A sulfide:quinone oxidoreductase from Chlorobaculum tepidum displays unusual kinetic properties.

Sulfide:quinone oxidoreductase (SQR) is the primary sulfide-oxidizing enzyme found in all three domains of life. Of the six phylogenetically distinct types of SQR, four have representatives that have been biochemically characterized. The genome of Chlorobaculum tepidum encodes three SQR homologs. One of these, encoded by CT1087, is a type VI SQR that has been previously shown to be required for growth at high sulfide concentrations and to be expressed in sulfide-dependent manner. Therefore, CT1087 was hypothesized to be a high sulfide adapted SQR. CT1087 was expressed in Escherichia coli with an N-terminal His-tag (CT1087NHis6) and purified by Ni-NTA chromatography. CT1087NHis6 was active and contained FAD as a strongly bound cofactor. The measured kinetic parameters for CT1087NHis6 indicate a low affinity for sulfide and a high enzymatic turnover rate consistent with the hypothesis for its function inferred from genetic and expression data. These are the first kinetic data for a type VI SQR and have implications for structure-function analyses of all SQR's.

Glyceraldehyde-3-phosphate dehydrogenase as a quinone reductase in the suppression of 1,2-naphthoquinone protein adduct formation.

1,2-Naphthoquinone (1,2-NQ) is electrophilic, and forms covalent bonds with protein thiols, but its two-electron reduction product 1,2-dihydroxynaphthalene (1,2-NQH(2)) is not, so enzymes catalyzing the reduction with reduced pyridine nucleotides as cofactors could protect cells from electrophile-based chemical insults. To assess this possibility, we examined proteins isolated from the 9000g supernatant from mouse liver for 1,2-NQ reductase activity using an HPLC assay procedure for the hydroquinone of 1,2-NQ and Cibacron Blue 3GA column chromatography and Western blot analysis with specific antibody to determine 1,2-NQ-bound proteins. Among the proteins with high affinities for pyridine nucleotides that also inhibited 1,2-NQ-protein adduct formation in the presence of NADH, a 37-kDa protein was found and identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Using recombinant human GAPDH, we found that this glycolytic enzyme indeed catalyzes the two-electron reduction of 1,2-NQ accompanied by extensive NADH consumption under 20% oxygen conditions. When either 1,2-NQH(2) or 1,2-NQ was incubated with GAPDH in the presence of NADH, minimal covalent bonding to the enzyme occurred compared to that in its absence. These results indicate that GAPDH can inhibit 1,2-NQ-based electrophilic protein modification by conversion to the nonelectrophilic 1,2-NQH(2) via an NADH-dependent process.

Biochemical and biophysical characterization of succinate: quinone reductase from Thermus thermophilus.

Enzymes serving as respiratory complex II belong to the succinate:quinone oxidoreductases superfamily that comprises succinate:quinone reductases (SQRs) and quinol:fumarate reductases. The SQR from the extreme thermophile Thermus thermophilus has been isolated, identified and purified to homogeneity. It consists of four polypeptides with apparent molecular masses of 64, 27, 14 and 15kDa, corresponding to SdhA (flavoprotein), SdhB (iron-sulfur protein), SdhC and SdhD (membrane anchor proteins), respectively. The existence of [2Fe-2S], [4Fe-4S] and [3Fe-4S] iron-sulfur clusters within the purified protein was confirmed by electron paramagnetic resonance spectroscopy which also revealed a previously unnoticed influence of the substrate on the signal corresponding to the [2Fe-2S] cluster. The enzyme contains two heme b cofactors of reduction midpoint potentials of -20mV and -160mV for b(H) and b(L), respectively. Circular dichroism and blue-native polyacrylamide gel electrophoresis revealed that the enzyme forms a trimer with a predominantly helical fold. The optimum temperature for succinate dehydrogenase activity is 70°C, which is in agreement with the optimum growth temperature of T. thermophilus. Inhibition studies confirmed sensitivity of the enzyme to the classical inhibitors of the active site, as there are sodium malonate, sodium diethyl oxaloacetate and 3-nitropropionic acid. Activity measurements in the presence of the semiquinone analog, nonyl-4-hydroxyquinoline-N-oxide (NQNO) showed that the membrane part of the enzyme is functionally connected to the active site. Steady-state kinetic measurements showed that the enzyme displays standard Michaelis-Menten kinetics at a low temperature (30°C) with a K(M) for succinate of 0.21mM but exhibits deviation from it at a higher temperature (70°C). This is the first example of complex II with such a kinetic behavior suggesting positive cooperativity with k' of 0.39mM and Hill coefficient of 2.105. While the crystal structures of several SQORs are already available, no crystal structure of type A SQOR has been elucidated to date. Here we present for the first time a detailed biophysical and biochemical study of type A SQOR-a significant step towards understanding its structure-function relationship.

