NADH-CoQ-reductase

Rotenone NADH-CoQ reductase Blocks oxidation of NADH (site I). NADH will become reduced Substrates such as succinate that enter via FADH will still be oxidized and make 2 ATPs/mol. 

Fig. 1. Gold stain of 2D separation of proteins from beef heart mitochondria. Arrow indicates the 49-kDa protein of NADH CoQ reductase (see Figs. 2 and 3).

Figure l shows the gold stain of a section of a 2D gel of beef heart mitochondria proteins separated by isoelectric focusing and then by SDS-PAGE, and transferred to nitrocellulose. The filter was then probed with antibody specific to the 49-kDa iron sulfur protein of NADH CoQ reductase. The blot was photographed with (Fig. 2) and without (Fig. 3) a red filter following development with 4-chloro-1 -naphthol. The position of the 49-kDa protein can then be determined by back reference to Fig. 1 (see arrow). 

Although DNA mutations in nuclear DNA may cause mitochondrial dysfunction, the majority of genetically defined mitochondrial diseases are caused by mutations in mtDNA (M15, PI, S4). Point mutations and deletions of mtDNA have been reported to be associated with or responsible for mitochondrial myopathies and/or encephalomyopathies (M15, PI, S4). Patients with such diseases usually manifest major clinical symptoms early in life and at a later stage may develop additional multisystem disorders such as encephalopathy and/or peripheral neuropathy. Most of the mitochondrial myopathies occur sporadically and are often caused by large-scale mtDNA deletions (PI). However, there are several reports on maternally inherited mitochondrial myopathy and familial mitochondrial myopathy. These patients usually harbor a specific mtDNA mutation and often exhibit defects in NADH-CoQ reductase and/or cytochrome c oxidase. 

Two reactions of the reverse electron transfer are of physiological significance. 1 mean (i) oxidation of succinate (redox potential, +0.03 V) by NAD (redox potential, -0.32 V) and (ii) oxidation of NADH by NADPH responsible for maintenance of [NADPH]/NADP ] [NADH]/[NAD ] in spite of the fact that redox potential of the NADPH/NADH pair is almost equal to that of NADH/NAD pair. The former process includes a reversal of NADH-CoQ reductase (Complex I of the respiratory chain). Usually it operates as a A 1h+ generator catalysing the downhill electron transfer from NADH to CoQ. However, when NAD is reduced by succinate, the same complex acts as a ApH consumer carrying out the uphill transfer of electrons from C0QH2 to NAD" [5]. 

Complex I catalyzes an NADH-CoQ reductase activity, and it contains the NADH dehydrogenase flavoprotein. It has two types of electron-carrying structures FMN and several iron-sulfur centers. FMN is a tightly bound prosthetic group of the dehydrogenase enzyme, and it is reduced to FMNH2 by the two reducing equivalents derived from NADH ... 

The electrons from FMNH2 are transferred to the next electron carrier, coenzyme Q, via the iron-sulfur centers of the NADH-CoQ reductase. The iron-sulfur centers consist of iron atoms paired with an equal number of acid-labile sulfur atoms. The respiratory chain iron-sulfur clusters are of the Fe2S2 or Fe4S4 type. The iron atom, present as nonheme iron, undergoes oxidation-reduction cycles (Fe + Fe + -t- e ). In the Fe4S4 complexes, the centers... 

Inhibitors of NADH-CoQ reductase rotenone (a toxic plant product), piericidin A (an antibiotic), and amytal (a barbiturate). 

Complex I NADH CoQ reductase Piericidin A Amytal Rotenone... 

A. Rotenone binds avidly to the flavoprotein NADH CoQ reductase, complex I (also called NADH dehydrogenase). The central portion of the rotenone structure resembles the isoalloxazine ring of the FMN molecule, and when it binds to complex I, rotenone prevents the transfer of electrons from NADH to coenzyme Q. 

Rotenone binds avidly to the flavoprotein NADH CoQ reductase, complex I (also called NADH dehydrogenase). 

A FIGURE 8-17 Overview of multiprotein complexes, bound prosthetic groups, and associated mobile carriers in the respiratory chain. Blue arrows indicate electron flow red arrows indicate proton translocation. Left) Pathway from NADH. A total of 10 protons are translocated per pair of electrons that flow from NADH to 02.The protons released into the matrix space during oxidation of NADH by NADH-CoQ reductase are consumed in the formation of water from O2 by cytochrome c oxidase, resulting in no net proton translocation from these reactions. Right) Pathway... 

NADH-CoQ Reductase (Complex I) Electrons are carried from NADH to CoQ by the NADH-CoQ reductase complex. NAD is exclusively a two-electron carrier it accepts or releases a pair of electrons at a time. In the NADH-CoQ reductase complex, electrons first flow from NADH to FMN (flavin mononucleotide), a cofactor related to FAD, then to an iron-sulfur cluster, and finally to CoQ (see Figure 8-17). FMN, like FAD, can accept two electrons, but does so one electron at a time. 

