BBA - Bioenergetics (v.1656, #2-3)

Cell surface oxygen consumption by mitochondrial gene knockout cells by Patries M Herst; An S Tan; Debbie-Jane G Scarlett; Michael V Berridge (79-87).
Mitochondrial gene knockout (ρ0) cells that depend on glycolysis for their energy requirements show an increased ability to reduce cell-impermeable tetrazolium dyes by electron transport across the plasma membrane. In this report, we show for the first time, that oxygen functions as a terminal electron acceptor for trans-plasma membrane electron transport (tPMET) in HL60ρ0 cells, and that this cell surface oxygen consumption is associated with oxygen-dependent cell growth in the absence of mitochondrial electron transport function. Non-mitochondrial oxygen consumption by HL60ρ0 cells was extensively inhibited by extracellular NADH and NADPH, but not by NAD+, localizing this process at the cell surface. Mitochondrial electron transport inhibitors and the uncoupler, FCCP, did not affect oxygen consumption by HL60ρ0 cells. Inhibitors of glucose uptake and glycolysis, the ubiquinone redox cycle inhibitors, capsaicin and resiniferatoxin, the flavin centre inhibitor, diphenyleneiodonium, and the NQO1 inhibitor, dicoumarol, all inhibited oxygen consumption by HL60ρ0 cells. Similarities in inhibition profiles between non-mitochondrial oxygen consumption and reduction of the cell-impermeable tetrazolium dye, WST-1, suggest that both systems may share a common tPMET pathway. This is supported by the finding that terminal electron acceptors from both pathways compete for electrons from intracellular NADH.
Keywords: Cell surface respiration; Non-mitochondrial oxygen consumption; WST-1; Trans-plasma membrane electron transport; HL60ρ0;

Thermoinactivaion analysis of vacuolar H+-pyrophosphatase by Su J Yang; Shih S Jiang; Yi Y Hsiao; Ru C Van; Yih J Pan; Rong L Pan (88-95).
Vacuolar H+-translocating pyrophosphatase (H+-PPase; EC 3.6.1.1) catalyzes both the hydrolysis of PPi and the electrogenic translocation of proton from the cytosol to the lumen of the vacuole. Vacuolar H+-PPase, purified from etiolated hypocotyls of mung bean (Vigna radiata L.), is a homodimer with a molecular mass of 145 kDa. To investigate the relationship between structure and function of this H+-translocating enzyme, thermoinactivation analysis was employed. Thermoinactivation studies suggested that vacuolar H+-PPase consists of two distinct states upon heat treatment and exhibited different transition temperatures in the presence and absence of ligands (substrate and inhibitors). Substrate protection of H+-PPase stabilizes enzyme structure by increasing activation energy from 54.9 to 70.2 kJ/mol. We believe that the conformation of this enzyme was altered in the presence of substrate to protect against the thermoinactivation. In contrast, the modification of H+-PPase by inhibitor (fluorescein 5′-isothiocyanate; FITC) augmented the inactivation by heat treatment. The native, substrate-bound, and FITC-labeled vacuolar H+-PPases possess probably distinct conformation and show different modes of susceptibility to thermoinactivation. Our results also indicate that the structure of one subunit of this homodimer exerts long distance effect on the other, suggesting a specific subunit–subunit interaction in vacuolar H+-PPase. A working model was proposed to interpret the relationship of the structure and function of vacuolar H+-PPase.
Keywords: Tonoplast; Vacuolar H+-pyrophosphatase; Thermoinactivation; Subunit interaction; Circular dichroism; Differential scanning calorimetry;

