Artificial Quinone Research Articles

Quinone-mediated extracellular electron transfer processes in ex situ biomethanation reactors

Redox mediators are used in a wide diversity of systems including biological ones. We investigated the effect of adding an artificial quinone (2,7-AQDS) as external redox molecule to an anaerobic digester system dominated by hydrogenotrophic methanogens. When oxidized AQDS was present, the methanogens diverted electrons from H2 to reduce AQDS instead of CO2. The AQDS reduction process was accompanied by a temporary CH4 inhibition, which was re-established several days after the full reduction of AQDS to AH2QDS. The presence of AQDS furthermore resulted in a community shift from Methanobacterium as the dominant methanogen to a more diverse community of methanogens. Protein expression profiles showed a shift in cofactor preference of the adapted community, as a potential response mechanism to AQDS inhibition. AH2QDS was only used as electron donor to a limited extent. Stable isotope incorporation experiments here indicated that the acetogen Acetoanaerobium used AH2QDS to reduce CO2 into acetate.

Acclimation of Chlamydomonas reinhardtii to extremely strong light

Most photosynthetic organisms are sensitive to very high light, although acclimation mechanisms enable them to deal with exposure to strong light up to a point. Here we show that cultures of wild-type Chlamydomonas reinhardtii strain cc124, when exposed to photosynthetic photon flux density 3000 μmol m−2 s−1 for a couple of days, are able to suddenly attain the ability to grow and thrive. We compared the phenotypes of control cells and cells acclimated to this extreme light (EL). The results suggest that genetic or epigenetic variation, developing during maintenance of the population in moderate light, contributes to the acclimation capability. EL acclimation was associated with a high carotenoid-to-chlorophyll ratio and slowed down PSII charge recombination reactions, probably by affecting the pre-exponential Arrhenius factor of the rate constant. In agreement with these findings, EL acclimated cells showed only one tenth of the 1O2 level of control cells. In spite of low 1O2 levels, the rate of the damaging reaction of PSII photoinhibition was similar in EL acclimated and control cells. Furthermore, EL acclimation was associated with slow PSII electron transfer to artificial quinone acceptors. The data show that ability to grow and thrive in extremely strong light is not restricted to photoinhibition-resistant organisms such as Chlorella ohadii or to high-light tolerant mutants, but a wild-type strain of a common model microalga has this ability as well.

Site-directed mutagenesis of two amino acid residues in cytochrome b559 α subunit that interact with a phosphatidylglycerol molecule (PG772) induces quinone-dependent inhibition of photosystem II activity

X-ray crystallographic analysis (1.9-Å resolution) of the cyanobacterial photosystem II (PSII) dimer showed the presence of five phosphatidylglycerol (PG) molecules per reaction center. One of the PG molecules, PG772, is located in the vicinity of the QB-binding site. To investigate the role of PG772 in PSII, we performed site-directed mutagenesis in the cytochrome (Cyt) b559 α subunit of Synechocystis sp. PCC 6803 to change two amino acids, Thr-5 and Ser-11, which interact with PG772. The photosynthetic activity of intact cells was slightly lower in all mutants than that of cells in the control strain; however, the oxygen-evolving PSII activity was decreased markedly in cells of mutants, as measured using artificial quinones (such as p-benzoquinone). Furthermore, electron transport from QA to QB was inhibited in mutants incubated with quinones, particularly under high-intensity light conditions. Lipid analysis of purified PSII showed approximately one PG molecule per reaction center, presumably PG772, was lost in the PSII dimer from the T5A and S11A mutants compared with that in the PSII dimer from the control strain. In addition, protein analysis of monomer and dimer showed decreased levels of PsbV and PsbU extrinsic proteins in the PSII monomer purified from T5A and S11A mutants. These results suggest that site-directed mutagenesis of Thr-5 and Ser-11, which presumably causes the loss of PG772, induces quinone-dependent inhibition of PSII activity under high-intensity light conditions and destabilizes the binding of extrinsic proteins to PSII.

