BBA - Bioenergetics (v.1757, #12)

Complex I of Escherichia coli is encoded by 13 consecutive genes, called the nuo operon. A chromosomal deletion of all nuo genes has been achieved by homologous recombination. A vector that encodes all of the nuo genes has been constructed, and it expresses a functional enzyme.
Keywords: Complex I; nuo genes; NADH dehydrogenase; Deletion strain; Complementation;

A region of subunit IV comprising residues 77–85 is identified as essential for interaction with the core complex to restore the bc 1 activity (reconstitutive activity). Recombinant subunit IV mutants with single or multiple alanine substitution at this region were generated and characterized to identify the essential amino acid residues. Residues 81–84, with sequence of YRYR, are required for reconstitutive activity of subunit IV, because a mutant with these four residues substituted with alanine has little activity, while a mutant with alanine substitution at residues 77–80 and 85 have the same reconstitutive activity as that of the wild-type IV. The positively charged group at R-82 and R-84 and both the hydroxyl group and aromatic group at Y-81 and Y-83 are essential. The interactions between these four residues of subunit IV and residues of core subunits are also responsible for the stability of the complex. However, these interactions are not essential for the incorporation of subunit IV into the bc 1 complex.
Keywords: Supernumerary subunit; Bacterial cytochrome bc 1 complex; The protein:protein interactions; Reconstitution; Alanine scan;

Using AS-30D rat ascites hepatoma cells, we studied the modulating action of various antioxidants, inhibitors of mitochondrial permeability transition pore and inhibitors of the respiratory chain on Cd2+-produced cytotoxicity. It was found that Cd2+ induced both necrosis and apoptosis in a time- and dose-dependent way. This cell injury involved dissipation of the mitochondrial transmembrane potential, respiratory dysfunction and initial increase of the generation of reactive oxygen species (ROS), followed by its decrease after prolonged incubation. Inhibitors of the mitochondrial permeability transition pore, cyclosporin A and bongkrekic acid, and inhibitors of respiratory complex III, stigmatellin and antimycin A, but not inhibitor of complex I, rotenone, partly prevented necrosis evoked by exposure of the cells to Cd2+. Apoptosis of the cells was partly prevented by free radical scavengers and by preincubation with N-acetylcysteine. Stigmatellin, antimycin A and cyclosporin A also abolished Cd2+-induced increase in ROS generation. It is concluded that Cd2+ toxicity in AS-30D rat ascites hepatoma, manifested by cell necrosis and/or apoptosis, involves ROS generation, most likely at the level of respiratory complex III, and is related to opening of the mitochondrial permeability transition pore.
Keywords: Cadmium; Apoptosis; Reactive oxygen species; Respiratory complex III; Permeability transition pore; Hepatoma AS-30D; Mitochondria;

NADH:ubiquinone oxidoreductase (complex I) is the largest and most complicated enzyme of aerobic electron transfer. The mechanism how it uses redox energy to pump protons across the bioenergetic membrane is still not understood. Here we determined the pumping stoichiometry of mitochondrial complex I from the strictly aerobic yeast Yarrowia lipolytica. With intact mitochondria, the measured value of 3.8 H → + / 2 e ¯ indicated that four protons are pumped per NADH oxidized. For purified complex I reconstituted into proteoliposomes we measured a very similar pumping stoichiometry of 3.6 H → + / 2 e ¯ . This is the first demonstration that the proton pump of complex I stayed fully functional after purification of the enzyme.
Keywords: Complex I; NADH:ubiquinone oxidoreductase; Proton pump; Energy transduction; Mitochondria; Proteoliposome;

