Redox-dependent change of nucleotide affinity to the active site of the mammalian complex I

Vera G. Grivennikova, Alexander Kotlyar*, Joel S. Karliner, Gary Cecchini, Andrei D. Vinogradov

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

36 Scopus citations

Abstract

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 μM 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 (∼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.

Original languageEnglish
Pages (from-to)10971-10978
Number of pages8
JournalBiochemistry
Volume46
Issue number38
DOIs
StatePublished - 25 Sep 2007

Funding

FundersFunder number
Fogarty International CenterR03TW007825

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