Hydrogen-oxidizing electron transport components in the hyperthermophilic archaebacterium Pyrodictium brockii.

The hyperthermophilic archaebacterium Pyrodictium brockii grows optimally at 105 degrees C by a form of metabolism known as hydrogen-sulfur autotrophy, which is characterized by the oxidation of H2 by S0 to produce ATP and H2S. UV-irradiated membranes were not able to carry out the hydrogen-dependent reduction of sulfur. However, the activity could be restored by the addition of ubiquinone Q10 or ubiquinone Q6 to the UV-damaged membranes. A quinone with thin-layer chromatography migration properties similar to those of Q6 was purified by thin-layer chromatography from membranes of P. brockii, but nuclear magnetic resonance analysis failed to confirm its identity as a ubiquinone. P. brockii quinone was capable of restoring hydrogen-dependent sulfur reduction to UV-irradiated membranes. Hydrogen-reduced-minus-air-oxidized absorption difference spectra on membranes revealed absorption peaks characteristic of c-type cytochromes. A c-type cytochrome with alpha, beta, and gamma peaks at 553, 522, and 421 nm, respectively, was solubilized from membranes with 0.5% Triton X-100. Pyridine ferrohemochrome spectra confirmed its identity as a c-type cytochrome, and heme staining of membranes loaded on sodium dodecyl sulfate gels revealed a single heme-containing component of 13 to 14 kDa. Studies with the ubiquinone analog 2-n-heptyl-4-hydroxyquinoline-N-oxide demonstrated that the P. brockii quinone is located on the substrate side of the electron transport chain with respect to the c-type cytochrome. These first characterizations of the strictly anaerobic, presumably primitive P. brockii electron transport chain suggest that the hydrogenase operates at a relatively high redox potential and that the H2-oxidizing chain more closely resembles those of aerobic eubacterial H2-oxidizing bacteria than those of the H2-metabolizing systems of anaerobes or the hyperthermophile Pyrococcus furiosus.     

Pleiotropic phenotypes of fission yeast defective in ubiquinone-10 production. A study from the abc1Sp (coq8Sp) mutant

We previously constructed two Schizosaccahromyces pombe ubiquinone-10 (or Coenzyme Q10) less mutants, which are either defective for decaprenyl diphosphate synthase or p-hydroxybenzoate polyprenyl diphosphate transferase. To further confirm the roles of ubiquinone in S. pombe, we examined the phenotype of the abc1Sp (coq8Sp) mutant, which is highly speculated to be defective in ubiquinone biosynthesis. We show here that the abc1Sp defective strain did not produce UQ-10 and could not grow on minimal medium. The abc1Sp-deficient strain required supplementation with antioxidants such as cysteine or glutathione to grow on minimal medium. In support of the antioxidant function of ubiquinone, the abc1Sp-deficient strain is sensitive to H2O2 and Cu2+. In addition, expression of the stress inducible ctt1 gene was much induced in the ubiquinone less mutant than wild type. Interestingly, we also found that the abc1-deficient strain as well as other ubiquinone less mutants produced a significant amount of H2S, which suggests that oxidation of sulfide by ubiquinone may be an important pathway for sulfur metabolism in S. pombe. Thus, analysis of the phenotypes of S. pombe ubiquinone less mutants clearly demonstrate that ubiquinone has multiple functions in the cell apart from being an integral component of the electron transfer system.     

Role of ubiquinone in the mitochondrial generation of hydrogen peroxide.

Antimycin-inhibited bovine heart submitochondrial particles generate O2- and H2O2 with succinate as electron donor. H2O2 generation involves the action of the mitochondrial superoxide dismutase, in accordance with the McCord & Fridovich [(1969) j. biol. Chem. 244, 6049-6055] reaction mechanism. Removal of ubiquinone by acetone treatment decreases the ability of mitochondrial preparations to generate O2- and H2O2, whereas supplementation of the depleted membranes with ubiquinone enhances the peroxide-generating activity in the reconstituted membranes. Addition of superoxide dismutase to ubiquinone-reconstituted membranes is essential in order to obtain maximal rates of H2O2 generation since the acetone treatment of the membranes apparently inactivates (or removes) the mitochondrial superoxide dismutase. Parallel measurements of H2O2 production, succinate dehydrogenase and succinate-cytochrome c reductase activities show that peroxide generation by ubiquinone-supplemented membranes is a monotonous function of the reducible ubiquinone content, whereas the other two measured activities reach saturation at relatively low concentrations of reducible quinone. Alkaline treatment of submitochondrial particles causes a significant decrease in succinate dehydrogenase activity and succinate-dependent H2O2 production, which contrasts with the increase of peroxide production by the same particles with NADH as electron donor. Solubilized succinate dehydrogenase generates H2O2 at a much lower rate than the parent submitochondrial particles. It is postulated that ubisemiquinone (and ubiquinol) are chiefly responsible for the succinate-dependent peroxide production by the mitochondrial inner membrane.     

