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Query: EC:1.6.5.3 (complex I)
 8,901 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bovine-heart NADH:ubiquinone oxidoreductase (EC 1.6.5.3; Complex I) is the first and most complicated enzyme in the mitochondrial respiratory chain. Biochemistry textbooks and virtually all literature on this enzyme state that it contains one FMN and at least four iron-sulfur clusters. We show here that this statement is incorrect as it is based on erroneous protein determinations. Quantitative amino acid analysis of the bovine Complex I, to our knowledge the first reported thus far, shows that the routine protein-determination methods used for the bovine Complex I overestimate its protein content by up to twofold. The FMN content of the preparations was determined to be at least 1.3-1.4 mol FMN/mol Complex I. The spin concentration of the electron paramagnetic resonance (EPR) signal ascribed to iron-sulfur cluster N2 was determined and accounted for 1.3-1.6 clusters per molecule of Complex I. These results experimentally confirm the hypothesis [FEBS Lett. 485 (2000) 1] that the bovine Complex I contains two FMN groups and two clusters N2. Also the protein content of preparations of the soluble NAD(+)-reducing [NiFe]-hydrogenase (EC 1.12.1.2) from Ralstonia eutropha, which shows clear evolutionary relationships with Complex I, scores too high by the colorimetric protein-determination methods. Determination of the FMN content and the spin concentration of the EPR signal of the [2Fe-2S] cluster shows that this hydrogenase also contains two FMN groups. A third enzyme (Ech), the membrane-bound [NiFe]-hydrogenase from Methanosarcina barkeri which shows an even stronger evolutionary relationship with Complex I, behaves rather normal in protein determinations and contains no detectable acid-extractable FMN in purified preparations.
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PMID:Quantitative amino acid analysis of bovine NADH:ubiquinone oxidoreductase (Complex I) and related enzymes. Consequences for the number of prosthetic groups. 1261 47

The rotenone sensitive NADH:menaquinone oxidoreductase (NDH-I or complex I) from the thermohalophilic bacterium Rhodothermus marinus has been purified and characterized. Three of its subunits react with antibodies against 78, 51, and 21.3c kDa subunits of Neurospora crassa complex I. The optimum conditions for NADH dehydrogenase activity are 50 degrees C and pH 8.1, and the enzyme presents a KM of 9 microM for NADH. The enzyme also displays NADH:quinone oxidoreductase activity with two menaquinone analogs, 1,4-naphtoquinone (NQ) and 2,3-dimethyl-1,4-naphtoquinone (DMN), being the last one rotenone sensitive, indicating the complex integrity as purified. When incorporated in liposomes, a stimulation of the NADH:DMN oxidoreductase activity is observed by dissipation of the membrane potential, upon addition of CCCP. The purified enzyme contains 13.5 +/- 3.5 iron atoms and approximately 3.7 menaquinone per FMN. At least five iron-sulfur centers are observed by EPR spectroscopy: two [2Fe-2S](2+/1+) and three [4Fe-4S](2+/1+) centers. By fluorescence spectroscopy a still unidentified chromophore was detected in R. marinus complex I.
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PMID:Purification and characterization of the complex I from the respiratory chain of Rhodothermus marinus. 1267 33

The proton-pumping NADH:ubiquinone oxidoreductase, also called respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. One FMN and up to 9 iron-sulfur (Fe/S) clusters participate in the redox reaction. There is discussion that the EPR-detectable Fe/S cluster N2 is involved in proton pumping. However, the assignment of this cluster to a distinct subunit of the complex as well as the number of Fe/S clusters giving rise to the EPR signal are still under debate. Complex I from Escherichia coli consists of 13 polypeptides called NuoA to N. Either subunit NuoB or NuoI could harbor Fe/S cluster N2. Whereas NuoB contains a unique motif for the binding of one Fe/S cluster, NuoI contains a typical ferredoxin motif for the binding of two Fe/S clusters. Individual mutation of all four conserved cysteine residues in NuoB resulted in a loss of complex I activity and of the EPR signal of N2 in the cytoplasmic membrane as well as in the isolated complex. Individual mutations of all eight conserved cysteine residues of NuoI revealed a variable phenotype. Whereas cluster N2 was lost in most NuoI mutants, it was still present in the cytoplasmic membranes of the mutants NuoI C63A and NuoI C102A. N2 was also detected in the complex isolated from the mutant NuoI C102A. From this we conclude that the Fe/S cluster N2 is located on subunit NuoB.
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PMID:Iron-sulfur cluster N2 of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I) is located on subunit NuoB. 1297 62

