The reaction of NADPH with bovine mitochondrial NADH:ubiquinone oxidoreductase revisited

The first purification of bovine NADH:ubiquinone oxidoreductase (Complex I) was reported nearly half a century ago (Hatefi et al. J Biol Chem 237:1676–1680, 1962). The pathway of electron-transfer through the enzyme is still under debate. A major obstacle is the assignment of EPR signals to the individual iron-sulfur clusters in the subunits. The preceding paper described a working model based on the kinetics with NADPH. This model is at variance with current views in the field. The present paper provides a critical overview on the possible causes for the discrepancies. It is concluded that the stability of all purified preparations described thus far, including Hatefi’s Complex I, is compromised due to removal of the enzyme from the protective membrane environment. In addition, most preparations described during the last two decades are purified by methods involving synthetic detergents and column chromatography. This results in delipidation, loss of endogenous quinones and loss of reactions with (artificial) quinones in a rotenone-sensitive way. The Fe:FMN ratio’s indicate that FMN-a is absent, but that all Fe-S clusters may be present. In contrast to the situation in bovine SMP and Hatefi’s Complex I, three of the six expected [4Fe-4S] clusters are not detected in EPR spectra. Qualitatively, the overall EPR lineshape of the remaining three cubane signals may seem similar to that of Hatefi’s Complex I, but quantitatively it is not. It is further proposed that point mutations in any of the TYKY, PSST, 49-kDa or 30-kDa subunits, considered to make up the delicate structural heart of Complex I, may have unpredictable effects on any of the other subunits of this quartet. The fact that most point mutations led to inactive enzymes makes a correct interpretation of such mutations even more ambiguous. In none of the Complex-I-containing membrane preparations from non-bovine origin, the pH dependencies of the NAD(P)H→O₂ reactions and the pH-dependent reduction kinetics of the Fe-S clusters with NADPH have been determined. This excludes a proper discussion on the absence or presence of FMN-a in native Complex I from other organisms.     

Post-translational Modifications near the Quinone Binding Site of Mammalian Complex I

Complex I (NADH:ubiquinone oxidoreductase) in mammalian mitochondria is an L-shaped assembly of 44 protein subunits with one arm buried in the inner membrane of the mitochondrion and the orthogonal arm protruding about 100 Å into the matrix. The protruding arm contains the binding sites for NADH, the primary acceptor of electrons flavin mononucleotide (FMN), and a chain of seven iron-sulfur clusters that carries the electrons one at a time from FMN to a coenzyme Q molecule bound in the vicinity of the junction between the two arms. In the structure of the closely related bacterial enzyme from Thermus thermophilus, the quinone is thought to bind in a tunnel that spans the interface between the two arms, with the quinone head group close to the terminal iron-sulfur cluster, N2. The tail of the bound quinone is thought to extend from the tunnel into the lipid bilayer. In the mammalian enzyme, it is likely that this tunnel involves three of the subunits of the complex, ND1, PSST, and the 49-kDa subunit. An arginine residue in the 49-kDa subunit is symmetrically dimethylated on the ω-N^G and ω-N^G′ nitrogen atoms of the guanidino group and is likely to be close to cluster N2 and to influence its properties. Another arginine residue in the PSST subunit is hydroxylated and probably lies near to the quinone. Both modifications are conserved in mammalian enzymes, and the former is additionally conserved in Pichia pastoris and Paracoccus denitrificans, suggesting that they are functionally significant.     

Pyridine nucleotide specificity and other properties of purified nitrate reductase from Chlorella variegata

Nitrate reductase (NR) (EC 1.6.6.2) from Chlorella variegata 211/10d has been purified by blue sepharose affinity chromatography. The enzyme can utilise NADH or NADPH for nitrate reduction with apparent K _m values of 11.5 M and 14.5 M, respectively. Apparent K _m values for nitrate are 0.13 mM (NADH-NR) and 0.14 mM (NADPH-NR). The diaphorase activity of the enzyme is inhibited strongly by parachloromercuribenzoic acid; NADH or NADPH protects the enzyme against this inhibition. NR proper activity of the enzyme is partially inactive after extraction and may be activated after the addition of ferricyanide. The addition of NAD(P)H and cyanide causes a reversible inactivation of the NR proper activity although preincubation with either NADH or NADH and ADP has no significant effect.Abbreviations NR Nitrate reductase - FAD Flavin-adenine dinucleotide - FMN Riboflavin 5-phosphate - p-CMB para-Chloromercuribenzoic - BV Benzyl viologen     