NADH-ubiquinone oxidoreductases of the Escherichia coli aerobic respiratory chain

Deamino-NADH/ubiquinone 1 oxidoreductase activity in membrane preparations from Escherichia coli GR19N is 20-50% of NADH/ubiquinone 1 oxidoreductase activity. In comparison, membranes from E. coli IY91, which contain amplified levels of NADH dehydrogenase, exhibit about 100-fold higher NADH/ubiquinone 1 reductase activity but about 20-fold less deamino-NADH/ubiquinone 1 reductase activity. Deamino-NADH/ubiquinone 1 reductase is more sensitive than NADH/ubiquinone 1 reductase activity to inhibition by 3-undecyl-2-hydroxyl-1,4-naphthoquinone, piericidin A, or myxothiazol. Furthermore, GR19N membranes exhibit two apparent Kms for NADH but only one for deamino-NADH. Inside-out membrane vesicles from E. coli GR19N generate a H+ electrochemical gradient (interior positive and acid) during electron transfer from deamino-NADH to ubiquinone 1 that is large and stable relative to that observed with NADH as substrate. Generation of the H+ electrochemical gradient in the presence of deamino-NADH is inhibited by 3-undecyl-2-hydroxy-1,4-naphthoquinone and is not observed in IY91 membrane vesicles or in vesicles from GR19N that are deficient in deamino-NADH/ubiquinone 1 reductase activity. The data provide a strong indication that the E. coli aerobic respiratory chain contains two species of NADH dehydrogenases: (i) an enzyme (NADH dh I) that reacts with deamino-NADH or NADH whose turnover leads to generation of a H+ electrochemical gradient at a site between the primary dehydrogenase and ubiquinone and (ii) an enzyme (NADH dh II) that reacts with NADH exclusively whose turnover does not lead to generation of a H+ electrochemical gradient between the primary dehydrogenase and ubiquinone 1.     

Phenotypes of fission yeast defective in ubiquinone production due to disruption of the gene for p-hydroxybenzoate polyprenyl diphosphate transferase

Ubiquinone is an essential component of the electron transfer system in both prokaryotes and eukaryotes and is synthesized from chorismate and polyprenyl diphosphate by eight steps. p-Hydroxybenzoate (PHB) polyprenyl diphosphate transferase catalyzes the condensation of PHB and polyprenyl diphosphate in ubiquinone biosynthesis. We isolated the gene (designated ppt1) encoding PHB polyprenyl diphosphate transferase from Schizosaccharomyces pombe and constructed a strain with a disrupted ppt1 gene. This strain could not grow on minimal medium supplemented with glucose. Expression of COQ2 from Saccharomyces cerevisiae in the defective S. pombe strain restored growth and enabled the cells to produce ubiquinone-10, indicating that COQ2 and ppt1 are functional homologs. The ppt1-deficient strain required supplementation with antioxidants, such as cysteine, glutathione, and alpha-tocopherol, to grow on minimal medium. This suggests that ubiquinone can act as an antioxidant, a premise supported by our observation that the ppt1-deficient strain is sensitive to H(2)O(2) and Cu(2+). Interestingly, we also found that the ppt1-deficient strain produced a significant amount of H(2)S, which suggests that oxidation of sulfide by ubiquinone may be an important pathway for sulfur metabolism in S. pombe. Ppt1-green fluorescent protein fusion proteins localized to the mitochondria, indicating that ubiquinone biosynthesis occurs in the mitochondria in S. pombe. Thus, analysis of the phenotypes of S. pombe strains deficient in ubiquinone production clearly demonstrates that ubiquinone has multiple functions in the cell apart from being an integral component of the electron transfer system.     

