties. Growth phenotype analyses suggest that the external NADH dehydrogenase activity of Ndh1p is important for optimum cellular growth with a number of nonfermentable carbon sources, including ethanol. Codisruption of NDH1 and genes encoding malate dehydrogenases essentially eliminates growth on nonfermentable carbon sources, suggesting that the external mitochondrial NADH dehydrogenase and the malate-aspartate shuttle may both contribute to reoxidation of cytosolic NADH under these growth conditions.     

Structural analysis and molybdenum-dependent expression of the pAO1-encoded nicotine dehydrogenase genes of Arthrobacter nicotinovorans

The genes of nicotine dehydrogenase (NDH) were identified, cloned and sequenced from the catabolic plasmid pA01 of Arthrobacter nicotinovorans. In immediate proximity to this gene cluster is the beginning of the 6-hydroxy-L-niotine oxidase (6-HLNO) gene. NDH is composed of three subunits (A, B and C) of M_r 30011, 14924 and 87677. It belongs to a family of bacterial hydroxylases with a similar subunit structure; they have molybdopterin dinucleotide, FAD and Fe-S clusters as cofactors. Here the first complete primary structure of a bacterial hydroxylase is provided. Sequence alignments of each of the NDH subunits show similarities to the sequences of eukaryotic xanthine dehydrogenase (XDH) but not to other known molybdenum-containing bacterial enzymes. Based on alignment with XDH it is inferred that the smallest subunit (NDHB) carries an iron-sulphur cluster, that the middle-sized subunit (NDHA) binds FAD, and that the largest NDH subunit (NDHC) corresponds to the molybdopterin-binding domain of XDH. Expression of both the ndh and the 6-hlno genes required the presence of nicotine and molybdenum in the culture medium. Tungsten inhibited enzyme activity but not the synthesis of the enzyme protein. The enzyme was found in A. nicotinovorans cells in a soluble form and in a membrane-associated form. In the presence of tungsten the fraction of membrane-associated NDH increased.     

The NdhV subunit is required to stabilize the chloroplast NADH dehydrogenase‐like complex in Arabidopsis

The chloroplast NADH dehydrogenase‐like (NDH) complex is involved in cyclic electron transport around photosystem I (PSI) and chlororespiration. Although the NDH complex was discovered more than 20 years ago, its low abundance and fragile nature render it recalcitrant to analysis, and it is thought that some of its subunits remain to be identified. Here, we identified the NDH subunit NdhV that readily disassociates from the NDH complex in the presence of detergent, salt and alkaline solutions. The Arabidopsis ndhv mutant is partially defective in the accumulation of NDH subcomplex A (SubA) and SubE, resulting in impaired NDH activity. NdhV was mainly detected in the wild‐type thylakoid membrane, and its accumulation in thylakoids strictly depended on the presence of the NDH complex. Quantitative immunoblot analysis revealed that NdhV and NdhN occur at close to equimolar concentrations. Furthermore, several NDH subunits were co‐immunopurified with NdhV using a combination of chemical crosslinking and an affinity chromatography assay. These data indicate that NdhV is an intrinsic subunit of NDH. We found that NdhV did not directly affect NDH activity, but that NDH SubA and SubE were more rapidly degraded in ndhv than in the wild type under high‐light treatment. We propose that NdhV is an NDH subunit that stabilizes this complex, especially under high‐light conditions.     

Malate Oxidation in Plant Mitochondria via Malic Enzyme and the Cyanide-insensitive Electron Transport Pathway

Malate oxidation in plant mitochondria proceeds through the activities of two enzymes: a malate dehydrogenase and a NAD⁺-dependent malic enzyme. In cauliflower, mitochondria malate oxidation via malate dehydrogenase is rotenone- and cyanide-sensitive. Addition of exogenous NAD⁺ stimulates the oxidation of malate via malic enzyme and generates an electron flux that is both rotenone- and cyanide-insensitive. The same effects of exogenous NAD⁺ are also observed with highly cyanide-sensitive mitochondria from white potato tubers or with mitochondria from spinach leaves. Both enzymes are located in the matrix, but some experimental data also suggest that part of malate dehydrogenase activity is also present outside the matrix compartment (adsorbed cytosolic malate dehydrogenase?). It is concluded that malic enzyme and a specific pool of NAD⁺/NADH are connected to the cyanide-insensitive alternative pathway by a specific rotenone-insensitive NADH dehydrogenase located on the inner face of the inner membrane. Similarly, malate dehydrogenase and another specific pool of NAD⁺/NADH are connected to the cyanide- (and antimycin-) sensitive pathway by a rotenone-sensitive NADH dehydrogenase located on the inner face of the inner membrane. A general scheme of electron transport in plant mitochondria for the oxidation of malate and NADH can be given, assuming that different pools of ubiquinone act as a branch point between various dehydrogenases, the cyanide-sensitive cytochrome pathway and the cyanide-insensitive alternative pathway.     

