HDQ (1-Hydroxy-2-dodecyl-4(1H)quinolone), a High Affinity Inhibitor for Mitochondrial Alternative NADH Dehydrogenase

Abstract

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...

Reaction Mechanism of Single Subunit NADH-Ubiquinone Oxidoreductase (Ndi1) from Saccharomyces cerevisiae

“…2). This result implies that exogenously bound UQ 6 is not bound to exactly the same site as endogenously bound UQ 8 . Moreover, these results suggest that Ndi1 forms a ternary complex with NADH and UQ in close vicinity of FAD and that UQ can withdraw an electron from reduced flavin, leading to the formation of the CT complex.…”

“…Indeed, the NADH binding site of the group A family of NDH-2 enzymes has been suggested to exist near the FAD binding site (2,20). In the case of NDH-2 from Yarrowia lipolytica, it was suggested that NADH and the quinone head group interact with the same binding pocket competitively (8). To obtain an understanding of the structure of the binding sites of the substrates in Ndi1, we referred to the x-ray crystal structures of other flavin enzymes: thioredoxin reductase of Drosophila melanogaster (DmTR) (21) and lipoamide dehydrogenase of Pseudomonas putida (PpLipDH) (22).…”

“…In addition to the preparation of UQ-free Ndi1 (TX enzyme), UQ-bound Ndi1 (containing endogenous quinone; UQ 8 if overexpressed in E. coli) was also prepared by a procedure using DM (5). To further increase the UQ content of purified Ndi1, the ionic strength of the buffers was raised from 200 to 500 mM.…”

“…C, UQ 8 content of each sample was calculated from the results of reversephase HPLC. * denotes an undetectable level of UQ 8 . Each value and vertical bar represent the mean Ϯ S.E.…”

“…To investigate this point, UQ 10 -enriched liposomes were fused with the Ndi1-overexpressing E. coli membrane containing endogenous UQ 8 . It was expected that if bound UQ of Ndi1 can reversibly dissociate from the enzyme in the membrane then bound endogenous UQ 8 of Ndi1 will be partly replaced by UQ 10 .…”