Phytomonas sp. are flagellated trypanosomatid plant parasites that cause diseases of economic importance in plantations of coffee, oil palm, cassava and coconuts. Here we investigated Ca2+ uptake by the vanadate-insensitive compartments using permeabilized Phytomonas serpens promastigotes. This uptake occurs at a rate of 1.13±0.23 nmol Ca2+ mg protein−1 min−1. It is completely abolished by the H+ ionophore FCCP and by valinomycin and nigericin. It is also inhibited by 2 μM ruthenium red, which, at this low concentration, is known to inhibit the mitochondrial calcium uniport. Furthermore, salicylhydroxamic acid (SHAM) and propylgallate, specific inhibitors of the alternative oxidase in plant and parasite mitochondria, are also effective as inhibitors of the Ca2+ transport. These compounds abolish the membrane potential that is monitored with safranine O. Rotenone, an inhibitor of NADH-CoQ oxidoreductase, can also dissipate 100% of the membrane potential. It is suggested that the mitochondria of P. serpens can be energized via oxidation of NADH in a pathway involving the NADH-CoQ oxidoreductase and the alternative oxidase to regenerate the ubiquinone. The electrochemical H+ gradient can be used to promote Ca2+ uptake by the mitochondria.
Keywords: Rotenone; Mitochondrial potential; Phytomonas serpens;

Energy transfer and trapping in the Photosystem I complex of Synechococcus PCC 7942 and in its supercomplex with IsiA by Elena G Andrizhiyevskaya; Dmitrij Frolov; Rienk van Grondelle; Jan P Dekker (104-113).
The cyanobacterium Synechococcus PCC 7942 grown under iron starvation assembles a supercomplex consisting of a trimeric Photosystem I (PSI) complex encircled by a ring of 18 CP43′ or IsiA complexes. It has previously been shown that PSI of Synechococcus PCC 7942 contains less special long-wavelength (‘red’) chlorophylls than PSI of most other cyanobacteria. Here we present a comparative analysis by time-resolved absorption difference and fluorescence spectroscopy of the processes of energy transfer and trapping in trimeric PSI and PSI–IsiA supercomplexes from Synechococcus PCC 7942. All experiments were performed with the primary electron donor of PSI (P700) in the oxidized state. Our data suggest that in the PSI complex the excitation energy is equilibrated with a lifetime of 0.6 ps among the so-called bulk chlorophylls, is distributed in 3–4 ps between the bulk and red chlorophylls, and is trapped in the reaction center in 19 ps. This trapping time is shorter than that observed for other cyanobacteria, which we attribute to the lower content of red chlorophylls in PSI of this organism. In the PSI–IsiA supercomplexes, the distribution of excited states is blue-shifted compared to that in PSI, leading to a lengthening of the equilibration processes. We attributed a phase of about 1 ps to initial energy equilibration steps among the IsiA and PSI core bulk chlorophylls, a 5–7 ps phase to equilibration between bulk and red chlorophylls within the PSI core, and a 38 ps phase to trapping in the reaction center. The data suggest that the excitation energy is equilibrated among the IsiA and PSI core antenna chlorophylls before trapping occurs. Data analysis based on a simple kinetic model revealed an intrinsic rate constant for energy transfer from IsiA to PSI in the range of 2±1 ps. Based on this value we suggest the presence of one or more linker chlorophylls between the IsiA and PSI core complexes. These results confirm that IsiA acts as an effective light-harvesting antenna for PSI.
Keywords: Photosystem I; PSI–IsiA; IsiA; Synechococcus PCC 7942; Transient absorption; Time-resolved fluorescence;

Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotrophic bacterium that can grow in the presence of either the weak reductant Fe2+, or reducing sulfur compounds that provide more energy for growth than Fe2+. We have previously shown that the uphill electron transfer pathway between Fe2+ and NAD+ involved a bc 1 complex that functions only in the reverse direction [J. Bacteriol. 182, (2000) 3602]. In the present work, we demonstrate both the existence of a bc 1 complex functioning in the forward direction, expressed when the cells are grown on sulfur, and the presence of two terminal oxidases, a bd and a ba 3 type oxidase expressed more in sulfur than in iron-grown cells, besides the cytochrome aa 3 that was found to be expressed only in iron-grown cells. Sulfur-grown cells exhibit a branching point for electron flow at the level of the quinol pool leading on the one hand to a bd type oxidase, and on the other hand to a bc 1ba 3 pathway. We have also demonstrated the presence in the genome of transcriptionally active genes potentially encoding the subunits of a bo 3 type oxidase. A scheme for the electron transfer chains has been established that shows the existence of multiple respiratory routes to a single electron acceptor O2. Possible reasons for these apparently redundant pathways are discussed.
Keywords: Acidithiobacillus ferrooxidans; Acidophile; Electron transfer chain; Iron and sulfur oxidation; bc 1 complex; Terminal oxidase;