Evaluation of photosynthetic activities in thylakoid membranes by means of Fourier transform infrared spectroscopy

Light-induced Fourier transformed infrared (FTIR) difference spectroscopy is a powerful method to study the structures and reactions of redox cofactors involved in the photosynthetic electron transport chain. So far, most of the FTIR studies of the reactions of oxygenic photosynthesis have been performed using isolated photosystem I (PSI) and photosystem II (PSII) preparations, which, however, could be modified during isolation procedures. In this study, we developed a methodology to evaluate the photosynthetic activities of thylakoids using FTIR spectroscopy. FTIR difference spectra upon successive flashes using thylakoids from spinach exhibited signals typical of the S-state cycle at the Mn4CaO5 cluster and QB reactions in PSII with period-four and -two oscillations, respectively. Similar measurement in the presence of an artificial quinone as an exogenous electron acceptor showed features specific to the S-state cycle. Simulations of the oscillation patterns provided the quantum efficiencies of the S-state cycle and electron transfer in PSII. Moreover, FTIR measurement under continuous illumination on thylakoids in the presence of DCMU showed signals due to QA reduction and P700 oxidation simultaneously. From the relative amplitudes of marker bands of QA− and P700+, the molar ratio of photoactive PSII and PSI centers in thylakoids was estimated. FTIR analyses of the photo-reactions in thylakoids, which are more intact than isolated photosystems, will be useful in investigations of the photosynthetic mechanism especially by genetic modification of photosystem proteins.

Structure, biochemical and kinetic properties of recombinant Pst2p from Saccharomyces cerevisiae, a FMN-dependent NAD(P)H:quinone oxidoreductase

The genome of the yeast Saccharomyces cerevisiae encodes four flavodoxin-like proteins, namely Lot6p, Pst2p, Rfs1p and Ycp4p. Thus far only Lot6p was characterized in detail demonstrating that the enzyme possesses NAD(P)H:quinone oxidoreductase activity. In the present study, we heterologously expressed PST2 in Escherichia coli and purified the produced protein to conduct a detailed biochemical and structural characterization. Determination of the three-dimensional structure by X-ray crystallography revealed that Pst2p adopts the flavodoxin-like fold and forms tetramers independent of cofactor binding. The lack of electron density for FMN indicated weak binding, which was confirmed by further biochemical analysis yielding a dissociation constant of 20±1μM. The redox potential of FMN bound to Pst2p was determined to −89±3mV and is thus 119mV more positive than that of free FMN indicating that reduced FMN binds ca. five orders of magnitude tighter to Pst2p than oxidized FMN. Due to this rather positive redox potential Pst2p is unable to reduce free FMN or azo dyes as reported for other members of the flavodoxin-like protein family. On the other hand, Pst2p efficiently catalyzes the NAD(P)H dependent two-electron reduction of natural and artificial quinones. The kinetic mechanism follows a ping-pong bi-bi reaction scheme. In vivo experiments with a PST2 knock out and overexpressing strain demonstrated that Pst2p enables yeast cells to cope with quinone-induced damage suggesting a role of the enzyme in managing oxidative stress.

Localization of Ubiquinone-8 in the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae

Na(+) is the second major coupling ion at membranes after protons, and many pathogenic bacteria use the sodium-motive force to their advantage. A prominent example is Vibrio cholerae, which relies on the Na(+)-pumping NADH:quinone oxidoreductase (Na(+)-NQR) as the first complex in its respiratory chain. The Na(+)-NQR is a multisubunit, membrane-embedded NADH dehydrogenase that oxidizes NADH and reduces quinone to quinol. Existing models describing redox-driven Na(+) translocation by the Na(+)-NQR are based on the assumption that the pump contains four flavins and one FeS cluster. Here we show that the large, peripheral NqrA subunit of the Na(+)-NQR binds one molecule of ubiquinone-8. Investigations of the dynamic interaction of NqrA with quinones by surface plasmon resonance and saturation transfer difference NMR reveal a high affinity, which is determined by the methoxy groups at the C-2 and C-3 positions of the quinone headgroup. Using photoactivatable quinone derivatives, it is demonstrated that ubiquinone-8 bound to NqrA occupies a functional site. A novel scheme of electron transfer in Na(+)-NQR is proposed that is initiated by NADH oxidation on subunit NqrF and leads to quinol formation on subunit NqrA.