Azoreductase from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> AS1.1737 is a flavodoxin that also functions as nitroreductase and flavin mononucleotide reductase

Previously reported azoreductase (AZR) from Rhodobacter sphaeroides AS1.1737 was shown to be a flavodoxin possessing nitroreductase and flavin mononucleotide (FMN) reductase activities. The structure model of AZR constructed with SWISS-MODEL displayed a flavodoxin-like fold with a three-layer α/β/α structure. With nitrofurazone as substrate, the optimal pH value and temperature were 7.0 and 50°C, respectively. AZR could reduce a number of nitroaromatic compounds including 2,4-dinitrotoluene, 2,6-dinitrotoluene, 3,5-dinitroaniline, and 2,4,6-trinitrotoluene (TNT). TNT resulted to be the most efficient nitro substrate and was reduced to hydroxylamino-dinitrotoluene. Both NADH and NADPH could serve as electron donors of AZR, where the latter was preferred. Externally added FMN was also reduced by AZR via ping-pong mechanism and was a competitive inhibitor of NADPH, methyl red, and nitrofurazone. AZR with broad substrate specificity is a member of a new nitro/FMN reductase family demonstrating potential application in bioremediation.     

Purification and characterization of the complex I from the respiratory chain of Rhodothermus marinus

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°C and pH 8.1, and the enzyme presents a K _M of 9 M 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 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.     

Evidence for two independent pathways of electron transfer in mitochondrial NADH:Q oxidoreductase. I. Pre-steady-state kinetics with NADPH

The reduction of NADH:Q oxidoreductase by NADPH occurring in submitochondrial particles has been studied with the freeze-quench technique. It was found that 50% of the Fe-S clusters 2, 3 and 4 could be reduced by NADPH within 30 ms at pH 6.5. The remainder of the clusters, including cluster 1, were reduced slowly and incompletely; it was concluded that these clusters play no role in the NADPH oxidase activity. Nearly the same results were obtained at pH 8 under anaerobic conditions, demonstrating that the rate of reaction of NADPH with the enzyme was essentially the same at both pH values. The rate and extent of reduction of half of the clusters 2 by NADPH at pH 8 were not affected by the presence of O2 of rotenone. This implies a pH-dependent oxidation of the enzyme as the cause for the absence of the NADPH oxidase activity at this pH. A dimeric model of the enzyme is proposed in which one protomer, containing FMN and the Fe-S clusters 1-4 in stoichiometric amounts, is responsible for NADH oxidation at pH 8. This protomer cannot react with NADPH. The other protomer, containing only FMN and the clusters 2, 3 and 4, is supposed to catalyse the oxidation of NADPH. The oxidation of this protomer by ubiquinone is expected to be strongly dependent on pH. This protomer might also catalyse NADH oxidation at pH 6-6.5.     

Function of conserved acidic residues in the PSST homologue of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica

Proton-translocating NADH:ubiquinone oxidoreductase (complex I) is the largest and least understood enzyme of the respiratory chain. Complex I from bovine mitochondria consists of more than forty different polypeptides. Subunit PSST has been suggested to carry iron-sulfur center N-2 and has more recently been shown to be involved in inhibitor binding. Due to its pH-dependent midpoint potential, N-2 has been proposed to play a central role both in ubiquinone reduction and proton pumping. To obtain more insight into the functional role of PSST, we have analyzed site-directed mutants of conserved acidic residues in the PSST homologous subunit of the obligate aerobic yeast Yarrowia lipolytica. Mutations D136N and E140Q provided functional evidence that conserved acidic residues in PSST play a central role in the proton translocating mechanism of complex I and also in the interaction with the substrate ubiquinone. When Glu(89), the residue that has been suggested to be the fourth ligand of iron-sulfur center N-2 was changed to glutamine, alanine, or cysteine, the EPR spectrum revealed an unchanged amount of this redox center but was shifted and broadened in the g(z) region. This indicates that Glu(89) is not a ligand of N-2. The results are discussedin the light of structural similarities to the homologous [NiFe] hydrogenases.     