Coupling site I and the rotenone-sensitive ubisemiquinone in tightly coupled submitochondrial particles

The rotenone-sensitive g = 2.00 low temperature EPR signal attributed to ubisemiquinone is observed in submitochondrial particles during coupled electron transfer from NADH to oxygen and from succinate to NAD+. The signal is seen only in the presence of oligomycin added to induce the respiratory control (7-9 with NADH and 3-4 with succinate) and it disappears in the presence of uncouplers (CCCP or gramicidin D). No reduction of the iron-sulfur center N-2 in the presence of 20 mM succinate and cyanide is observed, thus suggesting that N-2 is not in equilibrium with the ubiquinone pool. A hypothesis is proposed on delta mu H+ generation coupled with electron transfer between iron-sulfur center N-2 and the ubiquinone pool.     

D-lactate oxidation and generation of the proton electrochemical gradient in membrane vesicles from Escherichia coli GR19N and in proteoliposomes reconstituted with purified D-lactate dehydrogenase and cytochrome o oxidase

The respiratory chain in the cytochrome d deficient mutant Escherichia coli GR19N is a relatively simple, linear system consisting of primary dehydrogenases, ubiquinone 8, cytochrome b-556, and cytochrome o oxidase. By use of right-side-out and inside-out membrane vesicles from this strain, various oxidase activities and the generation of the H+ electrochemical gradient were studied. Oxidation of ubiquinol 1 or N,N,-N',N'-tetramethyl-p-phenylenediamine, which donate electrons directly to the terminal oxidase, generates a H+ electrochemical gradient comparable to that observed during D-lactate oxidation. In contrast, D-lactate/ubiquinone 1 or D-lactate/ferricyanide oxidoreductase activity does not appear to generate a membrane potential, suggesting that electron flow from D-lactate dehydrogenase to ubiquinone is not electrogenic. Moreover, proteoliposomes reconstituted with purified D-lactate dehydrogenase, ubiquinone 8, and purified cytochrome o catalyze D-lactate and ubiquinol 1 oxidation and generate a H+ electrochemical gradient similar to that observed in membrane vesicles. Strikingly, in inside-out vesicles, NADH oxidation generates a H+ electrochemical gradient that is very significantly greater than that produced by either D-lactate or ubiquinol 1; furthermore, NADH/ubiquinone 1 and NADH/ferricyanide oxidoreductase activities are electrogenic. It is suggested that the only component between D-lactate dehydrogenase or ubiquinol and oxygen in GR19N membranes that is directly involved in the generation of the H+ electrochemical gradient is cytochrome o, which functions as a "half-loop" (i.e., the oxidase catalyzes the scalar release of 2 H+ from ubiquinol on the outer surface of the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lateral diffusion of ubiquinone during electron transfer in phospholipid- and ubiquinone-enriched mitochondrial membranes

After fusion of small unilamellar phospholipid liposomes with mitochondrial inner membranes, the rate of electron transfer between membrane dehydrogenases and cytochrome c decreases as the average distance between integral membrane proteins increases, suggesting that electron transfer is mediated through a diffusional process in the membrane plane (Schneider, H., Lemasters, J. J., H?chli, M., and Hackenbrock, C. R. (1980)., J. Biol. Chem. 255, 3748-3756). The role of ubiquinone in this process was evaluated by fusing liposomes containing ubiquinone-10 or ubiquinone-6, with inner membranes. In control membranes enriched with phospholipid only, ubiquinol-cytochrome c reductase and NADH- and succinate-cytochrome c reductase activities decreased proportionally to the increase in bilayer lipid. These decreases were restored substantially in phospholipid plus ubiquinone-supplemented membranes. The degree to which restoration occurred was dependent upon the length of the isoprenoid side chain of the ubiquinone with the shorter chain length ubiquinone-6, always giving greater restoration than ubiquinone-10. It is concluded that electron transfer between flavin-linked dehydrogenases (Complexes I and II) and cytochrome bc1 (Complex III) occurs by independent, lateral diffusion of ubiquinone as well as independent, lateral diffusion of ubiquinone as well as the protein complexes within the plane of the membrane.     