Ca2+-binding and Ca2+-independent Respiratory NADH and NADPH Dehydrogenases of Arabidopsis thaliana

Type II NAD(P)H:quinone oxidoreductases are single polypeptide proteins widespread in the living world. They bypass the first site of respiratory energy conservation, constituted by the type I NADH dehydrogenases. To investigate substrate specificities and Ca(2+) binding properties of seven predicted type II NAD(P)H dehydrogenases of Arabidopsis thaliana we have produced them as T7-tagged fusion proteins in Escherichia coli. The NDB1 and NDB2 enzymes were found to bind Ca(2+), and a single amino acid substitution in the EF hand motif of NDB1 abolished the Ca(2+) binding. NDB2 and NDB4 functionally complemented an E. coli mutant deficient in endogenous type I and type II NADH dehydrogenases. This demonstrates that these two plant enzymes can substitute for the NADH dehydrogenases in the bacterial respiratory chain. Three NDB-type enzymes displayed distinct catalytic profiles with substrate specificities and Ca(2+) stimulation being considerably affected by changes in pH and substrate concentrations. Under physiologically relevant conditions, the NDB1 fusion protein acted as a Ca(2+)-dependent NADPH dehydrogenase. NDB2 and NDB4 fusion proteins were NADH-specific, and NDB2 was stimulated by Ca(2+). The observed activity profiles of the NDB-type enzymes provide a fundament for understanding the mitochondrial system for direct oxidation of cytosolic NAD(P)H in plants. Our findings also suggest different modes of regulation and metabolic roles for the analyzed A. thaliana enzymes.

Lot6p from Saccharomyces cerevisiae is a FMN‐dependent reductase with a potential role in quinone detoxification

NAD(P)H:quinone acceptor oxidoreductases are flavoenzymes expressed in the cytoplasm of many tissues and afford protection against the cytotoxic effects of electrophilic quinones by catalyzing a strict two-electron reduction. Such enzymes have been reported from several mammalian sources, e.g. human, mouse and rat, and from plant species. Here, we report identification of Lot6p (YLR011wp), the first soluble quinone reductase from the unicellular model organism Saccharomyces cerevisiae. Localization studies using an antibody raised against Lot6p as well as microscopic inspection of Lot6p-GFP demonstrated accumulation of the enzyme in the cytosol of yeast cells. Despite sharing only 23% similarity to type 1 human quinone reductase, Lot6p possesses biochemical properties that are similar to its human counterpart. The enzyme catalyzes a two-electron reduction of a series of natural and artificial quinone substrates at the expense of either NADH or NADPH. The kinetic mechanism follows a ping-pong bi-bi reaction scheme, with K(M) values of 1.6-11 microm for various quinones. Dicoumarol and Cibacron Marine, two well-known inhibitors of the quinone reductase family, bind to Lot6p and inhibit its activity. In vivo experiments demonstrate that the enzymatic activity of Lot6p is consistent with the phenotype of both Deltalot6 and Lot6p overexpressing strains, suggesting that Lot6p may play a role in managing oxidative stress in yeast.

An approach towards artificial quinone pools by use of photo- and redox-active dendritic molecules

Molecules with one porphyrin and multiple quinone groups are described. The molecules are based on dendritic frameworks, with the quinone groups attached at the “internal” positions and the porphyrin attached at the focal point, leading to a characteristic layer architecture. When irradiated with visible light in the presence of 4- tert-butylthiophenol, the quinones were converted to quinols. Such a behavior mimics the function of quinone pools in photosynthesis.