Specific substitutions of light‐harvesting complex I proteins associated with photosystem I are required for supercomplex formation with chloroplast NADH dehydrogenase‐like complex

In Arabidopsis, the chloroplast NADH‐dehydrogenase‐like (NDH) complex is sandwiched between two copies of photosystem I (PSI) supercomplex, consisting of a PSI core and four light‐harvesting complex I (LHCI) proteins (PSI‐LHCI) to form the NDH–PSI supercomplex. Two minor LHCI proteins, Lhca5 and Lhca6, contribute to the interaction of each PSI–LHCI copy with the NDH complex. Here, large‐pore blue‐native gel electrophoresis revealed that, in addition to this complex, there were at least two types of higher‐order association of more LHCI copies with the NDH complex. In single‐particle images, this higher‐order association of PSI–LHCI preferentially occurs at the left side of the NDH complex when viewed from the stromal side, placing subcomplex A at the top (Yadav et al., Biochim. Biophys. Acta ‐ Bioenerg., 1858, 2017, 12). The association was impaired in the lhca6 mutant but not in the lhca5 mutant, suggesting that the left copy of PSI–LHCI was linked to the NDH complex via Lhca6. From an analysis of subunit compositions of the NDH–PSI supercomplex in lhca5 and lhca6 mutants, we propose that Lhca6 substitutes for Lhca2 in the left copy of PSI–LHCI, whereas Lhca5 substitutes for Lhca4 in the right copy. In the lhca2 mutant, Lhca3 was specifically stabilized in the NDH–PSI supercomplex through heterodimer formation with Lhca6. In the left copy of PSI–LHCI, subcomplex B, Lhca6 and NdhD likely formed the core of the supercomplex interaction. In contrast, a larger protein complex, including at least subcomplexes B and L and NdhB, was needed to form the contact site with Lhca5 in the right copy of PSI–LHCI.     

Association of a missense nucleotide polymorphism in the MT‐ND2 gene with mitochondrial reactive oxygen species production in the Tibet chicken embryo incubated in normoxia or simulated hypoxia

NADH dehydrogenase (complex I) catalyzes the transfer of electrons from NADH to ubiquinone with pumping protons across the mitochondrial inner membrane and produces reactive oxygen species as a major source in mitochondria. A missense mutation in the mitochondrially encoded NADH dehydrogenase 2 (MT‐ND2) gene, which could produce a change in the protein's secondary structure, has been found in the Tibet chicken breed. In this study, breeding eggs of the Tibet chicken breed with the two genotypes were divided into two groups. One group was incubated in normoxia (20.9% oxygen concentration) and the other in simulated hypoxia (14.5% oxygen concentration). On the 16th day of incubation, complex I activity and mitochondrial reactive oxygen species production in the Tibet chicken embryonic liver with different genotypes in each group were measured. Results showed that: (1) hypoxia reduced complex I activity standardized and mitochondrial reactive oxygen species production significantly compared with normoxia and (2) the missense mutation in the MT‐ND2 gene was significantly associated with the production of reactive oxygen species in mitochondria while not associated with the standardized or unstandardized activity of complex I.     