Location and activity of ubiquinone 10 and ubiquinone analogues in model and biological membranes

Deuteriated analogues of ubiquinone 10 (Q10) have been dispersed with plasma membranes of Escherichia coli and with the inner membranes of beetroot mitochondria. Orientational order at various deuteriated sites was measured by solid-state deuterium nuclear magnetic resonance (2H NMR). Similar measurements were made, using the compounds dispersed in dimyristoylphosphatidylcholine (DMPC) and egg yolk lecithin and dispersions prepared from the lipid extracts of beetroot mitochondria. In all cases only a single unresolved 2H NMR spectrum (typically 1000-Hz full width at half-height) was observed at concentrations down to 0.02 mol % Q10 per membrane lipid. This result shows that most Q10 is in a mobile environment which is physically separate from the orientational constraints of the bilayer lipid chains. In contrast, a short-chain analogue of Q10, in which the 10 isoprene groups have been replaced by a perdeuteriated tridecyl chain, showed 2H NMR spectra with quadrupolar splittings typical of an ordered lipid that is intercalated into the bilayer. The NADH oxidase activity and O2 uptake in Escherichia coli and in mitochondria were independent of which analogue was incorporated into the membrane. Thus, despite the major difference in their physical association with membranes, or their lipid extracts, the electron transport function of the long- and short-chain ubiquinones is similar, suggesting that the bulk of the long-chain ubiquinone does not have a direct function in electron transporting activity. The physiologically active Q10 may only be a small fraction of the total ubiquinone, a fraction that is below the level of detection of the present NMR equipment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on electron paramagnetic resonance spectra manifested by a respiratory chain hydrogen carrier.

The high-potential iron-sulfur protein (HiPIP) center of succinate dehydrogenase has an electron paramagnetic resonance (epr) signal in the oxidized form, centered at g = 2.01, and under certain conditions this epr signal is accompanied by absorbances at g = 2.04, g = 1.99, and g = 1.96. These absorbances have been attributed to a spin-spin interaction of paramagnetic species, the semiquinone form of ubiquinone being involved (Ruzicka et al., Proc. Nat. Acad. Sci. USA72, 2886). In the present work this magnetic interaction is studied further; it is concluded that of the three possible species (HiPIP, Flavin H and UQ?H (ubiquinone)) which may interact with UQ?H; a second UQ? most likely partner for the interaction. Nonetheless, the HiPIP center of succinate dehydrogenase also plays a role in the interaction by acting as a “magnetic relaxer” of one or both of the interacting UQ?Hs. The physiological reaction of that part of the ubiquinone pool associated with the succinate dehydrogenase (on the matrix side of the inner mitochondrial membrane) is UQH₂ ? UQ?H + H⁺ + e^?. This is in line with recent postulates of the mechanism of ubiquinone mediation in electron transfer.     

Effect of inhibitors on the ubiquinone binding capacity of the primary energy conversion site in the Rhodobacter capsulatus cytochrome bc(1) complex.

A key issue concerning the primary conversion (Q(O)) site function in the cytochrome bc(1) complex is the stoichiometry of ubiquinone/ubihydroquinone occupancy. Previous evidence suggests that the Q(O) site is able to accommodate two ubiquinone molecules, the double occupancy model [Ding, H., Robertson, D. E., Daldal, F., and Dutton, P. L. (1992) Biochemistry 31, 3144-3158]. In the recently reported crystal structures of the cytochrome bc(1) complex, no electron density was identified in the Q(O) site that could be ascribed to ubiquinone. To provide further insight into this issue, we have manipulated the cytochrome bc(1) complex Q(O) site occupancy in photosynthetic membranes from Rhodobacter capsulatus by using inhibitor titrations and ubiquinone extraction to modulate the amount of ubiquinone bound in the site. The nature of the Q(O) site occupants was probed via the sensitivity of the reduced [2Fe-2S] cluster electron paramagnetic resonance (EPR) spectra to modulation of Q(O) site occupancy. Diphenylamine (DPA) and methoxyacrylate (MOA)-stilbene are known Q(O) site inhibitors of the cytochrome bc(1) complex. Addition of stoichiometric concentrations of MOA-stilbene or excess DPA to cytochrome bc(1) complexes with natural levels of ubiquinone elicits the same change in the [2Fe-2S] cluster EPR spectra; the g(x)() resonance broadens and shifts from 1. 800 to 1.783. This is exactly the same signal as that obtained when there is only one ubiquinone present in the Q(O) site. Furthermore, addition of MOA-stilbene or DPA to the cytochrome bc(1) complex depleted of ubiquinone does not alter the [2Fe-2S] cluster EPR spectral line shapes, which remain indicative of one ubiquinone or zero ubiquinones in the Q(O) site, with broad g(x)() resonances at 1. 783 or 1.765, respectively. The results are quite consistent with the Q(O) site double occupancy model, in which MOA-stilbene and DPA inhibit by displacing one, but not both, of the Q(O) site ubiquinones.     