Electron Transfer Ability from NADH to Menaquinone and from NADPH to Oxygen of Type II NADH Dehydrogenase ofCorynebacterium glutamicum

Type II NADH dehydrogenase of Corynebacterium glutamicum (NDH-2) was purified from an ndh overexpressing strain. Purification conferred 6-fold higher specific activity of NADH:ubiquinone-1 oxidoreductase with a 3.5-fold higher recovery than that previously reported (K. Matsushita et al., 2000). UV-visible and fluorescence analyses of the purified enzyme showed that NDH-2 of C. glutamicum contained non-covalently bound FAD but not covalently bound FMN. This enzyme had an ability to catalyze electron transfer from NADH and NADPH to oxygen as well as various artificial quinone analogs at neutral and acidic pHs respectively. The reduction of native quinone of C. glutamicum, menaquinone-2, with this enzyme was observed only with NADH, whereas electron transfer to oxygen was observed more intensively with NADPH. This study provides evidence that C. glutamicum NDH-2 is a source of the reactive oxygen species, superoxide and hydrogen peroxide, concomitant with NADH and NADPH oxidation, but especially with NADPH oxidation. Together with this unique character of NADPH oxidation, phylogenetic analysis of NDH-2 from various organisms suggests that NDH-2 of C. glutamicum is more closely related to yeast or fungal enzymes than to other prokaryotic enzymes.

Interaction of exogenous quinones with membranes of higher plant chloroplasts: modulation of quinone capacities as photochemical and non-photochemical quenchers of energy in Photosystem II during light–dark transitions

Light modulation of the ability of three artificial quinones, 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone (DBMIB), 2,6-dichloro- p-benzoquinone (DCBQ), and tetramethyl- p-benzoquinone (duroquinone), to quench chlorophyll (Chl) fluorescence photochemically or non-photochemically was studied to simulate the functions of endogenous plastoquinones during the thermal phase of fast Chl fluorescence induction kinetics. DBMIB was found to suppress by severalfold the basal level of Chl fluorescence ( F o) and to markedly retard the light-induced rise of variable fluorescence ( F v). After irradiation with actinic light, Chl fluorescence rapidly dropped down to the level corresponding to F o level in untreated thylakoids and then slowly declined to the initial level. DBMIB was found to be an efficient photochemical quencher of energy in Photosystem II (PSII) in the dark, but not after prolonged irradiation. Those events were owing to DBMIB reduction under light and its oxidation in the dark. At high concentrations, DCBQ exhibited quenching behaviours similar to those of DBMIB. In contrast, duroquinone demonstrated the ability to quench F v at low concentration, while F o was declined only at high concentrations of this artificial quinone. Unlike for DBMIB and DCBQ, quenched F o level was attained rapidly after actinic light had been turned off in the presence of high duroquinone concentrations. That finding evidenced that the capacity of duroquinone to non-photochemically quench excitation energy in PSII was maintained during irradiation, which is likely owing to the rapid electron transfer from duroquinol to Photosystem I (PSI). It was suggested that DBMIB and DCBQ at high concentration, on the one hand, and duroquinone, on the other hand, mimic the properties of plastoquinones as photochemical and non-photochemical quenchers of energy in PSII under different conditions. The first model corresponds to the conditions under which the plastoquinone pool can be largely reduced (weak electron release from PSII to PSI compared to PSII-driven electron flow from water under strong light and weak PSI photochemical capacity because of inactive electron transport on its reducing side), while the second one mimics the behaviour of the plastoquinone pool when it cannot be filled up with electrons (weak or moderate light and high photochemical competence of PSI).

Targeted disruption of psbX and biochemical characterization of photosystem II complex in the thermophilic cyanobacterium Synechococcus elongatus.