The plant pathogen Pectobacterium atrosepticum contains a functional formate hydrogenlyase‐2 complex

Pectobacterium atrosepticum SCRI1043 is a phytopathogenic Gram‐negative enterobacterium. Genomic analysis has identified that genes required for both respiration and fermentation are expressed under anaerobic conditions. One set of anaerobically expressed genes is predicted to encode an important but poorly understood membrane‐bound enzyme termed formate hydrogenlyase‐2 (FHL‐2), which has fascinating evolutionary links to the mitochondrial NADH dehydrogenase (Complex I). In this work, molecular genetic and biochemical approaches were taken to establish that FHL‐2 is fully functional in P. atrosepticum and is the major source of molecular hydrogen gas generated by this bacterium. The FHL‐2 complex was shown to comprise a rare example of an active [NiFe]‐hydrogenase‐4 (Hyd‐4) isoenzyme, itself linked to an unusual selenium‐free formate dehydrogenase in the final complex. In addition, further genetic dissection of the genes encoding the predicted membrane arm of FHL‐2 established surprisingly that the majority of genes encoding this domain are not required for physiological hydrogen production activity. Overall, this study presents P. atrosepticum as a new model bacterial system for understanding anaerobic formate and hydrogen metabolism in general, and FHL‐2 function and structure in particular.     

Unique coenzyme binding mode of hyperthermophilic archaeal sn‐glycerol‐1‐phosphate dehydrogenase from Pyrobaculum calidifontis

A gene encoding an sn‐glycerol‐1‐phosphate dehydrogenase (G1PDH) was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The gene was overexpressed in Escherichia coli, and its product was purified and characterized. In contrast to conventional G1PDHs, the expressed enzyme showed strong preference for NADH: the reaction rate (V_max) with NADPH was only 2.4% of that with NADH. The crystal structure of the enzyme was determined at a resolution of 2.45 Å. The asymmetric unit consisted of one homohexamer. Refinement of the structure and HPLC analysis showed the presence of the bound cofactor NADPH in subunits D, E, and F, even though it was not added in the crystallization procedure. The phosphate group at C2’ of the adenine ribose of NADPH is tightly held through the five biased hydrogen bonds with Ser40 and Thr42. In comparison with the known G1PDH structure, the NADPH molecule was observed to be pushed away from the normal coenzyme binding site. Interestingly, the S40A/T42A double mutant enzyme acquired much higher reactivity than the wild‐type enzyme with NADPH, which suggests that the biased interactions around the C2’‐phosphate group make NADPH binding insufficient for catalysis. Our results provide a unique structural basis for coenzyme preference in NAD(P)‐dependent dehydrogenases. Proteins 2016; 84:1786–1796. © 2016 Wiley Periodicals, Inc.     

Type‐II NADH:quinone oxidoreductase from Staphylococcus aureus has two distinct binding sites and is rate limited by quinone reduction

A prerequisite for any rational drug design strategy is understanding the mode of protein–ligand interaction. This motivated us to explore protein–substrate interaction in Type‐II NADH:quinone oxidoreductase (NDH‐2) from Staphylococcus aureus, a worldwide problem in clinical medicine due to its multiple drug resistant forms. NDHs‐2 are involved in respiratory chains and recognized as suitable targets for novel antimicrobial therapies, as these are the only enzymes with NADH:quinone oxidoreductase activity expressed in many pathogenic organisms. We obtained crystal and solution structures of NDH‐2 from S. aureus, showing that it is a dimer in solution. We report fast kinetic analyses of the protein and detected a charge‐transfer complex formed between NAD⁺ and the reduced flavin, which is dissociated by the quinone. We observed that the quinone reduction is the rate limiting step and also the only half‐reaction affected by the presence of HQNO, an inhibitor. We analyzed protein–substrate interactions by fluorescence and STD‐NMR spectroscopies, which indicate that NADH and the quinone bind to different sites. In summary, our combined results show the presence of distinct binding sites for the two substrates, identified quinone reduction as the rate limiting step and indicate the establishment of a NAD⁺‐protein complex, which is released by the quinone.     

Proteomic DIGE analysis of the mitochondria‐enriched fraction from aged rat skeletal muscle

Skeletal muscle aging is associated with a loss in tissue mass and contractile strength, as well as fiber type shifting and bioenergetic adaptation processes. Since mitochondria represent the primary site for energy generation via oxidative phosphorylation, we investigated potential changes in the expression pattern of the mitochondrial proteome using the highly sensitive DIGE approach. The comparative analysis of the mitochondria‐enriched fraction from young adult versus aged muscle revealed an age‐related change in abundance for 39 protein species. MS technology identified the majority of altered proteins as constituents of muscle mitochondria. An age‐dependent increase was observed for NADH dehydrogenase, the mitochondrial inner membrane protein mitofilin, peroxiredoxin isoform PRX‐III, ATPase synthase, succinate dehydrogenase, mitochondrial fission protein Fis1, succinate‐coenzyme A ligase, acyl‐coenzyme A dehydrogenase, porin isoform VDAC2, ubiquinol‐cytochrome c reductase core I protein and prohibitin. Immunoblotting, enzyme testing and confocal microscopy were used to validate proteomic findings. The DIGE‐identified increase in key mitochondrial elements during aging agrees with the concept that sarcopenia is associated with a shift to a slower contractile phenotype and more pronounced aerobic‐oxidative metabolism. This suggests that mitochondrial markers are reliable candidates that should be included in the future establishment of a biomarker signature of skeletal muscle aging.     