Evidence for two independent pathways of electron transfer in mitochondrial NADH:Q oxidoreductase. I. Pre-steady-state kinetics with NADPH

The reduction of NADH:Q oxidoreductase by NADPH occurring in submitochondrial particles has been studied with the freeze-quench technique. It was found that 50% of the Fe-S clusters 2, 3 and 4 could be reduced by NADPH within 30 ms at pH 6.5. The remainder of the clusters, including cluster 1, were reduced slowly and incompletely; it was concluded that these clusters play no role in the NADPH oxidase activity. Nearly the same results were obtained at pH 8 under anaerobic conditions, demonstrating that the rate of reaction of NADPH with the enzyme was essentially the same at both pH values. The rate and extent of reduction of half of the clusters 2 by NADPH at pH 8 were not affected by the presence of O₂ of rotenone. This implies a pH-dependent oxidation of the enzyme as the cause for the absence of the NADPH oxidase activity at this pH. A dimeric model of the enzyme is proposed in which one protomer, containing FMN and the Fe-S clusters 1–4 in stoichiometric amounts, is responsible for NADH oxidation at pH 8. This protomer cannot react with NADPH. The other protomer, containing only FMN and the clusters 2, 3 and 4, is supposed to catalyse the oxidation of NADPH. The oxidation of this protomer by ubiquinone is expected to be strongly dependent on pH. This protomer might also catalyse NADH oxidation at pH 6–6.5.     

Purification and molecular and enzymic properties of mitochondrial NADH dehydrogenase.

Mitochondrial NADH dehydrogenase has been purified to homogeneity by resolution of Complex I from beef heart mitochondria with the chaotrope NaClO₄ and precipitation of the enzyme with ammonium sulfate. The enzyme is water-soluble, has a molecular weight of 69,000 ± 1000 as determined by gel filtration on Sephadex G-100 and agarose 1.5 M. It is an iron-sulfur flavoprotein, with the ratio of flavin (FMN) to nonheme iron to labile sulfide being 1:5–6:5–6. The FMN content suggests a minimum molecular weight of 74,000 ± 3000 for the enzyme. NADH dehydrogenase is composed of three subunits with apparent M_r values, as determined by acrylamide gel electrophoresis as well as by gel filtration on agarose 5 M both in the presence of sodium dodecyl sulfate, of about 51,000, 24,000, and 9–10,000. Coomassie blue stain intensities of the subunits on acrylamide gels suggest that they are present in NADH dehydrogenase in equimolar amounts. However, summation of the apparent M_r values of the dodecyl sulfate-treated subunits appears to overestimate the molecular weight of the native enzyme. The amino acid compositions of NADH dehydrogenase and of each of the isolated and purified subunits have been determined. NADH dehydrogenase catalyzes the oxidation of NADH and NADPH by quinones, ferric compounds, and NAD (3-acetylpyridine adenine dinucleotide was used). All the activities of NADH dehydrogenase are greatly stimulated by addition of guanidine (up to 150 mm), alkylguanidines, arginine, and arginine methyl ester to the assay medium. Phosphoarginine had no effect. These results pointed to the importance of the positively charged guanido group, which appears to interact with and neutralize the negative charges on NAD(P)H and thereby allow for better enzyme-substrate interaction. In the absence of guanidine, NADPH is essentially unoxidized by the enzyme at pH values above 6.0. However, both NADPH dehydrogenase and NADPH → NAD transhydrogenase activities increase dramatically as the assay pH is lowered below pH = 6. Since the pK of the 2′-phosphate of NADPH is 6.1, it appears that the above pH effect is related to protonation of the 2′-phosphate, thus rendering NADPH a closer electronic analog of NADH, which is the primary substrate of the enzyme.     