Proton translocation coupled to quinone reduction by reduced nicotinamide–adenine dinucleotide in rat liver and ox heart mitochondria

Measurements were made of the stoicheiometry of proton-translocation coupled to NAD(P)H oxidation by several quinones (duroquinone, ubiquinone(0), ubiquinone(1), ubiquinone(2)) in mitochondria from rat liver and ox heart. Observed stoicheiometries of protons translocated per mol of NADH oxidized (-->H(+)/2e(-) ratios; Mitchell, 1966) ranged from 0.75 (rat liver mitochondria with ubiquinone(1)) to 1.55 (ox heart mitochondria with ubiquinone(1) or ubiquinone(2)). Only the rotenone-sensitive pathway of NADH oxidation by quinone was able to support proton translocation. Correction of the observed -->H(+)/2e(-) ratios for the loss of reducing equivalents to the rotenone-insensitive pathway increased their value to approx. 2.0. It is concluded that the rotenone-sensitive NADH- ubiquinone reductase activity of the respiratory chain may be organized in the mitochondrial membrane as a proton-translocating oxidoreduction loop. The number of such loops between NADH and ubiquinone is one, and not two, as initially proposed by Mitchell (1966).     

Farnesylacetone, a sesquiterpenic hormone of Crustacea, inhibits electron transport in isolated rat liver mitochondria

Farnesylacetone (C18 H30 0) is a male hormone extracted from the androgenic gland of crab, Carcinus maenas. Appropriate enzymatic assays, as well as spectrophotometric studies, indicate that micromolar concentrations of farnesylacetone interact with the electron transport pathway of rat liver mitochondria. By the use of artificial electron donors and electron acceptors, it is shown that farnesylacetone immediately inhibits the electron transfer within complex I (NADH ubiquinone reductase activity) and complex II (succinate ubiquinone reductase activity). It is proposed that farneylacetone could interact with these two complexes of the respiratory chain at the level of the iron-sulfur centers implicated in the dehydrogenase activities. These observations are compared with the results obtained with terpenic molecules which interact with mitochondrial respiration.     

Electron acceptors of bacterial photosynthetic reaction centers. II. H+ binding coupled to secondary electron transfer in the quinone acceptor complex.

The photoreduction of ubiquinone in the electron acceptor complex (QIQII) of photosynthetic reaction centers from Rhodopseudomonas sphaeroides, R26, was studied in a series of short, saturating flashes. The specific involvement of H+ in the reduction was revealed by the pH dependence of the electron transfer events and by net H+ binding during the formation of ubiquinol, which requires two turnovers of the photochemical act. On the first flash QII receives an electron via QI to form a stable ubisemiquinone anion (QII-); the second flash generates QI-. At low pH the two semiquinones rapidly disproportionate with the uptake of 2 H+, to produce QIIH2. This yields out-of-phase binary oscillations for the formation of anionic semiquinone and for H+ uptake. Above pH 6 there is a progressive increase in H+ binding on the first flash and an equivalent decrease in binding on the second flash until, at about pH 9.5, the extent of H+ binding is the same on all flashes. The semiquinone oscillations, however, are undiminished up to pH 9. It is suggested that a non-chromophoric, acid-base group undergoes a pK shift in response to the appearance of the anionic semiquinone and that this group is the site of protonation on the first flash. The acid-base group, which may be in the reaction center protein, appears to be subsequently involved in the protonation events leading to fully reduced ubiquinol. The other proton in the two electron reduction of ubiquinone is always taken up on the second flash and is bound directly to QII-. At pH values above 8.0, it is rate limiting for the disproportionation and the kinetics, which are diffusion controlled, are properly responsive to the prevailing pH. Below pH 8, however, a further step in the reaction mechanism was shown to be rate limiting for both H+ binding electron transfer following the second flash.     

