Interaction of the mitochondrial NADH-ubiquinone reductase with rotenone as related to the enzyme active/inactive transition

Abstract

The interaction of rotenone with active ('pulsed') and thermally de-activated ('resting') membrane-bound Complex I (Kotlyar, A.B. and Vinogradov, A.D. (1990) Biochim. Biophys. Acta 1019, 151-158) as revealed by inhibition of NADH-ubiquinone- and ubiquinol-NAD+ reductase activities was studied. Ki = 1 x 10(-9) M, k(on) = 5 x 10(7) M-1 min-1 and k(off) = 0.02 min-1 (inhibitory effect of rotenone on NADH oxidation) and Ki = 2 x 10(-8) M (inhibition of reverse electron transfer) were determined for pulsed enzyme. The equilibrium between de-activated and active enzyme is reached (K approximately 100) after the slow strongly temperature-dependent de-activation process has completed. Rotenone partially prevents and reverses the enzyme de-activation. About two order of magnitude difference in affinity of rotenone to the active and de-activated forms of the enzyme was demonstrated. The strong difference in rotenone sensitivity of the direct and reverse reactions can not be accounted for delta mu H(+)-dependence of rotenone binding. We propose that two rotenone-specific inhibitory sites exist in Complex I: one is involved in NADH oxidation by ubiquinone and the other is operating in ubiquinol-NAD+ reductase reaction. The affinities of rotenone for both sites are strongly altered upon the slow enzyme active/inactive transition.

A LON-ClpP Proteolytic Axis Degrades Complex I to Extinguish ROS Production in Depolarized Mitochondria

“…Deactive conformational change is largely restricted to Q-site architecture, exposing thiols in ND1, ND3, and FA9 (Galkin et al., 2008, Babot et al., 2014) to oxidation/redox modification (Gorenkova et al., 2013). Rotenone retards the deactive transition (Grivennikova et al., 1997) by probably binding peptide, which drives the conversion so complex I is jammed into a non-deactivate conformation. Accordingly, the deactive enzyme may form the degradative ground state by revealing degrons/exposing oxidizable peptide, which enables subsequent denaturation and damage contagion in a feedforward-like proteolytic mechanism.…”

Transient and partial mitochondrial inhibition for the treatment of postresuscitation injury: Getting it just right

“…This is most likely explained by the permanence offered by the binding affinity of rotenone to complex I. Unlike rhein tech and ␣-NADH, rotenone has been described in several studies to "glob" and "stick" to complex I irreversibly (22). Rotenone can shut down electron transport and abolish the damaging oxidant stress, beginning the rescue of ischemic cells; however, permanent inhibition of complex I is completely counterproductive and deleterious to cell viability.…”

Hypoxic Pulmonary Vasoconstriction

“…Rotenone has high affinity for the complex I NADH oxidoreductase, with a K i of ϳ1 nM in isolated mitochondria (667). At 100 nM, rotenone increased mitochondrial NADH autofluorescence maximally in pulmonary arteries (1041) and decreased O 2 consumption by ϳ80% in PASMC (2032).…”