The NADPH quinone reductase MdaB confers oxidative stress resistance to Helicobacter hepaticus.

An mdaB mutant strain in a quinone reductase (MdaB) of Helicobacter hepaticus type strain ATCC51449 was constructed by insertional mutagenesis, and the MdaB protein was purified and compared to the Helicobacter pylori enzyme. While wild type H. hepaticus cells could tolerate 6% O(2) for growth, the mdaB strain was clearly inhibited at this oxygen level. Disruption of the gene downstream of mdaB (HH1473) did not affect the oxidative stress phenotype of the strain. The mdaB mutant was also more sensitive to oxidative stress reagents such as H(2)O(2), cumene hydroperoxide, t-butyl hydroperoxide, and paraquat. All H. hepaticus mdaB strains isolated constitutively up-expressed another oxidative stress-combating enzyme, superoxide dismutase; this is in contrast to H. pylori mdaB strains. H. hepaticus MdaB is a flavoprotein catalyzing quinone reduction using a two-electron transfer mechanism from NAD(P)H to quinone. The H. hepaticus enzyme specific activity was far less than for the H. pylori enzyme purified in the same manner.

Purification and characterization of an intracellular NADH: quinone reductase from Trametes versicolor.

Intracellular NADH:quinone reductase involved in degradation of aromatic compounds including lignin was purified and characterized from white rot fungus Trametes versicolor. The activity of quinone reductase was maximal after 3 days of incubation in fungal culture, and the enzyme was purified to homogeneity using ion-exchange, hydrophobic interaction, and gel filtration chromatographies. The purified enzyme has a molecular mass of 41 kDa as determined by SDS-PAGE, and exhibits a broad temperature optimum between 20-40 degrees C , with a pH optimum of 6.0. The enzyme preferred FAD as a cofactor and NADH rather than NADPH as an electron donor. Among quinone compounds tested as substrate, menadione showed the highest enzyme activity followed by 1,4-benzoquinone. The enzyme activity was inhibited by CuSO(4), HgCl(2), MgSO(4), MnSO(4), AgNO(3), dicumarol, KCN, NaN(3), and EDTA. Its Km and Vmax with NADH as an electron donor were 23 microM and 101 mM/mg per min, respectively, and showed a high substrate affinity. Purified quinone reductase could reduce 1,4-benzoquinone to hydroquinone, and induction of this enzyme was higher by 1,4-benzoquinone than those of other quinone compounds.

Reductase-mediated metabolism of motexafin gadolinium (Xcytrin) in rat and human liver subcellular fractions and purified enzyme preparations.