A multifunctional hydroquinone oxidase of the external cell surface and sera.

A multifunctional cell surface protein with NADH oxidase (NOX) activity and capable of oxidizing hydroquinones is located at the exterior of the cell and is shed in soluble form into sera. The oxidase appears to function as a terminal oxidase of a trans plasma membrane electron transport chain consisting of a NAD(P)H-ubiquinone reductase at the cytosolic membrane surface, possibly a b-type cytochrome, ubiquinone and the oxidase. Hyperactivity or conditions that interrupt ordered 2H+ + 2e- transport from NAD(P)H or hydroquinone to molecular oxygen and other acceptors at the external cell surface may result in the generation of superoxide. The latter may serve to propagate aging-related redox changes both to adjacent cells and circulating blood components. A circulating NOX activity form associated with aging and the reduction of cytochrome c by sera of aged patients that is partially inhibited by ubiquinone are described.     

The function of ubiquinone in Escherichia coli

1. The function of ubiquinone in Escherichia coli was studied by using whole cells and membrane preparations of normal E. coli and of a mutant lacking ubiquinone. 2. The mutant lacking ubiquinone, strain AN59 (Ubi(-)), when grown under aerobic conditions, gave an anaerobic type of growth yield and produced large quantities of lactic acid, indicating that ubiquinone plays a vital role in electron transport. 3. NADH and lactate oxidase activities in membranes from strain AN59 (Ubi(-)) were greatly impaired and activity was restored by the addition of ubiquinone (Q-1). 4. Comparison of the percentage reduction of flavin, cytochrome b(1) and cytochrome a(2) in the aerobic steady state in membranes from the normal strain (AN62) and strain AN59 (Ubi(-)) and the effect of respiratory inhibitors on these percentages in membranes from strain AN62 suggest that ubiquinone functions at more than one site in the electron-transport chain. 5. Membranes from strain AN62, in the absence of substrate, showed an electron-spin-resonance signal attributed to ubisemiquinone. The amount of reduced ubiquinone (50%) found after rapid solvent extraction is consistent with the existence of ubiquinone in membranes as a stabilized ubisemiquinone. 6. The effects of piericidin A on membranes from strain AN62 suggest that this inhibitor acts at the ubiquinone sites: thus inhibition of electron transport is reversed by ubiquinone (Q-1); the aerobic steady-state oxidation-reduction levels of flavins and cytochrome b(1) in the presence of the inhibitor are raised to values approximating those found in the membranes of strain AN59 (Ubi(-)); the inhibitor rapidly eliminates the electron-spin-resonance signal attributed to ubisemiquinone and allows slow oxidation of endogenous ubiquinol in the absence of substrate and prevents reduction of ubiquinone in the presence of substrate. It is concluded that piericidin A separates ubiquinone from the remainder of the electron-transport chain. 7. A scheme is proposed in which ubisemiquinone, complexed to an electron carrier, functions in at least two positions in the electron-transport sequence.     

The proton-pumping NADH:ubiquinone oxidoreductase (complex I) of Aquifex aeolicus

The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first energy-transducing complex of many respiratory chains. Homologues of complex I are present in the three domains of life. Here, we report the properties of complex I in membranes of the hyperthermophilic bacterium Aquifex aeolicus. The complex reacted with NADH but not with NADPH and F(420)H(2) as electron donors. Short-chain analogues of ubiquinone like decyl-ubiquinone and ubiquinone-2 were suitable electron acceptors. The affinities towards NADH and ubiquinone-2 were comparable to the ones obtained with the Escherichia coli complex I. The reaction was inhibited by piericidin A at the same concentration as in E. coli. The complex showed an unusual pH optimum at pH 9 and a maximal rate at 80 degrees C. We found no evidence for the presence of an alternative, single subunit NADH dehydrogenase in A. aeolicus membranes. The NADH:ferricyanide reductase activity of detergent extracts of A. aeolicus membranes sedimented as a protein with a molecular mass of approximately 550 kDa. From the data we concluded that A. aeolicus contains a NADH:ubiquinone oxidoreductase resembling complex I of mesophilic bacteria.     