CO-dependent H2 evolution by Rhodospirillum rubrum: Role of CODH:CooF complex by Steven W. Singer; Marissa B. Hirst; Paul W. Ludden (1582-1591).
Upon exposure to CO during anaerobic growth, the purple phototrophic bacterium Rhodospirillum rubrum expresses a CO-oxidizing H2 evolving enzymatic system. The CO-oxidizing enzyme, carbon monoxide dehydrogenase (CODH), has been purified and extensively characterized. However the electron transfer pathway from CODH to the CO-induced hydrogenase that evolves H2 is not well understood. CooF is an Fe–S protein that is the proposed mediator of electron transfer between CODH and the CO-induced hydrogenase. Here we present the spectroscopic and biochemical properties of the CODH:CooF complex. The characteristic EPR signals observed for CODH are largely insensitive to CooF complexation. Metal analysis and EPR spectroscopy show that CooF contains 2 Fe4S4 clusters. The observation of 2 Fe4S4 clusters for CooF contradicts the prediction of 4 Fe4S4 clusters based on analysis of the amino acid sequence of CooF and structural studies of CooF homologs. Comparison of in vivo and in vitro CO-dependent H2 evolution indicates that ∼ 90% of the activity is lost upon cell lysis. We propose that the loss of two labile Fe–S clusters from CooF during cell lysis may be responsible for the low in vitro CO-dependent H2 evolution activity. During the course of these studies, a new assay for CODH:CooF was developed using membranes from an R. rubrum mutant that did not express CODH:CooF, but expressed high levels of the CO-induced hydrogenase. The assay revealed that the CO-induced hydrogenase requires the presence of CODH:CooF for optimal H2 evolution activity.
Keywords: Carbon monoxide dehydrogenase; Hydrogen; EPR; Hydrogenase;

Water as a hydrogen bonding bridge between a phenol and imidazole. A simple model for water binding in enzymes by Josefin E. Utas; Mikael Kritikos; Dick Sandström; Björn Åkermark (1592-1596).
The X-ray crystal structure of the mono-hydrate of 2,2-bis(imidazol-1-ylmethyl)-4-methylphenol has been determined. Three hydrogen bonds hold water very tightly in the crystal, as determined by deuterium solid-state NMR. The hydrogen bond between the phenolic hydroxyl and water appears to have about the same strength as the direct hydrogen bond to imidazole, suggesting that the structure can be a good model for hydrogen bonds that are mediated by a water molecule in enzymes.
Keywords: Hydrogen bonding; Water binding; 2H solid-state NMR; X-ray structure;

Different kinetics of the regulation of respiration in permeabilized cardiomyocytes and in HL-1 cardiac cells by Tiia Anmann; Rita Guzun; Nathalie Beraud; Sophie Pelloux; Andrey V. Kuznetsov; Lembi Kogerman; Tuuli Kaambre; Peeter Sikk; Kalju Paju; Nadja Peet; Enn Seppet; Carlos Ojeda; Yves Tourneur; Valdur Saks (1597-1606).
The aim of this study was to investigate the mechanism of cellular regulation of mitochondrial respiration in permeabilized cardiac cells with clearly different structural organization: (i) in isolated rat cardiomyocytes with very regular mitochondrial arrangement, (ii) in HL-1 cells from mouse heart, and (iii) in non-beating (NB HL-1 cells) without sarcomeres with irregular and dynamic filamentous mitochondrial network. We found striking differences in the kinetics of respiration regulation by exogenous ADP between these cells: the apparent Km for exogenous ADP was by more than order of magnitude (14 times) lower in the permeabilized non-beating NB HL-1 cells without sarcomeres (25 ± 4 μM) and seven times lower in normally cultured HL-1 cells (47 ± 15 μM) than in permeabilized primary cardiomyocytes (360 ± 51 μM). In the latter cells, treatment with trypsin resulted in dramatic changes in intracellular structure that were associated with 3-fold decrease in apparent Km for ADP in regulation of respiration. In contrast to permeabilized cardiomyocytes, in NB HL-1 cells creatine kinase activity was low and the endogenous ADP fluxes from MgATPases recorded spectrophotometrically by the coupled enzyme assay were not reduced after activation of mitochondrial oxidative phosphorylation by the addition of mitochondrial substrates, showing the absence of ADP channelling in the NB HL-1 cells. While in the permeabilized cardiomyocytes creatine strongly activated mitochondrial respiration even in the presence of powerful competing pyruvate kinase–phosphoenolpyruvate system, in the NB HL-1 cells the stimulatory effect of creatine was not significant. The results of this study show that in normal adult cardiomyocytes and HL-1 cells intracellular local restrictions of diffusion of adenine nucleotides and metabolic feedback regulation of respiration via phosphotransfer networks are different, most probably related to differences in structural organization of these cells.
Keywords: Respiration regulation; Primary cardiomyocyte; Non-beating HL-1 cell; Mitochondria; Creatine kinase;