PSII-X is a small hydrophobic protein, which is universally present in photosystem II (PSII) core complex among cyanobacteria and plants. The role of PSII-X was studied by directed mutagenesis and biochemical analysis in the thermophilic cyanobacterium Synechococcus elongatus. The psbX-disrupted mutant could grow photoautotrophically indicative of non-essential function, while it showed growth defect under low CO(2) conditions. An active O(2)-evolving PSII complex was successfully isolated from the mutant and wild type. Protein composition of the isolated PSII complex was the same as wild type except for the absence of PSII-X. O(2) evolution supported by artificial quinones was affected in the psbX-disrupted mutant. At high concentration of 2,6-dichlorobenzoquinone or 2,6-dimethylbenzoquinone, the mutant showed much lower activity than wild type, while not much difference was found at low concentration. These results imply that binding or turnover of quinones at the Q(B) site depends, at least in part, on PSII-X protein in the PSII complex. Gel filtration chromatography of the PSII complex revealed that the dimeric structure of the complex was not greatly affected in the psbX-disrupted mutant.

Photosystem I and II reaction centers of a new oxygenic organism Acaryochloris marina that use chlorophyll d.

PS I and II reaction centers of a cyanobacteria-like oxygenic organism Acaryochloris marina were studied. The organism has chlorophyll d (Chl d) that has a formyl group on chlorin ring and absorbs at 700-720 nm and Chl a in a molar ratio of 30:1. Isolated PS I contains one special pair of Chl d (P740), which shows an absorption change at 740 nm and has Em of +335 mV, in 145 chlorophyll d on RC polypeptides homologous to cyanobacteria1. Laser spectroscopy, ENDOR and FTIR studies indicated the structure and environments of P740 similar to but somewhat different from those of Chl a dimer P700. Electron acceptor phylloquinone was extracted with diethyl ether and its function was replaced by reconstituted artificial quinones. Absorption changes of Chls in PS II were also studied in isolated thylakoids after chemical oxidation of P740. The difference spectrum gave a peak at 720 nm with no peak at 680 nm suggesting PS II special pair of this organism to be Chl d. 1Q. Hu, H. Miyashita, I. Iwasaki, K. N. Kurano, S. Miyachi, M. Iwaki and S. Itoh, Proc. Natl. Acad. Sci., 95, 13319 (1998).

Selective extraction of antenna chlorophylls, carotenoids and quinones from photosystem I reaction center.

By the ether treatment of lyophilized PSI pigment-protein complexes, all the carotenoids and the secondary acceptor phylloquinone (A1), and more than 90% of the Chl were removed to yield the PSI complex with 9-11 molecules of Chl per reaction-center unit. The complexes retained the primary electron donor and acceptor (P700 and A0), in addition to three FeS clusters (F(X), F(A) and F(B)), and showed an activity of highly efficient electron transfer when phylloquinone was reconstituted. The methods for the preparation and the characterization of the ether-extracted PSI complexes are reviewed in this article. We also review the studies done with this PSI preparation on (1) the identification of the absorption and fluorescence spectra of P700, (2) the nano- and picosecond reaction of A0 and A1, (3) the energy-gap dependency of the reaction rate between A0 and the artificial quinones reconstituted at the A1 site, (4) the direct excitation of P700 followed by the ultra-fast electron transfer from P700 to A0, and (5) the de- and re-stabilization of the PSI structure by the removal and reconstitution, respectively, of antenna Chl in the presence of certain lipids.

Spin-Polarized Radical Pair in Photosystem I Reaction Center That Contains Different Quinones and Fluorenones as the Secondary Electron Acceptor

The spin-polarized radical pair of the primary donor chlorophyll P700+ and the secondary acceptor Q- was studied by the electron spin echo envelope modulation (ESEEM) technique in the spinach photosystem I reaction center in which artificial quinones and fluorenones were reconstituted as Q. From the dipole interaction between the spins on P700+ and Q-, the distance between P700 and anilinochloronaphthoquinone or hydroxyanthraquinone was estimated to be 25.6 ± 0.3 Å or 25.7 ± 0.3 Å, respectively, suggesting their proper binding at the original phylloquinone binding site. Distances of 26.0 Å with larger heterogeneity were estimated for (NO2)3- and (NO2)4-fluorenone compounds that have only one carbonyl group, suggesting their multiple orientations in the binding pocket. The lifetimes of the radical pairs estimated by the time dependency of the two-pulse ESE signals were almost temperature independent (30−80 μs at 4−80 K). In the three-pulse ESE measurement, the lifetimes were lengthened by 10−103 times and showed strong temperature dependencies.