Electron transport components involved in hydrogen oxidation in free-living Rhizobium japonicum.

Membranes from free-living Rhizobium japonicum were isolated to study electron transport components involved in H2 oxidation. The H2/O2 uptake rate ratio in membranes was approximately 2. The electron transport inhibitors antimycin A, cyanide, azide, hydroxylamine, and 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO) inhibited H2 uptake and H2-dependent O2 uptake significantly. H2-reduced minus O2-oxidized absorption difference spectra revealed peaks at 551.5, 560, and 603 nm, indicating the involvement of cytochromes c, b, and a-a3, respectively. H2-dependent cytochrome reduction was completely inhibited in the presence of 0.15 mM HQNO. This inhibition was relieved by the addition of 0.1 mM menadione. Evidence is presented for the involvement of two b-type cytochromes in H2 oxidation. One b-type cytochrome was not reduced by ascorbate and had an absorption peak at 560 nm. The reduction of this cytochrome by H2 was not inhibited by cyanide. A second b-type cytochrome, cytochrome b', was not reduced by H2 in the presence of cyanide. This cytochrome had an absorption peak at 558 nm. Carbon monoxide difference spectra with H2 as reductant provided evidence for the involvement of cytochrome o as well as cytochrome a3 in H2 oxidation. H2 uptake activity in cell-free extracts was inhibited by UV light irradiation. Most of the activity of the UV-treated extracts was restored with the addition of ubiquinone. The restored activity was inhibited by cyanide. A branched electron transport pathway from H2 to O2 is proposed.     

Evidence for a membrane bound NADH-plastoquinone-oxidoreductase in Chlamydomonas reinhardii CW-15

NADH-plastoquinone-oxidoreductase bound to the membrane fraction of Chlamydomonas reinhardii CW-15 has been solubilized with triton X-100. By treatment with high concentrations of MgCl₂ and KCl and (NH₄)₂SO₄ fractionation the enzyme could be enriched 8–10-fold. Spectral properties indicated a flavoprotein probably containing Fe–S groups. The enzyme oxidizes NADH and NADPH with various quinones as electron acceptors. Plastoquinone 1 is an effective electron acceptor, whereas ubiquinone 1 is only reduced with low activity. The enzyme is sensitive to rotenone and thenoyltrifluoroacetone, both inhibitors of ubiquinone reduction by mitochondrial dehydrogenases. As the bound enzyme is sensitive to inhibitors of photosynthetic electron flow, the enzyme is assumed to be responsible for light driven hydrogen evolution at the expense of NADH generating substrates.Abbreviations BQ benzoquinone - chl chlorophyll - DBMIB dibromothymoquinone - DNP-INT dinitro-phenylether of 2-iodo-4-nitrothymol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - UHDBT 5-n-undecyl-6-hydroxy-4,7-dioxy-benzothiazol - TTFA thenoyltri-fluoroacetone     

Role of subunit NuoL for proton translocation by respiratory complex I

The NADH:ubiquinone oxidoreductase, respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with a translocation of protons across the membrane. The complex consists of a peripheral arm catalyzing the electron transfer reaction and a membrane arm involved in proton translocation. The recently published X-ray structures of the complex revealed the presence of a unique 110 ? "horizontal" helix aligning the membrane arm. On the basis of this finding, it was proposed that the energy released by the redox reaction is transmitted to the membrane arm via a conformational change in the horizontal helix. The helix corresponds to the C-terminal part of the most distal subunit NuoL. To investigate its role in proton translocation, we characterized the electron transfer and proton translocation activity of complex I variants lacking either NuoL or parts of the C-terminal domain. Our data suggest that the H+/2e- stoichiometry of the ΔNuoL variant is 2, indicating a different stoichiometry for proton translocation as proposed from structural data. In addition, the same H+/e- stoichiometry is obtained with the variant lacking the C-terminal transmembraneous helix of NuoL, indicating its role in energy transmission.