Oxydation von reduziertem Nicotinamid-Adenin-Dinucleotid in Rhodospirillum rubrum

Zusammenfassung Es wird die 25–30 fache Anreicherung einer löslichen NADH-Dehydrogenase [NADH: (acceptor) oxidoreductase, EC 1.6.99.3.) aus R. rubrum durch Gelfiltration an Sephadex G 200 und Chromatographie an DEAE-Cellulose beschrieben. Das Enzym ist bei DEAE-Cellulosechromatographie nur in Gegenwart von FMN oder NADH stabil. Menadion, Ferricyanid, DCPIP, p-Benzochinon und Cytochrom c sind als Elektronenacceptoren wirksam. Cytochrom c ist ein schlechter Acceptor. Das pH-Optimum der Reaktion liegt bei 8,4. Km für NADH ist 3,0 · 10^-5 m. NADPH wird nur mit etwa 3–5% des Wertes von NADH umgesetzt. Die prosthetische Gruppe des Enzyms ist FMN, Km für FMN ist 0,3 · 10^-7 m. Das Enzymprotein wird bei Verdünnung in 0,05 m Puffer inaktiviert; FMN und FAD sowie NADH und NADPH haben einen stabilisierenden Effekt. Durch höhere Pufferkonzentrationen wird das Enzym zunehmend inaktiviert. Die Inaktivierung besteht in einer Labilisierung oder Abspaltung der prosthetischen Gruppe vom Enzymmolekül. Verschiedene Metalle inaktivieren das Enzym ebenfalls.

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Oxidation of reduced nicotinamide adenine dinucleotide in Rhodospirillum rubrum I. Characterization of a soluble NADH dehydrogenase

Summary A soluble NADH^* Dehydrogenase [NADH: (acceptor) oxidoreductase, EC 1.6.99.3.] has been purified 25–30 fold from R. rubrum by gelfiltration with Sephadex G 200 and ionexchange chromatography on DEAE-Cellulose. During the second purification step the enzyme is stable only in the presence of either FMN or NADH. Electronacceptors which were found to be effective include menadione, ferricyanide, DCPIP, p-benzoquinone and cytochrome c, the latter substance being a poor acceptor. The optimum pH of the reaction is at about 8.4. Km for NADH is 3.0×10^-5 m. NADPH is oxidized at only about 3–5% the rate of NADH. The active prosthetic group of the enzyme is FMN with an appearant Km of 0.3×10^-7 m. When diluted in 0.05 m buffer the enzyme becomes rapidly inactivated. FMN, FAD and also NADH and NADPH exhibit a stabilizing protective effect. Higher concentrations of buffer cause increasing inactivation. The mechanism of the inactivation is thought to be a labilization or detachment of the prosthetic group from the enzyme molecule. Several metals also inactivate the enzyme.

     

Kinetic and functional characterization of a membrane-bound NAD(P)H dehydrogenase located in the chloroplasts of Pleurochloris meiringensis (Xanthophyceae)

Using isolated chloroplasts or purified thylakoids from photoautotrophically grown cells of the chromophytic alga Pleurochloris meiringensis (Xanthophyceae) we were able to demonstrate a membrane bound NAD(P)H dehydrogenase activity. NAD(P)H oxidation was detectable with menadione, coenzyme Q₀, decylplastoquinone and decylubiquinone as acceptors in an in vitro assay. K _m-values for both pyridine nucleotides were in the molar range (K _m[NADH]=9.8 M, K _m[NADPH]=3.2 M calculated according to Lineweaver-Burk). NADH oxidation was optimal at pH 9 while pH dependence of NADPH oxidation showed a main peak at 9.8 and a smaller optimum at pH 7.5–8. NADH oxidation could be completely inhibited with rotenone, an inhibitor of mitochondrial complex I dehydrogenase, while NADPH oxidation revealed the typical inhibition pattern upon addition of oxidized pyridine nucleotides reported for ferredoxin: NADP⁺ reductase. Partly-denaturing gel electrophoresis followed by NAD(P)H dehydrogenase activity staining showed that NADPH and NADH oxidizing proteins had different electrophoretic mobilities. As revealed by denaturing electrophoresis, the NADH oxidizing enzyme had one main subunit of 22 kDa and two further polypeptides of 29 and 44 kDa, whereas separation of the NADPH depending protein yielded five bands of different molecular weight. Measurement of oxygen consumption due to PS I mediated methylviologen reduction upon complete inhibition of PS II showed that the NAD(P)H dehydrogenase is able to catalyze an input of electrons from NADH to the photosynthetic electron transport chain in case of an oxidized plastoquinone-pool. We suggest ferredoxin: NADP⁺ reductase to be the main NADPH oxidizing activity while a thylakoidal NAD(P)H: plastoquinone oxidoreductase involved in the chlororespiratory pathway in the dark acts mainly as an NADH oxidizing enzyme.Abbreviations Coenzyme Q₀-2,3-dimethoxy-5-methyl-1,4-benzoquinone - FNR ferredoxin: NADP⁺ reductase - MD menadione - MV methylviologen - NDH NAD(P)H dehydrogenase - PQ plastoquinone - PQ₁₀ decylplastoquinone - SDH succinate dehydrogenase - UQ₁₀ decylubiquinone (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone)     