Probing the structure of Lhca3 by mutation analysis by Milena Mozzo; Tomas Morosinotto; Roberto Bassi; Roberta Croce (1607-1613).
Lhc proteins constitute a family of transmembrane proteins which share homology in sequence and similarity in the general organisation although members can be strongly differentiated such as in the case of PsbS and ELIPs. In this work, we report on the structure of Lhca3, a pigment-protein subunit component of the antenna system of higher plants Photosystem I, through the effect of point mutations in critical sites. Based on the structure of PSI-LHCI (Ben Shem et al., PDB file 1QZV remark 999) it has been suggested that Lhca3 may have different folding as compared to other members of the Lhc family. In particular, it was proposed that the two central helices may be swapped and chlorophylls in sites 1013 and 1023 are not present. This different folding would imply that the chlorophylls coordinated to the two central helices have different ligands in Lhca3 with respect to the other Lhc complexes. The structural model was tested by substituting the putative binding residues with residues unable to coordinate chlorophyll and the spectroscopic properties of the individual pigments were used as structural probes. The results indicate that Lhca3 folds in the same way as the other antenna proteins. Moreover, the low-energy absorption form originates from interaction between chlorophylls in site 1015 and 1025, like for the other PSI antenna subunits. Evidence is also shown for the presence in Lhca3 of chlorophylls in sites 1013 and 1023.
Keywords: Photosystem I; Light-harvesting complex; Lhca3; Mutant; Fluorescence; Red form;

Respiratory system of Gluconacetobacter diazotrophicus PAL5 Evidence for a cyanide-sensitive cytochrome bb and cyanide-resistant cytochrome ba quinol oxidases by B. González; S. Martínez; J.L. Chávez; S. Lee; N.A. Castro; M.A. Domínguez; S. Gómez; M.L. Contreras; C. Kennedy; J.E. Escamilla (1614-1622).
In highly aerobic environments, Gluconacetobacter diazotrophicus uses a respiratory protection mechanism to preserve nitrogenase activity from deleterious oxygen. Here, the respiratory system was examined in order to ascertain the nature of the respiratory components, mainly of the cyanide sensitive and resistant pathways. The membranes of G. diazotrophicus contain Q10, Q9 and PQQ in a 13:1:6.6 molar ratios. UV360 nm photoinactivation indicated that ubiquinone is the electron acceptor for the dehydrogenases of the outer and inner faces of the membrane. Strong inhibition by rotenone and capsaicin and resistance to flavone indicated that NADH-quinone oxidoreductase is a NDH-1 type enzyme. KCN-titration revealed the presence of at least two terminal oxidases that were highly sensitive and resistant to the inhibitor. Tetrachorohydroquinol was preferentially oxidized by the KCN-sensitive oxidase. Neither the quinoprotein alcohol dehydrogenase nor its associated cytochromes c were instrumental components of the cyanide resistant pathway. CO-difference spectrum and photodissociation of heme-CO compounds suggested the presence of cytochromes b-CO and a 1-CO adducts. Air-oxidation of cytochrome b (432 nm) was arrested by concentrations of KCN lower than 25 μM while cytochrome a 1 (442 nm) was not affected. A KCN-sensitive (I 50  = 5 μM) cytochrome bb and a KCN-resistant (I 50  = 450 μM) cytochrome ba quinol oxidases were separated by ion exchange chromatography.
Keywords: Gluconacetobacter diazotrophicus; Cytochrome quinol oxidase; Respiratory chain; Cyanide; Acid acetic bacteria;

A conserved tryptophan residue located between the A1B and FX redox centres on the PsaB side of the Photosystem I reaction centre has been mutated to a glycine in Chlamydomonas reinhardtii, thereby matching the conserved residue found in the equivalent position on the PsaA side. This mutant (PsaB:W669G) was studied using EPR spectroscopy with a view to understanding the molecular basis of the reported kinetic differences in forward electron transfer from the A1A and the A1B phyllo(semi)quinones. The kinetics of A1 reoxidation due to forward electron transfer or charge recombination were measured by electron spin echo spectroscopy at 265 K and 100 K, respectively. At 265 K, the reoxidation kinetics are considerably lengthened in the mutant in comparison to the wild-type. Under conditions in which FX is initially oxidised the kinetics of charge recombination at 100 K are found to be biphasic in the mutant while they are substantially monophasic in the wild-type. Pre-reduction of FX leads to biphasic kinetics in the wild-type, but does not alter the already biphasic kinetic properties of the PsaB:W669G mutant. Reduction of the [4Fe–4S] clusters FA and FB by illumination at 15 K is suppressed in the mutant. The results provide further support for the bi-directional model of electron transfer in Photosystem I of C. reinhardtii, and indicate that the replacement of the tryptophan residue with glycine mainly affects the redox properties of the PsaB bound phylloquinone A1B.
Keywords: Photosystem I; Electron transfer reactions; Phylloquinone; Iron–sulphur centres;

