Kinetics of transhydrogenase reaction catalyzed by the mitochondrial NADH-ubiquinone oxidoreductase (Complex I) imply more than one catalytic nucleotide-binding sites.
(1/1100)
The steady-state kinetics of the transhydrogenase reaction (the reduction of acetylpyridine adenine dinucleotide (APAD+) by NADH, DD transhydrogenase) catalyzed by bovine heart submitochondrial particles (SMP), purified Complex I, and by the soluble three-subunit NADH dehydrogenase (FP) were studied to assess a number of the Complex I-associated nucleotide-binding sites. Under the conditions where the proton-pumping transhydrogenase (EC 1.6.1.1) was not operating, the DD transhydrogenase activities of SMP and Complex I exhibited complex kinetic pattern: the double reciprocal plots of the velocities were not linear when the substrate concentrations were varied in a wide range. No binary complex (ping-pong) mechanism (as expected for a single substrate-binding site enzyme) was operating within any range of the variable substrates. ADP-ribose, a competitive inhibitor of NADH oxidase, was shown to compete more effectively with NADH (Ki = 40 microM) than with APAD+ (Ki = 150 microM) in the transhydrogenase reaction. FMN redox cycling-dependent, FP catalyzed DD transhydrogenase reaction was shown to proceed through a ternary complex mechanism. The results suggest that Complex I and the simplest catalytically competent fragment derived therefrom (FP) possess more than one nucleotide-binding sites operating in the transhydrogenase reaction. (+info)
In the Nicotiana sylvestris CMSII mutant, a recombination-mediated change 5' to the first exon of the mitochondrial nad1 gene is associated with lack of the NADH:ubiquinone oxidoreductase (complex I) NAD1 subunit.
(2/1100)
We previously reported that the Nicotiana sylvestris CMSII mutant mitochondrial DNA carried a large deletion. Several expressed sequences, most of which are duplicated, and the unique copy of the nad7 gene encoding the NAD7 subunit of the NADH:ubiquinone oxidoreductase complex (complex I) are found in the deletion. Here, we show that the orf87-nad3-nad1/A cotranscription unit transcribed from a unique promoter element in the wild-type, is disrupted in CMSII. Nad3, orf87 and the promoter element are part of the deleted sequence, whilst the nad1/A sequence is present and transcribed from a new promoter brought by the recombination event, as indicated by Northern and primer extension experiments. However, Western analyses of mitochondrial protein fractions and of complex I purified using anti-NAD9 affinity columns, revealed that NAD1 is lacking in CMSII mitochondria. Our results suggest that translation of nad1 transcripts rather than transcription itself could be altered in the mutant. Consequences of lack of this submit belonging the membrane arm of complex I and thought to contain the ubiquinone-binding site, are discussed. (+info)
Mytilus mitochondrial DNA contains a functional gene for a tRNASer(UCN) with a dihydrouridine arm-replacement loop and a pseudo-tRNASer(UCN) gene.
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A 2500-nucleotide pair (ntp) sequence of F-type mitochondrial (mt) DNA of the Pacific Rim mussel Mytilus californianus (class Bivalvia, phylum Mollusca) that contains two complete (ND2 and ND3) and two partial (COI and COIII) protein genes and nine tRNA genes is presented. Seven of the encoded tRNAs (Ala, Arg, His, Met(AUA), Pro, Ser(UCN), and Trp) have the potential to fold into the orthodox four-armed tRNA secondary structure, while two [tRNASer(AGN) and a second tRNASer(UCN)] will fold only into tRNAs with a dihydrouridine (DHU) arm-replacement loop. Comparison of these mt-tRNA gene sequences with previously published, corresponding M. edulis F-type mtDNA indicates that similarity between the four-armed tRNASer(UCN) genes is only 63.8% compared with an average of 92.1% (range 86.2-98. 5%) for the remaining eight tRNA genes. Northern blot analysis indicated that mature tRNAs encoded by the DHU arm-replacement loop-containing tRNASer(UCN), tRNASer(AGN), tRNAMet(AUA), tRNATrp, and tRNAPro genes occur in M. californianus mitochondria, strengthening the view that all of these genes are functional. However, Northern blot and 5' RACE (rapid amplification of cDNA ends) analyses indicated that the four-armed tRNASer(UCN) gene is transcribed into a stable RNA that includes the downstream COI sequence and is not processed into a mature tRNA. On the basis of these observations the M. californianus and M. edulis four-armed tRNASer(UCN) sequences are interpreted as pseudo-tRNASer(UCN) genes. (+info)
-->H+/2e- stoichiometry in NADH-quinone reductase reactions catalyzed by bovine heart submitochondrial particles.
