Catalytic Activity of NADH-ubiquinone Oxidoreductase (Complex I) in Intact Mitochondria

Abstract

The mammalian purified dispersed NADH-ubiquinone oxidoreductase (Complex I) and the enzyme in insideout submitochondrial particles are known to be the slowly equilibrating mixture of the active and de-activated forms (Vinogradov, A. D. (1998) Biochim. Biophys. Acta 1364, 169 -185). We report here the phenomenon of slow active/de-active transition in intact mitochondria where the enzyme is located within its natural environment being exposed to numerous mitochondrial matrix proteins. A simple procedure for permeabilization of intact mitochondria by channel-forming antibiotic alamethicin was worked out for the "in situ" assay of Complex I activity. Alamethicin-treated mitochondria catalyzed the rotenone-sensitive NADH-quinone reductase reaction with exogenousely added NADH and quinoneacceptor at the rates expected if the enzyme active sites would be freely accessible for the substrates. The matrix proteins were retained in alamethicin-treated mitochondria as judged by their high rotenone-sensitive malate-cytochrome c reductase activity in the presence of added NAD ؉ . The sensitivity of Complex I to N-ethylmaleimide and to the presence of Mg 2؉ was used as the diagnostic tools to detect the presence of the de-activated enzyme. The NADH-quinone reductase activity of alamethicin-treated mitochondria was sensitive to neither N-ethylmaleimide nor Mg 2؉ . After exposure to elevated temperature (37°C, the conditions known to induce de-activation of Complex I) the enzyme activity became sensitive to the sulfhydryl reagent and/or Mg 2؉ . The sensitivity to both inhibitors disappeared after brief exposure of the thermally de-activated mitochondria with malate/glutamate, NAD ؉ , and cytochrome c (the conditions known for the turnover-induced reactivation of the enzyme). We conclude that the slow active/ de-active Complex I transition is a characteristic feature of the enzyme in intact mitochondria and discuss its possible physiological significance.In mammalian mitochondria NADH-ubiquinone oxidoreductase (Complex I, coupling Site 1, EC 1.6.99.3) functions as the main entry to the respiratory chain. The enzyme has an extremely complex structure being composed of more than 40 different subunits (1, 2). It contains multiple distinct redox components (FMN, a number of iron-sulfur clusters and tightly bound ubiquinones) operating in unknown sequence of the reactions coupled with vectorial translocation of protons from matrix to intermembraneous space. The functions of a vast majority of the enzyme subunits are not known. Most of the recent studies on Complex I and its simpler procaryotic counterparts (Type 1 NADH dehydrogenases) have focused on their structure (1-4), iron-sulfur clusters location (5, 6), possible mechanism of proton translocation (7-9), and the comparative molecular biology of the enzyme (10 -12).Very little is known about regulatory properties of Complex I. The bovine heart enzyme shows very complex kinetic behavior when assayed in either forward or reverse reactions. Following pioneering observations of Estabr...

A Novel, Enzymatically Active Conformation of the Escherichia coli NADH:Ubiquinone Oxidoreductase (Complex I)

“…An active and inactive form of complex I have also been described in mitochondria (39,40). It has been proposed that the transition between the two forms plays a role for the physiological regulation of the complex (40).…”

“…It has been proposed that the transition between the two forms plays a role for the physiological regulation of the complex (40). The molecular basis discriminating the two forms of the mitochondrial complex is, however, not understood.…”

The Inhibition of Mitochondrial Complex I (NADH:Ubiquinone Oxidoreductase) by Zn2+

“…Interestingly, the sensitivity of complex I to zinc probably increases under these conditions, when the enzyme is not fully catalytically active. Note that the existence of the deactive state remains to be demonstrated in vivo but that it has been reported in studies of anoxia using rat hearts (47) and in isolated mitochondria (44). Finally, comparison of the IC 50 values determined here with the zinc binding constants determined for the cytochrome bc 1 complex (0.…”

“…Complex I has been proposed to exist in two distinct forms, "active" and "deactive" (42)(43)(44). The enzyme deactivates slowly in the absence of turnover and is reactivated slowly when catalysis is initiated by the addition of substrates.…”

The Mitochondrial Complex I: Progress in Understanding of Catalytic Properties

“…This problem seems to be of special signicance for the biomedically oriented studies on Complex I when the amount of available material (mitochondria or tissue) is small and reliable and reproducible preparation of inside-out submitochondrial particles from the original samples is almost impossible. Recently, a procedure for the quantitative measurement of Complex I activity in mitochondria has been described (34). It is based on the effective and noninvasive (in terms of Complex I activity) permeabilization of the inner mitochondrial membrane by the channel-forming antibiotic alamethicin.…”