The biotransformation of motexafin gadolinium (MGd, Xcytrin) was investigated in subcellular rat and human liver fractions. Microsomal MGd metabolism was dependent on NADPH in both species. Cytosolic metabolism in rat and human livers was dependent on NADPH or NADH. Under anaerobic conditions, MGd metabolism increased in liver microsomes and purified enzyme preparations. Cytochrome P450 (CYP450) inhibitors ketoconazole, proadifen, and carbon monoxide increased NADPH-dependent MGd metabolism in microsomes. Treatment of rats with beta-naphthoflavone increased cytosolic metabolism of MGd twofold, but had no effect on microsomal metabolism. Conversely, in liver preparations from phenobarbital treated rats microsomal metabolism of MGd was enhanced twofold, but not in cytosolic preparations. Purified CYP450 reductase from phenobarbital-treated rabbit or untreated human livers metabolized MGd suggesting involvement of CYP450 reductase. Dicumarol, a potent DT-diaphorase inhibitor, inhibited MGd metabolism in both rat and human liver cytosol. These data suggest MGd metabolism in rat liver involves CYP450 reductase and/or DT-diaphorase. In human liver preparations only CYP450 reductase is directly involved in MGd metabolism. A metabolite identified in microsomes and cytosol is a metal-free, reduced form of MGd, indicating that both enzymes generate metabolite 1, which appears to be PCI-0108, a synthetic precursor to MGd.

Studies on the DT-diaphorase-catalysed reaction employing quinones as substrates: evidence for a covalent modification of DT-diaphorase by tetrachloro-p-benzoquinone.

In this study, the kinetic parameters, V(max) and K(m), of rat liver DT-diaphorase were determined for a series of p-benzoquinones, with methyl, methoxy, cyano, hydroxy and halo substituents. The results show that there is no correlation between the experimentally determined rates of p-benzoquinone reduction by DT-diaphorase and the calculated chemical reactivity of the examined substrates as expressed by the energy of the lowest unoccupied molecular orbital, E(LUMO). However, a reasonable correlation was found between the natural logarithm of V(max)/K(m) and the partition coefficient of the p-benzoquinones (r=0.81). Furthermore, tetrachloro-p-benzoquinone, one of the tested quinones is shown to be an inhibitor of rat DT-diaphorase. The presence of bovine serum albumin (BSA) in the incubation mixture protects DT-diaphorase against the inactivation by tetrachloro-p-benzoquinone, probably by interacting with the quinone. Maldi-Tof analysis of the incubation mixture of the purified DT-diaphorase and tetrachloro-p-benzoquinone showed that every subunit of the enzyme shifted about +414 amu, whereas the dimer shifted about +849 amu relative to control values. This indicates a covalent modification of the rat liver DT-diaphorase by tetrachloro-p-benzoquinone.

Alternative NAD(P)H dehydrogenases of plant mitochondria.

Plant mitochondria have a highly branched electron transport chain that provides great flexibility for oxidation of cytosolic and matrix NAD(P)H. In addition to the universal electron transport chain found in many organisms, plants have alternative NAD(P)H dehydrogenases in the first part of the chain and a second oxidase, the alternative oxidase, in the latter part. The alternative activities are nonproton pumping and allow for NAD(P)H oxidation with varying levels of energy conservation. This provides a mechanism for plants to, for example, remove excess reducing power and balance the redox poise of the cell. This review presents our current understanding of the alternative NAD(P)H dehydrogenases present in plant mitochondria.

Molecular cloning and characterization of an NADPH quinone oxidoreductase from Kluyveromyces marxianus.

NAD(P)H quinone oxidoreductase is a ubiquitous enzyme that is known to directly reduce quinone substrates to hydroquinones by a two-electron reaction. We report the identification of NADPH quinone oxidoreductase from Kluyveromyces marxianus (KmQOR), which reduces quinone substrates directly to hydroquinones. The KmQOR gene was sequenced, expressed in Escherichia coli, purified, and characterized. The open-reading frame of the KmQOR gene consists of 1143 nucleotides, encoding a 380 amino acid polypeptide. The nucleotide sequence of the KmQOR gene was assigned to EMBL under accession number AY040868. The M(r) that was determined by SDS-PAGE for the protein subunit was about 42 kDa, and the molecular mass of the native KmQOR was 84 kDa, as determined by column calibration, indicating that the native protein is a homodimer. The KmQOR protein efficiently reduced 1,4-benzoquinone, whereas no activities were found for menadiones and methoxyquinones. These observations, and the result of an extended sequence analysis of known NADPH quinone oxidoreductase, suggest that KmQOR possesses a different action mechanism.

