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.Alternative NADH dehydrogenases (NADH:ubiquinone oxidoreductases) are respiratory chain enzymes that carry out the same redox reaction as mitochondrial complex I. However, unlike this complicated multi-subunit enzyme, they do not contribute to the proton gradient across the respiratory membrane and are insensitive to complex I inhibitors like rotenone and piericidin A (for an overview see Ref. 1). Alternative NADH dehydrogenases are inhibited by flavones in the micromolar range. Acridones have been demonstrated to inhibit both complex I and alternative enzymes (2).Alternative NADH dehydrogenases are found in the respiratory chains of plants (3), fungi (4 -7), many eubacteria (8, 9), and archaebacteria (10 -12). They consist of a single polypeptide chain that exhibits no obvious transmembrane domains and contains one molecule of FAD with the exception of the archaebacterial enzymes that, presumably as an adaptation to thermic habitats, carry covalently attached FMN instead.In most plants and fungi, multiple isoforms of NADH dehydrogenases are expressed in the same species. The active site of the membrane-associated enzymes may be directed to the external or internal face of the mitochondrial inner membrane. In Saccharomyces cerevisiae, for example, two external (SCNDE1 and SCNDE2) and one internal (SCNDI1) alternative NADH dehydrogenases are found (13). The obligate aerobic yeast Yarrowia lipolytica has only a single external alternative enzyme, YLNDH2 (5). It has been demonstrated that this external enzyme can be transformed into an internal version simply by adding a targeting signal for the mitochondrial matrix (14). This suggests that there are no specific protein interaction partners for its membrane association. It remains unclear how the largely hydrophilic enzyme interacts with its highly hydrophobic substrate, ubiquinone. NADH, the other substrate of alternative NADH dehydrogenases, is highly hydrophilic.Inspection of the YLNDH2 sequence revealed two ␤␣␤-dinucleotide-binding domains that consist of two parallel ␤-strands connected by an ␣-helix (5). The sequence G(X)XGXXG, which marks the connection between the first ␤-strand and the connecting ␣-helix, makes close contact with the diphosphate moi...

Aerotaxis of two sulphate-reducing bacteria, the freshwater strain Desulfovibrio desulfuricans CSN (DSM 9104) and the marine strain Desulfovibrio oxyclinae N13 (DSM 11498), was studied using capillary microslides, microscopy and oxygen microsensors. The bacteria formed ring-shaped bands in oxygen diffusion gradients surrounding O2 bubbles, which were placed into anoxic sulphate-free cell suspensions in capillary microslides. The radial expansion of the oxic volume by diffusion was stopped by aerobic respiration. Bands were formed by cells avoiding high O2 levels near the O2 bubble, as well as by cells entering from the surrounding anoxic zone. At the inner edge of the bands, O2 levels of up to 20% air saturation (50 microM O2) were found, while the outer edge always coincided with the oxic-anoxic interface. Ring diameters and O2 concentrations at the inner edge of the band depended on the cell density and the strain used in the suspension. Band formation did not occur in the absence of an electron donor (5mM lactate) or when N2 gas bubbles were used. Both strains were highly motile with velocities of approximately equals 32 microm s(-1) during forward runs, and 7 microm s(-1) during backward runs respectively. Within the bands, cells moved in circles of about 20 microm diameter, while cells outside the band exhibited straighter or only slightly bent traces. It is concluded that the capacity of respiration at high rates and the positive and negative aerotactical responses of Desulfovibrio provide an efficient strategy for removing O2 from the habitat in situations where sufficient electron donors and high cell densities are present.

Growth and chemotactic behavior in oxic-anoxic gradients were studied with two freshwater and four marine strains of sulfate-reducing bacteria related to the genera Desulfovibrio, Desulfomicrobium or Desulfobulbus. Cells were grown in oxygen-sulfide counter-gradients within tubes filled with agar-solidified medium. The immobilized cells grew mainly in the anoxic zone, revealing a peak below the oxic-anoxic interface. All tested strains survived exposure to air for 8 h and all were capable of oxygen reduction with lactate. Most strains also oxidized sulfide with oxygen. Desulfovibrio desulfuricans responded chemotactically to lactate, nitrate, sulfate and thiosulfate, and even sulfide functioned as an attractant. In oxic-anoxic gradients the bacteria moved away from high oxygen concentrations and formed bands at the outer edge of the oxic zone at low oxygen concentration (<5% O2 saturation). They were able to actively change the extension and slope of the gradients by oxygen reduction with lactate or even sulfide as electron donor. Generally, the chemotactic behavior was in agreement with a defense strategy that re-establishes anoxic conditions, thus promoting anaerobic growth and, in a natural community, fermentative production of the preferred electron donors of the sulfate-reducing bacteria.

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-offunction 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.

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.