Mammalian NADH:ubiquinone oxidoreductase (Complex I) and nicotinamide nucleotide transhydrogenase (Nnt) together regulate the mitochondrial production of H<Subscript>2</Subscript>O<Subscript>2</Subscript>—Implications for their role in disease,especially cancer

Mammalian NADH:ubiquinone oxidoreductase (Complex I) in the mitochondrial inner membrane catalyzes the oxidation of NADH in the matrix. Excess NADH reduces nine of the ten prosthetic groups of the enzyme in bovine-heart submitochondrial particles with a rate of at least 3,300 s⁻¹. This results in an overall NADH→O₂ rate of ca. 150 s⁻¹. It has long been known that the bovine enzyme also has a specific reaction site for NADPH. At neutral pH excess NADPH reduces only three to four of the prosthetic groups in Complex I with a rate of 40 s⁻¹ at 22 °C. The reducing equivalents remain essentially locked in the enzyme because the overall NADPH→O₂ rate (1.4 s⁻¹) is negligible. The physiological significance of the reaction with NADPH is still unclear. A number of recent developments has revived our thinking about this enigma. We hypothesize that Complex I and the Δp-driven nicotinamide nucleotide transhydrogenase (Nnt) co-operate in an energy-dependent attenuation of the hydrogen-peroxide generation by Complex I. This co-operation is thought to be mediated by the NADPH/NADP⁺ ratio in the vicinity of the NADPH site of Complex I. It is proposed that the specific H₂O₂ production by Complex I, and the attenuation of it, is of importance for apoptosis, autophagy and the survival mechanism of a number of cancers. Verification of this hypothesis may contribute to a better understanding of the regulation of these processes.     