The membrane-associated methane monooxygenase (pMMO) and pMMO-NADH:quinone oxidoreductase complex from Methylococcus capsulatus Bath.

Improvements in purification of membrane-associated methane monooxygenase (pMMO) have resulted in preparations of pMMO with activities more representative of physiological rates: i.e., >130 nmol.min(-1).mg of protein(-1). Altered culture and assay conditions, optimization of the detergent/protein ratio, and simplification of the purification procedure were responsible for the higher-activity preparations. Changes in the culture conditions focused on the rate of copper addition. To document the physiological events that occur during copper addition, cultures were initiated in medium with cells expressing soluble methane monooxygenase (sMMO) and then monitored for morphological changes, copper acquisition, fatty acid concentration, and pMMO and sMMO expression as the amended copper concentration was increased from 0 (approximately 0.3 microM) to 95 microM. The results demonstrate that copper not only regulates the metabolic switch between the two methane monooxygenases but also regulates the level of expression of the pMMO and the development of internal membranes. With respect to stabilization of cell-free pMMO activity, the highest cell-free pMMO activity was observed when copper addition exceeded maximal pMMO expression. Optimization of detergent/protein ratios and simplification of the purification procedure also contributed to the higher activity levels in purified pMMO preparations. Finally, the addition of the type 2 NADH:quinone oxidoreductase complex (NADH dehydrogenase [NDH]) from M. capsulatus Bath, along with NADH and duroquinol, to enzyme assays increased the activity of purified preparations. The NDH and NADH were added to maintain a high duroquinol/duroquinone ratio.

Regeneration of lipophilic antioxidants by NAD(P)H:quinone oxidoreductase 1.

The aim of this work was to study the activity of NAD(P)H:(quinone acceptor) oxidoreductase 1 (EC 1.6.99.2) in the regeneration of lipophilic antioxidants, alpha-tocopherol, and reduced-coenzyme Q analogs. First, we tested whether or not two isoforms of the NAD(P)H:(quinone acceptor) oxidoreductase 1 designated as "hydrophilic" and "hydrophobic" (H. J. Prochaska and P. Talalay, Journal of Biological Chemistry 261: 1372-1378, 1986) show differential enzyme activities towards hydrophilic or hydrophobic ubiquinone homologs. By chromatography on phenyl Sepharose, we purified the two isoforms from pig liver cytosol and measured their reduction of several ubiquinone homologs of different side chain length. We also studied by electron paramagnetic resonance the effect of NAD(P)H:(quinone acceptor) oxidoreductase 1 on steady-state levels of chromanoxyl radicals generated by linoleic acid and lipooxygenase and confirmed the enzyme's ability to protect alpha-tocopherol against oxidation induced with H(2)O(2)-Fe(2+). Our results demonstrated that the different hydrophobicities of the isoforms do not reflect different reactivities towards ubiquinones of different side chain length. In addition, electron paramagnetic resonance studies showed that in systems containing the reductase plus NADH, levels of chromanoxyl radicals were dramatically reduced. Morever, in the presence of oxidants, alpha-tocopherol was preserved by NAD(P)H:(quinone acceptor) oxidoreductase 1, supporting our hypothesis that regeneration of alpha-tocopherol may be one of the physiologic functions of this enzyme.

Discovery of novel targets of quinoline drugs in the human purine binding proteome.