Redox potentials of the blue copper sites of bilirubin oxidases by Andreas Christenson; Sergey Shleev; Nicolas Mano; Adam Heller; Lo Gorton (1634-1641).
The redox potentials of the multicopper redox enzyme bilirubin oxidase (BOD) from two organisms were determined by mediated and direct spectroelectrochemistry. The potential of the T1 site of BOD from the fungus Myrothecium verrucaria was close to 670 mV, whereas that from Trachyderma tsunodae was > 650 mV vs. NHE. For the first time, direct electron transfer was observed between gold electrodes and BODs. The redox potentials of the T2 sites of both BODs were near 390 mV vs. NHE, consistent with previous finding for laccase and suggesting that the redox potentials of the T2 copper sites of most blue multicopper oxidases are similar, about 400 mV.
Keywords: Bilirubin oxidase; Copper enzyme; Redox potential; T1; T2; T3 sites; Redox titration; Spectroelectrochemistry;

Thermal stability of trimeric light-harvesting chlorophyll a/b complex (LHCIIb) in liposomes of thylakoid lipids by Chunhong Yang; Stephanie Boggasch; Winfried Haase; Harald Paulsen (1642-1648).
The major light-harvesting chlorophyll a/b complex (LHCIIb) of photosystem (PS) II functions by harvesting light energy and by limiting and balancing the energy flow directed towards the PSI and PSII reaction centers. The complex is predominantly trimeric; however, the monomeric form may play a role in one or several of the regulatory functions of LHCIIb. In this work the dissociation temperature was measured of trimeric LHCIIb isolated from Pisum thylakoids and inserted into liposomes made of various combinations of thylakoid lipids at various protein densities. Dissociation was measured by monitoring a trimer-specific circular dichroism signal in the visible range. The LHCIIb density in the membrane significantly affected the trimer dissociation temperature ranging from 70 °C at an LHCIIb concentration comparable to or higher than the one in thylakoid grana, to 65 °C at the density estimated in stromal lamellae. Omitting one thylakoid lipid from the liposomes had virtually no effect on the thermal trimer stability in most cases except when digalactosyl diacylglycerol (DGDG) was omitted which caused a drop in the apparent dissociation temperature by 2 °C. In liposomes containing only one lipid species, DGDG and, even more so, monogalactosyl diacylglycerol (MGDG) increased the thermal stability of LHCIIb trimers whereas phosphatidyl diacylglycerol (PG) significantly decreased it. The lateral pressure exerted by the non-bilayer lipid MGDG did not significantly influence LHCII trimer stability.
Keywords: Light harvesting complexes of photosystem II (LHCIIb); Liposome; Thermal stability; Thylakoid lipids; Trimer;

Functions of carotenoids in xanthorhodopsin and archaerhodopsin, from action spectra of photoinhibition of cell respiration by Vladimir A. Boichenko; Jennifer M. Wang; Josefa Antón; Janos K. Lanyi; Sergei P. Balashov (1649-1656).
The recent discovery of a carotenoid light-harvesting antenna in xanthorhodopsin, a retinal-based proton pump in Salinibacter ruber, made use of photoinhibition of respiration in whole cells to obtain action spectra [Balashov et al. Science 309, (2005) 2061–2064]. Here we provide further details of this phenomenon, and compare action spectra in three different systems where carotenoids have different functions or efficiencies of light-harvesting. The kinetics of light-induced inhibition of respiration in Salinibacter ruber was determined with single short flashes, and the photochemical cross section of the photoreaction was estimated. These measurements confirm that the xanthorhodopsin complex includes no more than a few, and most likely only one, carotenoid molecule, which is far less than the core complex antenna of photosynthetic bacteria. Although the total cross-section of light absorption in the purple bacterium Rhodospirillum rubrum greatly exceeds that in Salinibacter, the cross-sections are roughly equivalent in the shared wavelength range. We show further that despite interaction of bacterioruberin with archaerhodopsin, another retinal-based proton pump, there is no significant energy transfer from this carotenoid. This emphasizes the uniqueness of the salinixanthin–retinal interaction in xanthorhodopsin, and indicates that bacterioruberin in Halorubrum species has a structural or photoprotective rather than energetic role.
Keywords: Xanthorhodopsin; Salinixanthin; Energy transfer; Archaerhodopsin; Bacterioruberin; Salinibacter ruber;