Enzymatic Activity in Mitochondria from Orobanche

Mitochondria from Orobanche were analysed for the activities of aconitate hydratase, isocitrate dehydrogenase, succinate dehydro-genase, fumarate hydratase, malate dehydrogenase, NADH oxidase, substrate-cytochrome c oxidoreductases, glutamate dehydrogenase, aminotransferases, ATPase and “malic” enzyme. The specific activities of isocitrate dehydrogenase, NADH oxidase, substrate-cytochrome c oxidoreductases and glutamate dehydrogenase in the mitochondria) fraction from parasite tissue compared favourably with those reported for most of the mitochondria from growing and storage tissues. Succinate dehydrogenase, fumarate hydratase and aspartate aminotransferase were of intermediate activity, while aconitate hydratase and malate dehydrogenase had rather low activity, and “malic” enzyme had very low activity in comparison with other preparations. The relevance of these findings in relation to mitochondrial metabolism in the parasite is discussed. No evidence was obtained to suggest any basic abnormality in the biochemical properties of the mitochondria from Orobanche centua which may be correlated with its obligatorily parasitic existence.     

The NAD(P)H Dehydrogenase in Barley Thylakoids Is Photoactivatable and Uses NADPH as well as NADH

An improved light-dependent assay was used to characterize the NAD(P)H dehydrogenase (NDH) in thylakoids of barley (Hordeum vulgare L.). The enzyme was sensitive to rotenone, confirming the involvement of a complex I-type enzyme. NADPH and NADH were equally good substrates for the dehydrogenase. Maximum rates of activity were 10 to 19 μmol electrons mg⁻¹ chlorophyll h⁻¹, corresponding to about 3% of linear electron-transport rates, or to about 40% of ferredoxin-dependent cyclic electron-transport rates. The NDH was activated by light treatment. After photoactivation, a subsequent light-independent period of about 1 h was required for maximum activation. The NDH could also be activated by incubation of the thylakoids in low-ionic-strength buffer. The kinetics, substrate specificity, and inhibitor profiles were essentially the same for both induction strategies. The possible involvement of ferredoxin:NADP⁺ oxidoreductase (FNR) in the NDH activity could be excluded based on the lack of preference for NADPH over NADH. Furthermore, thenoyltrifluoroacetone inhibited the diaphorase activity of FNR but not the NDH activity. These results also lead to the conclusion that direct reduction of plastoquinone by FNR is negligible.     

NAD(P)H-ubiquinone oxidoreductases in plant mitochondria

Plant (and fungal) mitochondria contain multiple NAD(P)H dehydrogenases in the inner membrane all of which are connected to the respiratory chain via ubiquinone. On the outer surface, facing the intermembrane space and the cytoplasm, NADH and NADPH are oxidized by what is probably a single low-molecular-weight, nonproton-pumping, unspecific rotenone-insensitive NAD(P)H dehydrogenase. Exogenous NADH oxidation is completely dependent on the presence of free Ca²⁺ with aK _0.5 of about 1 µM. On the inner surface facing the matrix there are two dehydrogenases: (1) the proton-pumping rotenone-sensitive multisubunit Complex I with properties similar to those of Complex I in mammalian and fungal mitochondria. (2) a rotenone-insensitive NAD(P)H dehydrogenase with equal activity with NADH and NADPH and no proton-pumping activity. The NADPH-oxidizing activity of this enzyme is completely dependent on Ca²⁺ with aK _0.5 of 3 µM. The enzyme consists of a single subunit of 26 kDa and has a native size of 76 kDa, which means that it may form a trimer.     