Mobility of the primary electron-accepting plastoquinone QA of photosystem II in a Synechocystis sp. PCC 6803 strain carrying mutations in the D2 protein.

Upon introduction of random mutations in a region of the psbDI gene that encodes the D2 protein in the cyanobacterium Synechocystis sp. PCC 6803, an obligate photoheterotrophic mutant was isolated that contained three mutations: V247M, A249T, and M329I. This mutant evolved oxygen in the absence of added electron acceptors, but oxygen evolution was inhibited by micromolar concentrations of several artificial quinones. Complementation analysis showed that the V247M and/or A249T mutations were responsible for this phenotype. Using fluorescence induction and decay measurements, the site of inhibition by the quinones was found to be at the level of the primary electron-accepting quinone in photosystem II, QA. Duroquinone inhibited by blocking reduction of QA, and in the presence of other quinones such as 2,5-dichloro-p-benzoquinone, 2, 5-dimethyl-p-benzoquinone, and p-benzoquinone, QA could be reduced but could not efficiently transfer an electron to QB. To distinguish the effects of the V247M and A249T mutations, single mutants were created. V247M was photoautotrophic and had an essentially normal phenotype. The A249T mutant, although photoautotrophic, was affected by artificial quinones, but less than the mutant carrying both the V247M and A249T changes. The results indicate a decreased plastoquinone affinity at the QA site in the strains carrying a A249T mutation, such that after dark-adaptation a significant percentage of the QA sites is empty or is occupied by an artificial quinone. In light, the percentage of photosystem II centers with plastoquinone bound at the QA site appears to increase, which may be due in part to an increased affinity of the semiquinone versus that of the quinone at the QA site.

Purification and Characterization of an NAD(P)H:Quinone Oxidoreductase fromGlycine MaxSeedlings

An NAD(P)H:(quinone acceptor) oxidoreductase (EC 1.6.99.2) was purified from Glycine max seedlings by means of chromatographic procedures. After 1371-fold purification, the enzyme showed a single band in IEF corresponding to an isoelectric point of 6.1. A single band was also found in native-PAGE both by activity staining and Coomassie brilliant blue staining. The molecular mass determined in SDS-PAGE was 21900 Da, while in HPLC gel-filtration it was 61000 Da. The NAD(P)H:quinone oxidoreductase was able to use NADH or NADPH as the electron donor. Among the artificial quinones which are reduced by this enzyme, 6-hydroxydopa- and 6-hydroxydopamine-quinone are of particular interest because of their neurotoxic effects.

Competition among plastoquinol and artificial quinone / quinol couples at the quinol oxidizing site of the cytochrome bf complex