Although isopentenyl diphosphate-dimethylallyl diphosphate isomerase is thought to be essential for archaea because they use the mevalonate pathway, its corresponding activity has not been detected in any archaea. A novel type of the enzyme, which has no sequence similarity to the known, well-studied type of enzymes, was recently reported in some bacterial strains. In this study, we describe the cloning of a gene of a homologue of the novel bacterial isomerase from a thermoacidophilic archaeon Sulfolobus shibatae. The gene was heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. The thermostable archaeal enzyme is tetrameric, and requires NAD(P)H and Mg2+ for activity, similar to its bacterial homologues. Using its apoenzyme, we were able to confirm that the archaeal enzyme is strictly dependent on FMN. Moreover, we provide evidence to show that the enzyme also has NADH dehydrogenase activity although it catalyzes the isomerase reaction without consuming any detectable amount of NADH.
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PMID:Type 2 isopentenyl diphosphate isomerase from a thermoacidophilic archaeon Sulfolobus shibatae. 1500 87

Type II NADH dehydrogenase of Corynebacterium glutamicum (NDH-2) was purified from an ndh overexpressing strain. Purification conferred 6-fold higher specific activity of NADH:ubiquinone-1 oxidoreductase with a 3.5-fold higher recovery than that previously reported (K. Matsushita et al., 2000). UV-visible and fluorescence analyses of the purified enzyme showed that NDH-2 of C. glutamicum contained non-covalently bound FAD but not covalently bound FMN. This enzyme had an ability to catalyze electron transfer from NADH and NADPH to oxygen as well as various artificial quinone analogs at neutral and acidic pHs respectively. The reduction of native quinone of C. glutamicum, menaquinone-2, with this enzyme was observed only with NADH, whereas electron transfer to oxygen was observed more intensively with NADPH. This study provides evidence that C. glutamicum NDH-2 is a source of the reactive oxygen species, superoxide and hydrogen peroxide, concomitant with NADH and NADPH oxidation, but especially with NADPH oxidation. Together with this unique character of NADPH oxidation, phylogenetic analysis of NDH-2 from various organisms suggests that NDH-2 of C. glutamicum is more closely related to yeast or fungal enzymes than to other prokaryotic enzymes.
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PMID:Electron transfer ability from NADH to menaquinone and from NADPH to oxygen of type II NADH dehydrogenase of Corynebacterium glutamicum. 1566 80

In this report, we have quantified the superoxide and H(2)O(2) production rates of intact rat brain and skeletal muscle mitochondria under condition of oxygen saturation applying p-hydroxyphenylacetate as fluorescent probe for H(2)O(2) generation and hydroethidine as probe for superoxide formation. The localisation of superoxide producing sites was determined by evaluating the effects of SOD addition. At comparable respiration rates and functional quality of mitochondria, we detected in brain mitochondria, a high reversed electron flow-dependent H(2)O(2) generation while the bc(1)-complex-dependent H(2)O(2) generation in the presence of succinate+antimycin was low. On the other hand, the reversed electron flow-dependent superoxide generation rate was small while the bc(1)-complex-dependent superoxide production was considerable. In contrast, isolated skeletal muscle mitochondria of comparable quality showed at almost comparable reversed electron flow-dependent H(2)O(2) generation more than 10-fold higher bc(1)-complex-dependent H(2)O(2) generation. Our data are compatible with the following suppositions: (i) The major ROS generation site in complex I visible during reversed electron flow (very likely the FMN moiety) is liberating superoxide predominantly to the mitochondrial matrix space. (ii) Similarly, the bc(1)-complex-dependent superoxide generation site (the semiquinone at center 'o') liberates superoxide with preference to the cytosolic space and (iii) Muscle mitochondria, most probably due to their higher endogenous CoQ content, generate at comparable maximal rates of respiration considerable larger amounts of superoxide at center 'o' of complex III.
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PMID:Characterization of superoxide production sites in isolated rat brain and skeletal muscle mitochondria. 1586 10

Generation of reactive oxygen species (ROS) is increasingly recognized as an important cellular process involved in numerous physiological and pathophysiological processes. Complex I (NADH:ubiquinone oxidoreductase) is considered as one of the major sources of ROS within mitochondria. Yet, the exact site and mechanism of superoxide production by this large membrane-bound multiprotein complex has remained controversial. Here we show that isolated complex I from Yarrowia lipolytica forms superoxide at a rate of 0.15% of the rate measured for catalytic turnover. Superoxide production is not inhibited by ubiquinone analogous inhibitors. Because mutant complex I lacking a detectable iron-sulfur cluster N2 exhibited the same rate of ROS production, this terminal redox center could be excluded as a source of electrons. From the effect of different ubiquinone derivatives and pH on this side reaction of complex I we concluded that oxygen accepts electrons from FMNH2 or FMN semiquinone either directly or via more hydrophilic ubiquinone derivatives.
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PMID:Superoxide radical formation by pure complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica. 1598 26