The quinolines have been used in the treatment of malaria, arthritis, and lupus for many years, yet the precise mechanism of their action remains unclear. In this study, we used a functional proteomics approach that exploited the structural similarities between the quinoline compounds and the purine ring of ATP to identify quinoline-binding proteins. Several quinoline drugs were screened by displacement affinity chromatography against the purine binding proteome captured with gamma-phosphate-linked ATP-Sepharose. Screening of the human red blood cell purine binding proteome identified two human proteins, aldehyde dehydrogenase 1 (ALDH1) and quinone reductase 2 (QR2). In contrast, no proteins were detected upon screening of the Plasmodium falciparum purine binding proteome with the quinolines. In a complementary approach, we passed cell lysates from mice, red blood cells, or P. falciparum over hydroxychloroquine- or primaquine-Sepharose. Consistent with the displacement affinity chromatography screen, ALDH and QR2 were the only proteins recovered from mice and human red blood cell lysate and no proteins were recovered from P. falciparum. Furthermore, the activity of QR2 was potently inhibited by several of the quinolines in vitro. Our results show that ALDH1 and QR2 are selective targets of the quinolines and may provide new insights into the mechanism of action of these drugs.

Purification and characterization of NAD(P)H quinone reductase from the latex of Hevea brasiliensis Müll.-Arg. (Euphorbiaceae).

NAD(P)H quinone reductase [NAD(P)H-QR] present in the latex of Hevea brasiliensis Müll.-Arg. (Euphorbiaceae) was purified to homogeniety from the B-serum fraction obtained by freeze-thawing of the bottom fraction of ultracentrifuged fresh latex. The purification protocol involved acetone fractionation, heat treatment, ion exchange chromatography and affinity chromatography. The M(r) determined by SDS-PAGE for the protein subunit was 21 kDa, and the molecular mass of the native enzyme estimated by gel filtration was 83 kDa, indicating that the native enzyme is a homotetramer. The enzyme showed pH stability over a range of 6 to at least 10 (with an optimum at pH 8) and thermal stability up to 80 degrees C. High NAD(P)H-QR activity (70%) was still retained after 10 h of preincubation at 80 degrees C. A comparable substrate specificity for this enzyme was observed among menadione, p-benzoquinone, juglone, and plumbagin, with only duroquinone generating a lower activity. Positive correlations between latex NAD(P)H-QR activity and rubber yield per tapping [fresh latex (r=0.89, P<0.01), dry rubber (r=0.81, P<0.01)] together with flow time (r=0.85, P<0.01) indicated that enzyme activity could possibly be used as a marker to predict the yield potential of selected clones.

Identification of zeta-crystallin/NADPH:quinone reductase as a renal glutaminase mRNA pH response element-binding protein.

Increased renal ammoniagenesis and bicarbonate synthesis from glutamine during chronic metabolic acidosis facilitate the excretion of acids and partially restore normal acid-base balance. This adaptation is sustained, in part, by a cell-specific stabilization of the glutaminase mRNA that leads to an increased synthesis of the mitochondrial glutaminase. A direct repeat of an 8-base AU sequence within the 3'-nontranslated region of the glutaminase mRNA binds a unique protein with high affinity and specificity. Expression of various chimeric mRNAs in LLC-PK(1)-FBPase(+) cells demonstrated that a single 8-base AU sequence is both necessary and sufficient to function as a pH response element (pH RE). A biotinylated oligoribonucleotide containing the direct repeat was used as an affinity ligand to purify the pH RE-binding protein from a cytosolic extract of rat renal cortex. The purified binding activity retained the same specific binding properties as observed with crude extracts and correlated with the elution of a 36-kDa protein. Microsequencing by mass spectroscopy and Western blot analysis were used to identify this protein as zeta-crystallin/NADPH:quinone reductase. The purified protein contained eight tryptic peptides that were identical to sequences found in mouse zeta-crystallin and three peptides that differed by only a single amino acid. The observed differences may represent substitutions found in the rat homolog. A second protein purified by this protocol was identified as T-cell-restricted intracellular antigen-related protein (TIAR). However, the purified TIAR neither bound nor affected the binding of zeta-crystallin/NADPH:quinone reductase to the pH RE. Furthermore, specific antibodies to zeta-crystallin, but not TIAR, blocked the formation of the complex between the pH RE and either the crude cytosolic extract or the purified protein. Thus, zeta-crystallin/NADPH:quinone reductase is a pH response element-binding protein.