We performed picosecond time-resolved fluorescence spectroscopy in spinach photosystem II (PS II) particles at 4, 40, and 77 K and identified a new fluorescence band, F689. F689 was identified in addition to the well-known F685 and F695 bands in both analyses of decay-associated spectra and global Gaussian deconvolution of time-resolved spectra. Its fast decay suggests the energy transfer directly from F689 to the reaction center chlorophyll P680. The contribution of F689, which increases only at low temperature, explains the unusually broad and variable bandwidth of F695 at low temperature. Global analysis revealed the three types of excitation energy transfer/dissipation processes: (1) energy transfer from the peripheral antenna to the three core antenna bands F685, F689, and F695 with time constants of 29 and 171 ps at 77 and 4 K, respectively; (2) between the three core bands (0.18 and 0.82 ns); and (3) the decays of F689 (0.69 and 3.02 ns) and F695 (2.18 and 4.37 ns). The retardations of these energy transfer rates and the slow F689 decay rate produced the strong blue shift of the PS II fluorescence upon the cooling below 77 K.
Keywords: Photosystem II; Excitation energy transfer; Time-resolved fluorescence spectroscopy; Chlorophyll fluorescence; Global analysis; Temperature dependency;

We have described a direct, high-performance liquid chromatography-based method of estimation of the total level of plastoquinone (PQ) in leaves, the redox state of total (photoactive and non-photoactive) PQ, as well as the redox state of the PQ-pool that is applicable to any illumination conditions. This method was applied to Arabidopsis thaliana leaves but it can be applied to any other plant species. The obtained results show that the level of total PQ was 25 ± 3 molecules/1000 chlorophyll (Chl) molecules in relation to foliar total Chl content. The level of the photoactive PQ, i.e., the PQ-pool, was about 31% of the total PQ present in Arabidopsis leaves that corresponds to about 8 PQ molecules/1000 Chl molecules. The reduction level of the non-photoactive PQ fraction, present outside thylakoids in chloroplasts, was estimated to account for about 49%. The measurements of the redox state of the PQ-pool showed that the pool was reduced during the dark period in about 24%, and during the light period (150 μmol/m2·s) the reduction of the PQ-pool increased to nearly 100%. The obtained results were discussed in terms of the activity of chlororespiration pathways in Arabidopsis and the regulatory role of the redox state of PQ-pool in various physiological and molecular processes in plants.
Keywords: Plastoquinone; Arabidopsis; Redox state; Chlororespiration; Redox signalling; HPLC;

Tight binding of NADPH to the 39-kDa subunit of complex I is not required for catalytic activity but stabilizes the multiprotein complex by Albina Abdrakhmanova; Klaus Zwicker; Stefan Kerscher; Volker Zickermann; Ulrich Brandt (1676-1682).
In addition to the 14 central subunits, respiratory chain complex I from the aerobic yeast Yarrowia lipolytica contains at least 24 accessory subunits, most of which are poorly characterized. Here we investigated the role of the accessory 39-kDa subunit which belongs to the heterogeneous short-chain dehydrogenase/reductase (SDR) enzyme family and contains non-covalently bound NADPH. Deleting the chromosomal copy of the gene that codes for the 39-kDa subunit drastically impaired complex I assembly in Y. lipolytica. We introduced several site-directed mutations into the nucleotide binding motif that severely reduced NADPH binding. This effect was most pronounced when the arginine at the end of the second β-strand of the NADPH binding Rossman fold was replaced by leucine or aspartate. Mutations affecting nucleotide binding had only minor or moderate effects on specific catalytic activity in mitochondrial membranes but clearly destabilized complex I. One mutant exhibited a temperature sensitive phenotype and significant amounts of three different subcomplexes were observed even at more permissive temperature. We concluded that the 39-kDa subunit of Y. lipolytica plays a critical role in complex I assembly and stability and that the bound NADPH serves to stabilize the subunit and complex I as a whole rather than serving a catalytic function.
Keywords: Mitochondria; Complex I; NADH:ubiquinone oxidoreductase; 39-kDa subunit; Short-chain dehydrogenases/reductases; NADPH; Yarrowia lipolytica;