In Rhodobacter sphaeroides reaction centers (RCs) containing the mutation Ala M260 to Trp (AM260W), transmembrane electron transfer along the full-length of the A-branch of cofactors is prevented by the loss of the QA ubiquinone, but it is possible to generate the radical pair P+HA by A-branch electron transfer or the radical pair P+QB by B-branch electron transfer. In the present study, FTIR spectroscopy was used to provide direct evidence for the complete absence of the QA ubiquinone in mutant RCs with the AM260W mutation. Light-induced FTIR difference spectroscopy of isolated RCs was also used to probe the neutral QB and the semiquinone QB states in two B-branch active mutants, a double AM260W–LM214H mutant, denoted WH, and a quadruple mutant, denoted WAAH, in which the AM260W, LM214H, and EL212A–DL213A mutations were combined. The data were compared to those obtained with wild-type (Wt) RCs and the double EL212A–DL213A (denoted AA) mutant which exhibit the usual A-branch electron transfer to QB. The QB /QB spectrum of the WH mutant is very close to that of Wt RCs indicating similar bonding interactions of QB and QB with the protein in both RCs. The QB /QB spectra of the AA and WAAH mutants are also closely related to one another, but are very different to that of the Wt complex. Isotope-edited IR fingerprint spectra were obtained for the AA and WAAH mutants reconstituted with site-specific 13C-labeled ubiquinone. Whilst perturbations of the interactions of the semiquinone QB with the protein are observed in the AA and WAAH mutants, the FTIR data show that the bonding interaction of neutral QB in these two mutants are essentially the same as those for Wt RCs. Therefore, it is concluded that QB occupies the same binding position proximal to the non-heme iron prior to reduction by either A-branch or B-branch electron transfer.
Keywords: Photosynthetic reaction center; Electron transfer; B-branch; Mutagenesis; FTIR; Quinone reduction;

Yessotoxin, a shellfish biotoxin, is a potent inducer of the permeability transition in isolated mitochondria and intact cells by Cristina Bianchi; Romana Fato; Alessia Angelin; Fabiana Trombetti; Vittoria Ventrella; Anna Rosa Borgatti; Ernesto Fattorusso; Patrizia Ciminiello; Paolo Bernardi; Giorgio Lenaz; Giovanna Parenti Castelli (139-147).
The diarrhetic poisoning by bivalve molluscs, diarrhetic shellfish poisoning, is due to consumption of mussels containing biotoxins produced by some Dinoflagellate species. Toxic effects of yessotoxin (YTX) include morphological alterations of mitochondria from heart and liver but the biochemical basis for these alterations is completely unknown.This paper demonstrates that YTX is a very powerful compound that opens the permeability transition pore (PTP) of the inner mitochondrial membrane of rat liver mitochondria at nanomolar concentrations. The effect requires the presence of a permissive level of calcium, by itself incapable of opening the pore. The direct effect of YTX on PTP is further confirmed by the inhibition exerted by cyclosporin A (CsA) that is known as a powerful inhibitor of PTP opening. Moreover, YTX induces membrane depolarization as shown by the quenching of tetramethylrhodamine methyl ester (TMRM), also prevented by the addition of CsA. YTX caused PTP opening in Morris Hepatoma 1C1 cells, as shown by the occurrence of CsA-sensitive depolarization within minutes of the addition of submicromolar concentrations of the toxin. These results provide a biochemical basis for the mitochondrial alterations observed in the course of intoxication with YTX, offering the first clue into the pathogenesis of diseases caused by YTX, and providing a novel tool to study the PTP in situ.
Keywords: Yessotoxin; Mitochondrial permeability transition; Rat liver mitochondria;