Identification of the melatonin-binding site MT3 as the quinone reductase 2.

The regulation of the circadian rhythm is relayed from the central nervous system to the periphery by melatonin, a hormone synthesized at night in the pineal gland. Besides two melatonin G-coupled receptors, mt(1) and MT(2), the existence of a novel putative melatonin receptor, MT(3), was hypothesized from the observation of a binding site in both central and peripheral hamster tissues with an original binding profile and a very rapid kinetics of ligand exchange compared with mt(1) and MT(2). In this report, we present the purification of MT(3) from Syrian hamster kidney and its identification as the hamster homologue of the human quinone reductase 2 (QR(2), EC ). Our purification strategy included the use of an affinity chromatography step which was crucial in purifying MT(3) to homogeneity. The protein was sequenced by tandem mass spectrometry and shown to align with 95% identity with human QR(2). After transfection of CHO-K1 cells with the human QR(2) gene, not only did the QR(2) enzymatic activity appear, but also the melatonin-binding sites with MT(3) characteristics, both being below the limit of detection in the native cells. We further confronted inhibition data from MT(3) binding and QR(2) enzymatic activity obtained from samples of Syrian hamster kidney or QR(2)-overexpressing Chinese hamster ovary cells, and observed an overall good correlation of the data. In summary, our results provide the identification of the melatonin-binding site MT(3) as the quinone reductase QR(2) and open perspectives as to the function of this enzyme, known so far mainly for its detoxifying properties.

Characterization and partial purification of microsomal NAD(P)H:quinone oxidoreductases.

Quinone oxidoreductases are flavoproteins that catalyze two-electron reduction and detoxification of quinones. This leads to the protection of cells against toxicity, mutagenicity, and cancer due to exposure to environmental and synthetic quinones and its precursors. Two cytosolic forms of quinone oxidoreductases [NAD(P)H:quinone oxidoreductase 1 (NQO1) and NRH:quinone oxidoreductase 2 (NQO2)] were previously identified, purified, and cloned. A role of cytosolic NQO1 in protection of cells from oxidative stress, cytotoxicity, and mutagenicity of quinones was established. Currently, we have characterized and partially purified the NQO activity from rat liver microsomes. This activity was designated as microsomal NQO (mNQO). The mNQO activity showed significantly higher affinity for NADH than NADPH as electron donors and catalyzed reduction of 2,6-dichlorophenolindophenol and menadione. The mNQO activity was insensitive to dicoumarol, a potent inhibitor of cytosolic NQO1. Western analysis of microsomal proteins revealed 29- and 18-kDa bands that cross-reacted with polyclonal antibodies raised against cytosolic NQO1. The mNQO activity was partially purified by solubilization of microsomes with detergent Chaps, ammonium sulfate fractionation, and DEAE-Sephacel column chromatography. The microsomal mNQO proteins are expected to provide additional protection after cytosolic NQOs against quinone toxicity and mutagenicity.

One-step purification of the NADH dehydrogenase fragment of the Escherichia coli complex I by means of Strep-tag affinity chromatography.

The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first energy-transducing complex of many respiratory chains. Complex I of Escherichia coli can be split into three fragments. One of these fragments, the soluble NADH dehydrogenase fragment, represents the electron input part of complex I. It comprises the subunits NuoE, F and G and harbors one flavin mononucleotide and up to six iron-sulfur clusters. Here, we report the one-step purification of this fragment by means of affinity chromatography on StrepTactin. This was achieved by fusing the Strep-tag II peptide to the C-terminus of NuoF or NuoG. Fusion of this peptide to the N-terminus of either NuoE or NuoF disturbed the assembly of the NADH dehydrogenase fragment.