(4/1100)
Tightly coupled bovine heart submitochondrial particles treated to activate complex I and to block ubiquinol oxidation were capable of rapid uncoupler-sensitive inside-directed proton translocation when a limited amount of NADH was oxidized by the exogenous ubiquinone homologue Q1. External alkalization, internal acidification and NADH oxidation were followed by the rapidly responding (t1/2 < or = 1 s) spectrophotometric technique. Quantitation of the initial rates of NADH oxidation and external H+ decrease resulted in a stoichiometric ratio of 4 H+ vectorially translocated per 1 NADH oxidized at pH 8.0. ADP-ribose, a competitive inhibitor of the NADH binding site decreased the rates of proton translocation and NADH oxidation without affecting -->H+/2e- stoichiometry. Rotenone, piericidin and thermal deactivation of complex I completely prevented NADH-induced proton translocation in the NADH-endogenous ubiquinone reductase reaction. NADH-exogenous Q1 reductase activity was only partially prevented by rotenone. The residual rotenone- (or piericidin-) insensitive NADH-exogenous Q1 reductase activity was found to be coupled with vectorial uncoupler-sensitive proton translocation showing the same -->H+/2e- stoichiometry of 4. It is concluded that the transfer of two electrons from NADH to the Q1-reactive intermediate located before the rotenone-sensitive step is coupled with translocation of 4 H+. (+info)
A single external enzyme confers alternative NADH:ubiquinone oxidoreductase activity in Yarrowia lipolytica.
(5/1100)
NADH:ubiquinone oxidoreductases catalyse the first step within the diverse pathways of mitochondrial NADH oxidation. In addition to the energy-conserving form commonly called complex I, fungi and plants contain much simpler alternative NADH:ubiquinone oxido-reductases that catalyze the same reaction but do not translocate protons across the inner mitochondrial membrane. Little is known about the distribution and function of these enzymes. We have identified YLNDH2 as the only gene encoding an alternative NADH:ubiquinone oxidoreductase (NDH2) in the obligate aerobic yeast Yarrowia lipolytica. Cells carrying a deletion of YLNDH2 were fully viable; full inhibition by piericidin A indicated that complex I activity was the sole NADH:ubiquinone oxidoreductase activity left in the deletion strains. Studies with intact mitochondria revealed that NDH2 in Y. lipolytica is oriented towards the external face of the mitochondrial inner membrane. This is in contrast to the situation seen in Saccharomyces cerevisiae, Neurospora crassa and in green plants, where internal alternative NADH:ubiquinone oxidoreductases have been reported. Phylogenetic analysis of known NADH:ubiquinone oxidoreductases suggests that during evolution conversion of an ancestral external alternative NADH:ubiquinone oxidoreductase to an internal enzyme may have paved the way for the loss of complex I in fermenting yeasts like S. cerevisiae. (+info)
Type 2 NADH dehydrogenases in the cyanobacterium Synechocystis sp. strain PCC 6803 are involved in regulation rather than respiration.