Structural Basis for the Mechanism of Respiratory Complex I

Complex I plays a central role in cellular energy production, coupling electron transfer between NADH and quinone to proton translocation. The mechanism of this highly efficient enzyme is currently unknown. Mitochondrial complex I is a major source of reactive oxygen species, which may be one of the causes of aging. Dysfunction of complex I is implicated in many human neurodegenerative diseases. We have determined several x-ray structures of the oxidized and reduced hydrophilic domain of complex I from Thermus thermophilus at up to 3.1 Å resolution. The structures reveal the mode of interaction of complex I with NADH, explaining known kinetic data and providing implications for the mechanism of reactive oxygen species production at the flavin site of complex I. Bound metals were identified in the channel at the interface with the frataxin-like subunit Nqo15, indicating possible iron-binding sites. Conformational changes upon reduction of the complex involve adjustments in the nucleotide-binding pocket, as well as small but significant shifts of several α-helices at the interface with the membrane domain. These shifts are likely to be driven by the reduction of nearby iron-sulfur clusters N2 and N6a/b. Cluster N2 is the electron donor to quinone and is coordinated by unique motif involving two consecutive (tandem) cysteines. An unprecedented “on/off switch” (disconnection) of coordinating bonds between the tandem cysteines and this cluster was observed upon reduction. Comparison of the structures suggests a novel mechanism of coupling between electron transfer and proton translocation, combining conformational changes and protonation/deprotonation of tandem cysteines.Complex I (NADH:ubiquinone oxidoreductase, EC 1.6.5.3) is the first enzyme of the mitochondrial and bacterial respiratory chains. It catalyzes the transfer of two electrons from NADH to quinone, coupled to the translocation of approximately four protons across the membrane, contributing to the proton-motive force required for the synthesis of ATP (1, 2). The mitochondrial enzyme consists of 45 subunits (3) with a combined mass of ∼980 kDa. The prokaryotic enzyme is simpler, consisting of ∼14 subunits conserved from bacteria to humans, and has a total mass of ∼550 kDa (2). The mitochondrial and bacterial enzymes contain equivalent redox components and have a similar L-shaped structure, with the hydrophobic arm embedded in the membrane and the hydrophilic peripheral arm protruding into the mitochondrial matrix or the bacterial cytoplasm (2, 4). Thus, the bacterial enzyme represents a “minimal” model of complex I. Because of the central role of complex I in respiration, mutations in individual subunits can lead to many human neurodegenerative diseases (5). Complex I, along with complex III (bc₁), has been suggested to be a major source of reactive oxygen species (ROS)² in mitochondria, which can damage mitochondrial DNA and may be one of the causes of aging (6). Parkinson disease, at least in its sporadic form (which represents ∼95% of cases), may be caused by increased ROS production from malfunctioning complex I (7).We have previously determined the crystal structure of the hydrophilic domain (eight different subunits of 280 kDa total mass) of complex I from Thermus thermophilus, establishing the electron transfer pathway from the primary electron acceptor flavin mononucleotide (FMN) through seven conserved iron-sulfur clusters to the quinone-binding site (Q-site) at the interface with the membrane domain (8). Two additional iron-sulfur clusters, which are not part of the main redox chain, may represent an evolutionary remnant (cluster N7) and a possible anti-oxidant (cluster N1a; cluster names are assigned to structural motifs as in Ref. 8). The membrane-spanning part of the enzyme lacks covalently bound prosthetic groups (9) but must contain essential components of the proton translocating machinery. Its atomic structure is currently unknown.The mechanism of the highly efficient coupling between electron transfer and proton pumping, conserving nearly 100% of the available energy, remains a mystery. Two models are being discussed: direct (redox-driven through chemical intermediates, usually employing modifications of the Q cycle, with quinol as a mobile proton/electron carrier) (10) and indirect or conformation-driven coupling (2, 4, 11, 12). Sequence comparisons indicate that the three largest hydrophobic subunits of complex I, Nqo12, 13, and 14 (Thermus nomenclature), are homologous to each other and to the antiporter family (Mrp) (13, 14) and so are likely to participate in proton translocation. Two of these subunits, Nqo12 and Nqo13, are located ∼100 Å away from the Q-site (15), which implies the need for conformational coupling as at least a part of the mechanism. We have now determined several structures of the oxidized and reduced hydrophilic domain of complex I from T. thermophilus, which show how NADH interacts with the complex and provide novel insights into the coupling mechanism.     

The reconstitution of the mitochondrial energy-linked transhydrogenase.

Complex I (NADH-ubiquinone reductase) catalyzes pyridine nucleotide transhydrogenase at rates several fold higher than those found in submitochondrial particles from bovine heart. An ATP-dependent reduction of NADP⁺ by NADH was demonstrated after combination of Complex I with phospholipids, hydrophobic proteins derived from bovine heart mitochondria, and mitochondrial ATPase (F₁)¹. The reaction was inhibited by oligomycin, uncoupling agents and low concentrations of Triton X-100.     

Molecular study of the rat liver NADH: Cytochrome c oxidoreductase complex during development and aging

The mechanisms involved in ageing are yet to be fully understood but it is thought that changes produced in energy transfer pathways occurring in the mitochondria may be responsible for the lack of energy typical of the later stages of life. The aim of the present investigation was to determine the enzymatic activity of the liver NADH cytochrome c oxidorectuctase complex (Complex I-III) in mitochondria isolated from the liver of rats of 3 different age groups: lactating, animals (15-17 days), adult females (3-5 months) and old animals (26-30 months). The activities of the unbound Complexes I and III were also determined.An increase in Complex I-III activity was detected during development (142 ± 10 vs. 447 ± 23 mol cyt. c/mg/min, p < 0.001) ang ageing (447 ± 23 vs. 713 ± 45 mol cyt. c/mg//min, p < 0.001). However, unbound Complex I showed a reduction in activity during the ageing period whilst Complex III activity moderately increased. Immunological studies indicated only a moderate increase in the amount of Complex I-III and studies on the purified complex suggested that the increase in activity was due to effects other than an increase in enzyme quantity. The analysis of protein bands and the quantification of prosthetic groups showed particular reductions in the relative concentrations of Complex I subunits including the 51 kDa unit, which binds FMN, confirmed by a similar reduction in levels of the nucleotide. In contrast, 4 of the 5 subunits which increased during the lifetime of the animals corresponded to those of Complex III. These subunits are responsible for the binding of catalytic groups. The results suggest that, in addition to the increase in the amount of enzyme, binding factors between Complexes I and III may also play an important role in the observed increase in Complex I-III activity.     