Reduction of the indoloquinone anticancer drug EO9 by purified DT-diaphorase: a detailed kinetic study and analysis of metabolites.

DT-diaphorase has been implicated in the activation and mechanism of cytotoxicity of the investigational indoloquinone anticancer drug EO9. Here, we have used a highly purified DT-diaphorase isolated from rat Walker tumour cells to provide unambiguous evidence for the ability of this enzyme to catalyze reduction of EO9 and to provide a more detailed characterization of the reaction. Under the conditions used hypoxia had no effect on the initial rate of this reduction but did effect the nature and stability of metabolites formed. Electron spin resonance (ESR) spectrometry studies showed that DT-diaphorase reduced EO9 to a highly oxygen-sensitive metabolite that is probably the hydroquinone. In the presence of air, this metabolite is auto-oxidized to generate both drug- and oxygen-based radicals. Comproportionation:disproportionation reactions may also be involved in the generation of these radical species. The identification of these metabolites may contribute to the understanding of the molecular mechanism of DNA damage and cytotoxicity exerted by EO9.

Properties of the respiratory NAD(P)H dehydrogenase isolated from the cyanobacterium Synechocystis PCC6803.

Activity staining with NADPH-nitroblue tetrazolium after native-PAGE of membrane proteins of Synechocystis PCC6803, solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), revealed four NAD(P)H dehydrogenase (NDH) activities; an NDH complex of the respiratory chain, a ferredoxin NADP+ reductase (FNR), a drgA product which oxidized both NADH and NADPH, and an uncharacterized NADH-specific enzyme. The NDH complex was purified with anion exchange and gel filtration chromatographies. The purified complex had a molecular mass of 376 kDa and was composed of 9 subunits. Western analysis showed that the complex contained the NDH-H subunit, but not NDH-A or B. The enzyme reduced ferricyanide much faster than plastoquinone and used NADPH as its preferred electron donor rather than NADH. The enzymatic activity was inhibited by diphenyleneiodonium chloride and salicylhydroxamic acid, but not by rotenone, p-chloromercuribenzoate, N-ethylmaleimide, flavon, dicumarol, or antimycin A. These results suggest that the purified complex is a hydrophilic subcomplex which contains an NADPH binding site and flavin, and is dissociated from a hydrophobic subcomplex, which contains quinone binding site.

The 24-kDa subunit of the bovine mitochondrial NADH:ubiquinone oxidoreductase is a G protein.

Based on the results obtained from GTP overlay assay, immunoprecipitation, two dimensional electrophoresis and radiolabeled GTP binding, we provide evidence that the bona fide subunit of Complex I, the long known 24 kDa protein is a G protein. Bacterially expressed 24 kDa protein with additional N-terminal methionine and alanine residues or naturally expressed truncated isoform fail to bind GTP suggesting that secondary modification/ processed N-terminal end is necessary for GTP binding. Competitive inhibition of binding of radiolabeled GTP to electroblotted 24 kDa protein with unlabelled nucleotides showed that the protein binds GTP and GDP with high affinity in presence of Mg2+, and has decreased to very low affinity for ITP, CTP, GMP and UTP. A comparative binding of [gamma-35S]-GTP to Complex I and 24 kDa protein (electroblotted) suggests that the GTP binding in the native Complex is solely due to 24 kDa protein. Further, four fold difference in the binding affinities between native Complex I and 24 kDa protein (electroblotted) as seen by Scatchard analysis of the binding data indicates that protein undergoes structural rearrangement in Complex I bound form, that presumably triggers divalent cation dependent GTPase activity in native complex. We were unable to detect the effect of GTP/ GDP on the ubiquinone/ferricyanide reductase activity. Since the subunit is found missing in tissues affected by mitochondrial respiratory chain diseases, we presume that the subunit has regulatory role in the Complex I function in the electron transport chain.