(6/1100)
Analysis of the genome of Synechocystis sp. strain PCC 6803 reveals three open reading frames (slr0851, slr1743, and sll1484) that may code for type 2 NAD(P)H dehydrogenases (NDH-2). The sequence similarity between the translated open reading frames and NDH-2s from other organisms is low, generally not exceeding 30% identity. However, NAD(P)H and flavin adenine dinucleotide binding motifs are conserved in all three putative NDH-2s in Synechocystis sp. strain PCC 6803. The three open reading frames were cloned, and deletion constructs were made for each. An expression construct containing one of the three open reading frames, slr1743, was able to functionally complement an Escherichia coli mutant lacking both NDH-1s and NDH-2s. Therefore, slr0851, slr1743, and sll1484 have been designated ndbA, ndbB, and ndbC, respectively. Strains that lacked one or more of the ndb genes were created in wild-type and photosystem (PS) I-less backgrounds. Deletion of ndb genes led to small changes in photoautotrophic growth rates and respiratory activities. Electron transfer rates into the plastoquinone pool in thylakoids in darkness were consistent with the presence of a small amount of NDH-2 activity in thylakoids. No difference was observed between wild-type and the Ndb-less strains in the banding patterns seen on native gels when stained for either NADH or NADPH dehydrogenase activity, indicating that the Ndb proteins do not accumulate to high levels. A striking phenotype of the PS I-less background strains lacking one or more of the NDH-2s is that they were able to grow at high light intensities that were lethal to the control strain but they retained normal PS II activity. We suggest that the Ndb proteins in Synechocystis sp. strain PCC 6803 are redox sensors and that they play a regulatory role responding to the redox state of the plastoquinone pool. (+info)
Mitochondrial electron transport complex I is a potential source of oxygen free radicals in the failing myocardium.
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Oxidative stress in the myocardium may play an important role in the pathogenesis of congestive heart failure (HF). However, the cellular sources and mechanisms for the enhanced generation of reactive oxygen species (ROS) in the failing myocardium remain unknown. The amount of thiobarbituric acid reactive substances increased in the canine HF hearts subjected to rapid ventricular pacing for 4 weeks, and immunohistochemical staining of 4-hydroxy-2-nonenal ROS-induced lipid peroxides was detected in cardiac myocytes but not in interstitial cells of HF animals. The generation of superoxide anion was directly assessed in the submitochondrial fractions by use of electron spin resonance spectroscopy with spin trapping agent, 5, 5'-dimethyl-1-pyrroline-N-oxide, in the presence of NADH and succinate as a substrate for NADH-ubiquinone oxidoreductase (complex I) and succinate-ubiquinone oxidoreductase (complex II), respectively. Superoxide production was increased 2.8-fold (P<0.01) in HF, which was due to the functional block of electron transport at complex I. The enzymatic activity of complex I decreased in HF (274+/-13 versus 136+/-9 nmol. min(-1). mg(-1) protein, P<0.01), which may thus have caused the functional uncoupling of the respiratory chain and the deleterious ROS production in HF mitochondria. The present study provided direct evidence for the involvement of ROS in the mitochondrial origin of HF myocytes, which might be responsible for both contractile dysfunction and structural damage to the myocardium. (+info)
Chloromethyltetramethylrosamine (Mitotracker Orange) induces the mitochondrial permeability transition and inhibits respiratory complex I. Implications for the mechanism of cytochrome c release.
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We have investigated the interactions with isolated mitochondria and intact cells of chloromethyltetramethylrosamine (CMTMRos), a probe (Mitotracker Orange) that is increasingly used to monitor the mitochondrial membrane potential (Deltapsi(m)) in situ. CMTMRos binds to isolated mitochondria and undergoes a large fluorescence quenching. Most of the binding is energy-independent and can be substantially reduced by sulfhydryl reagents. A smaller fraction of the probe is able to redistribute across the inner membrane in response to a membrane potential, with further fluorescence quenching. Within minutes, however, this energy-dependent fluorescence quenching spontaneously reverts to the same level obtained by treating mitochondria with the uncoupler carbonylcyanide-p-trifluoromethoxyphenyl hydrazone. We show that this event depends on inhibition of the mitochondrial respiratory chain at complex I and on induction of the permeability transition pore by CMTMRos, with concomitant depolarization, swelling, and release of cytochrome c. After staining cells with CMTMRos, depolarization of mitochondria in situ with protonophores is accompanied by changes of CMTMRos fluorescence that range between small and undetectable, depending on the probe concentration. A lasting decrease of cellular CMTMRos fluorescence associated with mitochondria only results from treatment with thiol reagents, suggesting that CMTMRos binding to mitochondria in living cells largely occurs at SH groups via the probe chloromethyl moiety irrespective of the magnitude of Deltapsi(m). Induction of the permeability transition precludes the use of CMTMRos as a reliable probe of Deltapsi(m) in situ and demands a reassessment of the conclusion that cytochrome c release can occur without membrane depolarization and/or onset of the permeability transition. (+info)