Redox-dependent change of nucleotide affinity to the active site of the mammalian complex I.
A very potent and specific inhibitor of mitochondrial NADH:ubiquinone oxidoreductase (complex I), a derivative of NADH (NADH-OH) has recently been discovered (Kotlyar, A. B., Karliner, J. S., and Cecchini, G. (2005) FEBS Lett. 579, 4861-4866). Here we present a quantitative analysis of the interaction of NADH-OH and other nucleotides with oxidized and reduced complex I in tightly coupled submitochondrial particles. Both the rate of the NADH-OH binding and its affinity to complex I are strongly decreased in the presence of succinate. The effect of succinate is completely reversed by rotenone, antimycin A, and uncoupler. The relative affinity of ADP-ribose, a competitive inhibitor of NADH oxidation, is also shown to be significantly affected by enzyme reduction (KD of 30 and 500 microM for oxidized and the succinate-reduced enzyme, respectively). Binding of NADH-OH is shown to abolish the succinate-supported superoxide generation by complex I. Gradual inhibition of the rotenone-sensitive uncoupled NADH oxidase and the reverse electron transfer activities by NADH-OH yield the same final titration point (approximately 0.1 nmol/mg of protein). The titration of NADH oxidase appears as a straight line, whereas the titration of the reverse reaction appears as a convex curve. Possible models to explain the different titration patterns for the forward and reverse reactions are briefly discussed.
Grivennikova
Vera G
VG
Department of Biochemistry, School of Biology, Moscow State University, Moscow 119992, Russian Federation.
Kotlyar
Alexander B
AB
Karliner
Joel S
JS
Cecchini
Gary
G
Vinogradov
Andrei D
AD
D000246:Adenosine Diphosphate Ribose / Q000494:pharmacology; D000465:Algorithms; D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects*; D002417:Cattle; D004579:Electron Transport; D042967:Electron Transport Complex I / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008929:Mitochondria, Heart / Q000187:drug effects / Q000201:enzymology; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects*; D019163:Reducing Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D012402:Rotenone / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013367:Submitochondrial Particles / Q000187:drug effects / Q000201:enzymology; D019802:Succinic Acid / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D014475:Uncoupling Agents / Q000494:pharmacology
D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D009711:Nucleotides; D019163:Reducing Agents; D014475:Uncoupling Agents; D012402:Rotenone; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D000968:Antimycin A; D019802:Succinic Acid; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I