Current evidence suggests that a total of 10 protons are transported from the matrix space across the inner mitochondrial membrane for every electron pair that is transferred from NADH to O2 (see Figure 8-17). Since the succinate-CoQ reductase complex does not transport protons, only six protons are transported across the membrane for every electron pair that is transferred from succinate (or FADH2) to O2. Relatively little is known about the coupling of electron flow and proton translocation by the NADH-CoQ reductase complex. More is known about operation of the cytochrome c oxidase complex, which we discuss here. The coupled electron and proton movements mediated by the CoQH2-cytochrome c reductase complex, which involves a unique mechanism, are described separately. 

C0QH2 is generated both by the NADH-CoQ reductase and succinate-CoQ reductase complexes and, as we shall see, by the CoQH2-cytochrome c reductase complex Itself. In all cases, a molecule from the pool of reduced C0QH2 in the membrane binds to the Qo site on the intermembrane... 

The major components of the mitochondrial respiratory chain are four inner membrane multiprotein complexes NADH-CoQ reductase (I), succinate-CoQ reductase (II), CoQH2-cytochrome c reductase (III), and cytochrome c oxidase (IV). The last complex transfers electrons to O2 to form H2O. 

The function of the enzymes of the mitochondrial respiratory chain is to transform the energy of redox reactions into an electrochemical proton gradient across the hydrophobic barrier of a coupling membrane. Isolated oligoenzyme complexes of the respiratory chain of mitochondria, cytochrome c oxidase, succinate ytochrome c reductase, and NADH CoQ reductase, are able to catalyze charge transfer in model systems, e.g., at a water/octane interface, which can be followed by a change in the interfacial potential at this interface [20-... 

Photodependent Inhibition of NADH-CoQ Reductase by Arylazido-p-alanine NADH... 

NADH-CoQ reductase after a 4-min period of photoirradiation, and an equal concentration of arylazido-j8-alanine NAD results in 75% inhibition. The photodependent inhibition is a function of both the time of irradiation and the concentration of arylazido-/3-alanine NADH. 

Arylazido-)8-alanine NAD+ can bring about a sizable inhibition of NADH-CoQ reductase activity when incubated with the enzyme complex in the dark without prior photoirradiation. In this case the addition of higher substrate concentrations (NADH) results in a reversal of this inhibition. Reduced NAD at 53 iiM is able to completely reverse the inhibitory influence of 11 yM arylazido- 8-alanine NAD+. On the other hand, when complex I is subjected to photoirradiation in the presence of 11 fiM arylazido-j8-alanine NAD, the addition of higher concentrations of the natural substrate (NADH) is not able to reverse the inhibition. It would thus appear that the arylazido-j8-alanine NAD acts as a competitive inhibitor of NADH-CoQ reductase in the dark and is converted to a noncompetitive irreversible inhibitor upon light irradiation in the presence of the protein complex. 

Isolated oligoenzyne complexes of the respiratory chain of mitochondria - cytochrome oxidase, succinate-cytochrome c reductase, and NADH-CoQ reductase -catalyze the transfer of charges between water and octane that can be recorded from the change in the potential shift at the octane/water phase separation boundary by the vibrating plate method. A necessary condition for the appearance of this effect has proved [18,62] to be the presence of the corresponding enzymes in the oxidation substrates in the aqueous phase and also the presence of a charge acceptor in the octane phase [10, 18, 59]. 

Fig. 16. Transfer of negative charges from water into octane catalyzed by NADH-CoQ reductase as a function of the concentration of enzymes. Incubation medium ...

The half-saturation of the system with cytochrome c was achieved at a concentration of about 10 M (Fig. 15a, b). In all cases, the effect was reversed and prevented by antimycin - an inhibitor of the succinate-cytochrome c reductase activity of this enzyme complex - but was not suppressed by cyanide. In concluding this series of experiments, we investigated the isolated NADH-CoQ reductase complex of the respiratory chain of mitochondria. As can be seen from the results given in Fig. 16, an increase in the concentration of the enzyme complex was accompanied by an increase in the negative charge of the octane phase. 

Fig. 18. Catalysis of the transfer of positive charges from water into octane by NADH-CoQ reductase (1) and by succinate-cytochrome c reductase (2) as a function of the concentration of DNP. Incubation medium l)20mU tris-HCl (pH 7.4) 0.2 mM NADH and 25 ig of NADH-CoQ reductase protein per ml 2)20 mM tris-HCl (pH 4.7), 7 mM succinate, 0.2 mM cytochrome c, and 40 pg of succinate-cytochrome c reductase protein per ml. In both experiments, the medium contained 1 mM ferricyanide [18]...

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