We have investigated the interaction of plastoquinol (PQH 2), duroquinol (DQH 2) and duroquinone (DQ) with the PQH 2 oxidizing site (Q o site) of the chloroplast bf complex. In the absence of exogenous quinones or quinols, with an essentially fully reduced PQ pool, the half-time for reduction of cyt b upon a single-turnover actinic flash was approx. 2–2.5 ms, with an initial rate of about 250 s −1. The same rate was found with an approx. 20% oxidized PQ pool, indicating that this rate is most probably at or near the V max for the reaction. When the PQ pool was reduced by addition of 100 μM DQH 2, the rate of cyt b reduction was considerably slower, with a half-time of about 6 ms and an intial rate of about 100 s −1. Only at much higher concentrations of DQH 2 did the initial rate of cyt b reduction approach that found in samples where PQH 2 was the sole reductant. We concluded that, in the presence of O 2, a fraction of the added DQH 2 was oxidized to DQ which acted as a competitive inhibitor of the Q o site. The slowed cyt b reduction kinetics were not observed when the production of DQ was prevented by excluding oxygen from the sample or by pre-reduction of the PQ pool by sodium dithionite. In strictly anaerobic samples titrated with a series of DQ and DQH 2 concentrations, where the PQH 2 pool was at all times essentially completely reduced, we were able to demonstrate a competitive interaction at the Q o site among PQH 2, DQH 2 and DQ. By using an appropriate kinetic model - consisting of an enzyme (the bf complex), two alternate substrates (PQH 2 and DQH 2) and one competitive inhibitor (DQ) - we were able to simulate the pre-steady state kinetics of cyt b reduction that resulted from this competition. From these simulations, we concluded that the V max for oxidation of DQH 2 and PQH 2 were both about 250 s −1. The predicted binding constants for the species at the Q o site depended on the assumed values of the partition coefficients of DQ and DQH 2 into the thylakoid membrane. When estimated partition coefficients for the DQ and DQH 2 were introduced into the simulations, we were able to estimate that the binding constant of PQH 2 to the Q o site was ≥ 2 · 10 4 M −1. We concluded that, in native thylakoids, with a completely reduced PQ pool, essentially all Q o sites were occupied with PQH 2, consistent with a relatively tight binding. Approx. 3 μM of added DQ is expected to displace PQH 2 from half of the Q o sites. DQH 2 can displace nearly all of the DQ from the sites, but only at high added concentrations. At the high concentrations of DQH 2 typically employed in electron transfer assays - typically 0.5–1 mM - nearly all turnovers of the complex occur at the expense of DQH 2. At lower concentrations, in the presence of O 2, competitive inhibition by DQ can severely affect experimental results. We have also found that decyl-ubiquinol (dUQH 2) is a substrate for the bf complex, but with the product of partition coefficient into the membrane and binding constant into the Q o site greater than that of DQH 2 and DQ.

Light saturation curves show competence of the water splitting complex in inactive Photosystem II reaction centers

Photosystem II complexes of higher plants are structurally and functionally heterogeneous. While the only clearly defined structural difference is that Photosystem II reaction centers are served by two distinct antenna sizes, several types of functional heterogeneity have been demonstrated. Among these is the observation that in dark-adapted leaves of spinach and pea, over 30% of the Photosystem II reaction centers are unable to reduce plastoquinone to plastoquinol at physiologically meaningful rates. Several lines of evidence show that the impaired reaction centers are effectively inactive, because the rate of oxidation of the primary quinone acceptor, QA, is 1000 times slower than in normally active reaction centers. However, there are conflicting opinions and data over whether inactive Photosystem II complexes are capable of oxidizing water in the presence of certain artificial electron acceptors. In the present study we investigated whether inactive Photosystem II complexes have a functional water oxidizing system in spinach thylakoid membranes by measuring the flash yield of water oxidation products as a function of flash intensity. At low flash energies (less that 10% saturation), selected to minimize double turnovers of reaction centers, we found that in the presence of the artificial quinone acceptor, dichlorobenzoquinone (DCBQ), the yield of proton release was enhanced 20±2% over that observed in the presence of dimethylbenzoquinone (DMBQ). We argue that the extra proton release is from the normally inactive Photosystem II reaction centers that have been activated in the presence of DCBQ, demonstrating their capacity to oxidize water in repetitive flashes, as concluded by Graan and Ort (Biochim Biophys Acta (1986) 852: 320-330). The light saturation curves indicate that the effective antenna size of inactive reaction centers is 55±12% the size of active Photosystem II centers. Comparison of the light saturation dependence of steady state oxygen evolution in the presence of DCBQ or DMBQ support the conclusion that inactive Photosystem II complexes have a functional water oxidation system.

Quinone compounds are able to replace molecular oxygen as terminal electron acceptor in phytoene desaturation in chromoplasts of Narcissus pseudonarcissus L.

The desaturation of phytoene to zeta-carotene and of zeta-carotene to lycopene employs molecular oxygen as the terminal electron acceptor. 2,3,5,6-Tetramethyl-1,4-benzoquinone (duroquinone) and other artificial quinones are able to replace oxygen, which demonstrates that oxygen does not act in a mixed-function oxygenase-like mechanism at the desaturase itself, but at a spatially separate site. Evidence for additional redox elements mediating between desaturase and oxygen is presented.