The NADH:menaquinone oxidoreductase (Nqo) is one of the enzymes present in the respiratory chain of the thermohalophilic bacterium Rhodothermus marinus. The genes coding for the R. marinus Nqo subunits were isolated and sequenced, clustering in two operons [nqo1 to nqo7 (nqoA) and nqo10 to nqo14 (nqoB)] and two independent genes (nqo8 and nqo9). Unexpectedly, two genes encoding homologues of a NhaD Na+/H+ antiporter (NhaD) and of a pterin-4alpha-carbinolamine dehydratase (PCD) were identified within nqoB, flanked by nqo13 and nqo14. Eight conserved motives to harbour iron-sulphur centres are identified in the deduced primary structures, as well as two consensus sequences to bind nucleotides, in this case NADH and FMN. Moreover, the open-reading-frames of the putative NhaD and PCD were shown to be co-transcribed with the other complex I genes encoded by nqoB. The possible role of these two genes in R. marinus complex I is discussed.
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PMID:A nhaD Na+/H+ antiporter and a pcd homologues are among the Rhodothermus marinus complex I genes. 1602 73

Two distinguishable activity bands for dye-linked l-proline dehydrogenase (PDH1 and PDH2) were detected when crude extract of the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 was run on a polyacrylamide gel. After purification, PDH1 was found to be composed of two different subunits with molecular masses of 56 and 43 kDa, whereas PDH2 was composed of four different subunits with molecular masses of 52, 46, 20 and 8 kDa. The native molecular masses of PDH1 and PDH2 were 440 and 101 kDa, respectively, indicating that PDH1 has an alpha4beta4 structure, while PDH2 has an alphabetagammadelta structure. PDH2 was found to be similar to the dye-linked l-proline dehydrogenase complex from Thermococcus profundus, but PDH1 is a different type of enzyme. After production of the enzyme in Escherichia coli, high-performance liquid chromatography showed the PDH1 complex to contain the flavins FMN and FAD as well as ATP. Gene expression and biochemical analyses of each subunit revealed that the beta subunit bound FAD and exhibited proline dehydrogenase activity, while the alpha subunit bound ATP, but unlike the corresponding subunit in the T. profundus enzyme, it exhibited neither proline dehydrogenase nor NADH dehydrogenase activity. FMN was not bound to either subunit, suggesting it is situated at the interface between the alpha and beta subunits. A comparison of the amino-acid sequences showed that the ADP-binding motif in the alpha subunit of PDH1 clearly differs from that in the alpha subunit of PDH2. It thus appears that a second novel dye-linked l-proline dehydrogenase complex is produced in P. horikoshii.
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PMID:A second novel dye-linked L-proline dehydrogenase complex is present in the hyperthermophilic archaeon Pyrococcus horikoshii OT-3. 1609 88

Mitochondrial superoxide (O(2)(.)) production is an important mediator of oxidative cellular injury. While NADH dehydrogenase (NDH) is a critical site of this O(2)(.) production; its mechanism of O(2)(.) generation is not known. Therefore, the catalytic function of NDH in the mediation of O(2)(.) generation was investigated by EPR spin-trapping. In the presence of NADH, O(2)(.) generation from NDH was observed and was inhibited by diphenyleneiodinium chloride (DPI), indicating involvement of the FMN-binding site of NDH. Addition of FMN increased O(2)(.) production. Destruction of the cysteine ligands of iron-sulfur clusters decreased O(2)(.) generation, suggesting a secondary role of this site. This inhibitory effect was reversed by addition of FMN. However, FMN addition could not reverse the inhibition of NDH by either DPI or heat denaturation, demonstrating involvement of both FMN and its FMN-binding protein moiety in the catalysis of O(2)(.) generation. O(2)(.) production by NDH also induced self-inactivation. Immunospin-trapping with anti-DMPO antibody and subsequent mass spectrometry was used to define the sites of oxidative damage of NDH. A DMPO adduct was detected on the 51-kDa subunit and was O(2)(.)-dependent. Alkylation of the cysteine residues of NDH significantly inhibited NDH-DMPO spin adduct formation, indicating involvement of protein thiyl radicals. LC/MS/MS analysis of a tryptic digest of the 51-kDa polypeptide revealed that cysteine (Cys(206)) and tyrosine (Tyr(177)) were specific sites of NDH-derived protein radical formation. Thus, two domains of the 51-kDa subunit, Gly(200)-Ala-Gly-Ala-Tyr-Ile-Cys(206)-Gly-Glu-Glu-Thr-Ala-Leu-Ile-Glu-Ser-Ile-Glu-Gly-Lys(219) and Ala(176)-Tyr(177)-Glu-Ala-Gly-Leu-Ile-Gly-Lys(184), were demonstrated to be susceptible to oxidative attack, and their oxidative modification results in decreased electron transfer activity.
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PMID:Superoxide generation from mitochondrial NADH dehydrogenase induces self-inactivation with specific protein radical formation. 1615 Jul 35


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