Heterotrophic arsenite oxidation by Hydrogenophaga sp. str. NT-14 is coupled to the reduction of oxygen and appears to yield energy for growth. Purification and partial characterization of the arsenite oxidase revealed that it (1) contains two heterologous subunits, AroA (86 kDa) and AroB (16 kDa), (2) has a native molecular mass of 306 kDa suggesting an α3β3 configuration, and (3) contains molybdenum and iron as cofactors. Although the Hydrogenophaga sp. str. NT-14 arsenite oxidase shares similarities to the arsenite oxidases purified from NT-26 and Alcaligenes faecalis, it differs with respect to activity and overall conformation. A c-551-type cytochrome was purified from Hydrogenophaga sp. str. NT-14 and appears to be the physiological electron acceptor for the arsenite oxidase. The cytochrome can also accept electrons from the purified NT-26 arsenite oxidase. A hypothetical electron transport chain for heterotrophic arsenite oxidation is proposed.
Keywords: Arsenite; Arsenite oxidase; Heterotroph; Cytochrome; Electron transport;

The membrane-bound proton-pumping inorganic pyrophosphatase from Rhodospirillum rubrum was heterologously expressed in Escherichia coli C43(DE3) cells and was inhibited by 4-bromophenacyl bromide (BPB), N,N′-dicyclohexylcarbodiimid (DCCD), diethyl pyrocarbonate (DEPC) and fluorescein 5′-isothiocyanate (FITC). In each case, the enzyme activity was rather well protected against inhibitory action by the substrate Mg2PPi. Site-directed mutagenesis was employed in attempts to identify target residues for these inhibitors. D217 and K469 appear to be the prime targets for DCCD and FITC, respectively, and may thus be involved in substrate binding. No major effect on enzyme activities was seen when any one of the four histidine residues present in the enzyme were substituted. Nevertheless, a mutant with all of the four charged histidine residues replaced retained only less than 10% of the hydrolysis and proton-pumping activities. Substitution of D217 with A or H yielded an enzyme with at least an order of magnitude lower hydrolysis activity. In contrast with the wild-type, these variants showed higher hydrolysis rates at lower concentrations of Mg2+, possibly reflecting a change in substrate preference from Mg2PPi to MgPPi. BPB is a H+-pyrophosphatase inhibitor that apparently has not been used previously as an inhibitor of these enzymes.
Keywords: Proton-pumping pyrophosphatase; Rhodospirillum rubrum; Site-specific mutant; Inhibition; Substrate preference;

Cyclic electron flow under saturating excitation of dark-adapted Arabidopsis leaves by Pierre Joliot; Daniel Béal; Anne Joliot (166-176).
The rate of cyclic electron flow measured in dark-adapted leaves under aerobic conditions submitted to a saturating illumination has been performed by the analysis of the transmembrane potential changes induced by a light to dark transfer. Using a new highly sensitive spectrophotometric technique, a rate of the cyclic flow of ∼130 s−1 has been measured in the presence or absence of 3-(3,4-dichloro-phenyl)-1,1-dimethylurea (DCMU). This value is ∼1.5 times larger than that previously reported [Proc. Natl. Acad. Sci. U. S. A. 99 (2001) 10209]. We have characterized in the presence or absence of DCMU charge recombination process (t 1/2∼60 μs) that involves P700 + and very likely the reduced form of the iron sulfur acceptor FX. This led to conclude that, under saturating illumination, the PSI centers involved in the cyclic pathway have most of the iron sulfur acceptors FA and FB reduced. In the proposed mechanism, electrons are transferred from a ferredoxin bound to a site localized on the stromal side of the cytochrome b 6 f complex to the Qi site. Two possible models of the organization of the membrane complexes are discussed, in which the cyclic and linear electron transfer chains are isolated one from the other.
Keywords: Cyclic electron flow; Photosystem I; Cytochrome b 6 f; Arabidopsis;