A single amino acid residue controls ROS production in the respiratory Complex I from Escherichia coli

Reactive oxygen species (ROS) production by respiratory Complex I from Escherichia coli was studied in bacterial membrane fragments and in the isolated and purified enzyme, either solubilized or incorporated in proteoliposomes. We found that the replacement of a single amino acid residue in close proximity to the nicotinamide adenine dinucleotide (NADH)‐binding catalytic site (E95 in the NuoF subunit) dramatically increases the reactivity of Complex I towards dioxygen (O₂). In the E95Q variant short‐chain ubiquinones exhibit strong artificial one‐electron reduction at the catalytic site, also leading to a stronger increase in ROS production. Two mechanisms can contribute to the observed kinetic effects: (a) a change in the reactivity of flavin mononucleotide (FMN) towards dioxygen at the catalytic site, and (b) a change in the population of the ROS‐generating state. We propose the existence of two (closed and open) states of the NAD⁺‐bound enzyme as one feature of the substrate‐binding site of Complex I. The analysis of the kinetic model of ROS production allowed us to propose that the population of Complex I with reduced FMN is always low in the wild‐type enzyme even at low ambient redox potentials, minimizing the rate of reaction with O₂ in contrast to E95Q variant.     

\vec H^ + /2\bar e Stoichiometry of the NADH:Ubiquinone Reductase Reaction Catalyzed by Submitochondrial Particles

Mitochondrial NADH:ubiquinone-reductase (Complex I) catalyzes proton translocation into inside-out submitochondrial particles. Here we describe a method for determining the stoichiometric ratio (n) for the coupled reaction of NADH oxidation by the quinone acceptors. Comparison of the initial rates of NADH oxidation and alkalinization of the surrounding medium after addition of small amounts of NADH to coupled particles in the presence of Q₁ gives the value of n = 4. Thermally induced deactivation of Complex I [1,2] results in complete inhibition of the NADH oxidase reaction but only partial inhibition of the NADH:Q₁-reductase reaction. N-Ethylmaleimide (NEM) prevents reactivation and thus completely blocks the thermally deactivated enzyme. The residual NADH:Q₁-reductase activity of the deactivated, NEM-treated enzyme is shown to be coupled with the transmembraneous proton translocation (n = 4). Thus, thermally induced deactivation of Complex I as well as specific inhibitors of the endogenous ubiquinone reduction (rotenone, piericidin A) do not inhibit the proton translocating activity of the enzyme.     

Quantitative amino acid analysis of bovine NADH:ubiquinone oxidoreductase (Complex I) and related enzymes. Consequences for the number of prosthetic groups

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.     

A central functional role for the 49-kDa subunit within the catalytic core of mitochondrial complex I

We have analyzed a series of eleven mutations in the 49-kDa protein of mitochondrial complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica to identify functionally important domains in this central subunit. The mutations were selected based on sequence homology with the large subunit of [NiFe] hydrogenases. None of the mutations affected assembly of complex I, all decreased or abolished ubiquinone reductase activity. Several mutants exhibited decreased sensitivities toward ubiquinone-analogous inhibitors. Unexpectedly, seven mutations affected the properties of iron-sulfur cluster N2, a prosthetic group not located in the 49-kDa subunit. In three of these mutants cluster N2 was not detectable by electron-paramagnetic resonance spectroscopy. The fact that the small subunit of hydrogenase is homologous to the PSST subunit of complex I proposed to host cluster N2 offers a straightforward explanation for the observed, unforeseen effects on this iron-sulfur cluster. We propose that the fold around the hydrogen reactive site of [NiFe] hydrogenase is conserved in the 49-kDa subunit of complex I and has become part of the inhibitor and ubiquinone binding region. We discuss that the fourth ligand of iron-sulfur cluster N2 missing in the PSST subunit may be provided by the 49-kDa subunit.