Direct evidence for the presence of a rotenone-resistant NADH dehydrogenase on the inner surface of the inner membrane of plant mitochondria

Submitochondrial particles (SMP) were produced from Jerusalem artichoke (Helianthus tuberosus L.) mitochondria by sonication and differential centrifugation. The SMP were about 50% inside-out as measured by the access of reduced cytochrome c to cytochrome c oxidase. Uncoupled NADH oxidation (1 mM NADH) by the SMP was 120 nmol O₂ min^?1mg^?1, which was reduced to 98 nmol O₂ min^?1 (mg mitochondrial protein)^?1 in the presence of EGTA. In contrast, the oxidation of NADH by intact mitochondria was completely inhibited by EGTA (from 182 to 14 nmol O₂ min^?1mg^?1). The EGTA-resistant NADH oxidation by the SMP is ascribed to the NADH dehydrogenase(s) on the inside of the inner membrane and exposed to the medium in the inside-out SMP. In the presence of EGTA it could be shown that two NADH dehydrogenase activities were present in the SMP. One had an apparent K_m of 7 μM for NADH, a V_max of 80 nmol NADH min^?1mg^?1, and was rotenone-sensitive. This dehydrogenase is equivalent to the mammalian Complex I NADH dehydrogenase. The other dehydrogenase, which was rotenone-resistant, had a K_m of 80 μM and a V_max of 131 nmol NADH min^?1mg^?1; it is probably responsible for the rotenone-resistant oxidation of organic acids often observed in plant mitochondria. The redox poise of the pyridine nucleotides had only a small effect on the relative rates of the two internal dehydrogenases. Electron flow through these dehydrogenases appears, therefore, to be regulated mainly by the concentration of NADH in the matrix of the mitochondria.     

The branched mitochondrial respiratory chain from Debaryomyces hansenii: Components and supramolecular organization

The branched respiratory chain in mitochondria from the halotolerant yeast Debaryomyces hansenii contains the classical complexes I, II, III and IV plus a cyanide-insensitive, AMP-activated, alternative-oxidase (AOX). Two additional alternative oxidoreductases were found in this organism: an alternative NADH dehydrogenase (NDH2e) and a mitochondrial isoform of glycerol-phosphate dehydrogenase (_MitGPDH). These monomeric enzymes lack proton pump activity. They are located on the outer face of the inner mitochondrial membrane. NDH2e oxidizes exogenous NADH in a rotenone-insensitive, flavone-sensitive, process. AOX seems to be constitutive; nonetheless, most electrons are transferred to the cytochromic pathway. Respiratory supercomplexes containing complexes I, III and IV in different stoichiometries were detected. Dimeric complex V was also detected. In-gel activity of NADH dehydrogenase, mass spectrometry, and cytochrome c oxidase and ATPase activities led to determine the composition of the putative supercomplexes. Molecular weights were estimated by comparison with those from the yeast Y. lipolytica and they were IV₂, I–IV, III₂–IV₄, V₂, I–III₂, I–III₂–IV, I–III₂–IV₂, I–III₂–IV₃ and I–III₂–IV₄. Binding of the alternative enzymes to supercomplexes was not detected. This is the first report on the structure and organization of the mitochondrial respiratory chain from D. hansenii.     

During the stationary growth phase, Yarrowia lipolytica prevents the overproduction of reactive oxygen species by activating an uncoupled mitochondrial respiratory pathway

In the branched mitochondrial respiratory chain from Yarrowia lipolytica there are two alternative oxido-reductases that do not pump protons, namely an external type II NADH dehydrogenase (NDH2e) and the alternative oxidase (AOX). Direct electron transfer between these proteins is not coupled to ATP synthesis and should be avoided in most physiological conditions. However, under low energy-requiring conditions an uncoupled high rate of oxygen consumption would be beneficial, as it would prevent overproduction of reactive oxygen species (ROS). In mitochondria from high energy-requiring, logarithmic-growth phase cells, most NDH2e was associated to cytochrome c oxidase and electrons from NADH were channeled to the cytochromic pathway. In contrast, in the low energy requiring, late stationary-growth phase, complex IV concentration decreased, the cells overexpressed NDH2e and thus a large fraction of this enzyme was found in a non-associated form. Also, the NDH2e-AOX uncoupled pathway was activated and the state IV external NADH-dependent production of ROS decreased. Association/dissociation of NDH2e to/from complex IV is proposed to be the switch that channels electrons from external NADH to the coupled cytochrome pathway or allows them to reach an uncoupled, alternative, ΔΨ-independent pathway.