Stark effect measurements on monomers and trimers of reconstituted light-harvesting complex II of plants by Miguel A. Palacios; Stefano Caffarri; Roberto Bassi; Rienk van Grondelle; Herbert van Amerongen (177-188).
The electric-field induced absorption changes (Stark effect) of reconstituted light-harvesting complex II (LHCII) in different oligomerisation states—monomers and trimers—with different xanthophyll content have been probed at 77 K. The Stark spectra of the reconstituted control samples, containing the xanthophylls lutein and neoxanthin, are very similar to previously reported spectra of native LHCII. Reconstituted LHCII, containing lutein but no neoxanthin, shows a similar electrooptical response in the Chl a region, but the Stark signal of Chl b around 650 nm amounts to at most ∼25% of that of the control samples. We conclude that neoxanthin strongly modifies the electronic states of the nearby Chl b molecules causing a large electrooptical response at 650 nm stemming from one or more Chls b in the control samples. Ambiguities about the assignment of several bands in the Soret region [Biochim. Biophys. Acta 1605 (2003) 83] are resolved and the striking difference in electric field response between the two lutein molecules is confirmed. The Stark effect in the carotenoid spectral region in both control and neoxanthin-deficient samples is almost identical, showing that the neoxanthin Stark signal is small and much less intense than the lutein Stark signal.
Keywords: LHCII; Xanthophyll; Lutein; Neoxanthin; Charge-transfer state; Nonphotochemical quenching; Exciton interaction;

Proton transfer/translocation is studied in cytochrome c oxidase (CcO) by a combination of quantum mechanics (QM) for the transferring protons and classical molecular dynamics (MD) for the protein and solvent. The possibility of a glutamate, Glu286 in the Rhodobacter sphaeroides numbering scheme, acting as a rely point for proton translocation is investigated. The MD finds a hydrogen-bonded cycle of two waters and the carboxylate oxygens of Glu286. The possibility of protonating Glu286 to form neutral GluH is studied and we find that, as experimentally inferred, this glutamate can spend most of its time as GluH. Since translocation relies on the presence of water chains within CcO channels, MD is used to assess their formation. Glu286 and Mg2+ can be connected by continuous hydrogen-bonded chains that are robust, though transient, and the protein appears spongy above (toward the outer membrane) the Mg2+. In contrast, the D-channel spanning Asp132, close to the inner membrane surface, to Glu286, forms water chains that are much sparser and do not continuously connect these residues. Rather, there are chains spanning Glu286 to the vicinity of Asn140, and other more robust and ramified water structures that connect Asp132 with waters close to Asn140.
Keywords: Proton transfer and translocation; Cytochrome c oxidase;

Photoaccumulation of two ascorbyl free radicals per photosystem I at 200 K by Pierre Sétif; Karen Meimberg; Ulrich Mühlenhoff; Alain Boussac (203-213).
Illumination of photosystem I (PSI) from the cyanobacterium Synechocystis sp. PCC 6803 at 200 K in the presence of ascorbate leads to the formation of two ascorbyl radicals per PSI, which are formed by P700+ reduction by ascorbate. During photoaccumulation, one half of the ascorbyl radicals is formed with a halftime of 1 min and the other half with a halftime of 7 min. Pulsed electron paramagnetic resonance (EPR) experiments with protonated/deuterated PSI show that a PSI proton/deuteron is strongly coupled to the ascorbyl radical. Our data indicate that reactive ascorbate molecules bind to PSI at two specific locations, which might be symmetrically located with respect to the pseudo-C2 axis of symmetry of the heterodimeric core of PSI. Reduction of P700+ by ascorbate leads to multiple turnover of PSI photochemistry, resulting in partial photoaccumulation of the doubly reduced species (FA , FB ). A modified form of FB —in accordance with Chamorovsky and Cammack [Biochim. Biophys. Acta 679 (1982) 146–155], but not of FA , is observed by EPR after illumination at 200 K, which indicates that reduction of FB at 200 K is followed by some relaxation process, in line with this cluster being the most exposed to the solvent.
Keywords: Photosynthesis; Ascorbate; EPR; HYSCORE; Protein relaxation; Iron–sulfur cluster; Hydrogen bond;