Identification of ubiquinone as the secondary electron acceptor in the photosynthetic apparatus of Chromatium vinosum

Extracting Chromatium vinosum chromatophores with light petroleum destroys their ability to perform photochemistry on the second of two closely-spaced actinic flashes, without affecting photochemistry on the first flash. Extraction also increases the likelihood of a back-reaction in which an electron returns from the primary electron acceptor directly to P₈₇₀. These effects probably reflect the removal of a secondary electron acceptor. Extraction does not appear to interfere with the primary photochemical reaction. Reconstituting the extracted chromatophores with the lipid extract or with pure ubiquinone (Q) completely reverses the effects of the extraction. Chromatography of the lipid extract shows that Q is the only active material that it contains in detectable quantity. These observations support the conclusion that Q is the secondary electron acceptor.

Piericidin A, certain alkyl-substituted quinolinequinones, and a substituted 4,7-dioxobenzothiazole inhibit electron transfer between the primary and secondary acceptors. The sensitivity to these inhibitors, and the participation of Q and non-heme iron suggest that the secondary electron-transfer reaction resembles the reactions catalyzed by respiratory dehydrogenases.

The proton uptake that follows flash excitation does not seem to be tightly linked to the reduction of the secondary electron acceptor. It still occurs (though with decreased amplitude) in extracted chromatophores, and even in the presence of inhibitors of the secondary electron-transfer reaction.     

Characterization of the complex interaction between the electron acceptor silicomolybdate and Photosystem II

Silicomolybdate (SiMo) and its effects on thylakoids have been characterized to evaluate its use as a probe for Photosystem II (PS II). It can accept electrons at two places in the electron transport chain: one at PS II and the other at PS I. In the presence of 1 M 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) only the site at PS II is available. It is suggested that SiMo must disp;ace bicarbonate from its binding site to be able to function as an electron acceptor. This displacement is non-competitive. The binding of SiMo is inhibited differentially by PS II inhibitors: dinoseb>ioxynil> diuron. This difference is determined by the different positions of the inhibitors within the Q_B binding niche and their interaction with bicarbonate. The experimental results show that the SiMo-binding niche is located between the parallel helices of the D1 and D2 proteins of PS II, close to the non-heme iron. We conclude that SiMo is an electron acceptor with unique characteristics useful as a probe of the acceptor side of PS II.     

Hg ++ - a DCMU independent electron acceptor of photosystem II

Mercuric chloride functions as a direct electron acceptor from the quencher of fluorescence in Photosystem II. The photoreduction of ferricyanide, dichlorophenol-indophenol or methyl viologen is inhibited by mercuric ion while oxygen evolution is uneffected. Mercuric chloride supported oxygen evolution (mercury Hill reaction) is not prevented by DCMU or other similar electron transport inhibitors.     

Inhibition of mitochondrial ATP-synthetase by redox-agents]

Effects of phenol and phenothiazine on ATP synthesis and electron transport in submitochondrial particles were studied. Nitrophenols and phenothiazines inhibited ATP synthesis without notable effect on electron transport. On the contrary chlorphenols equally decreased the velocities of electron transport and ATP synthesis. The inhibitors studied showed the properties of electron acceptors in relation to the radicals, their acceptor properties corresponding to their ability to inhibit ATP synthesis.     

Probing the primary quinone environment in photosynthetic bacterial reaction centers by light-induced FTIR difference spectroscopy

The photoreduction of the primary electron acceptor, QA, has been characterized by light-induced Fourier transform infrared difference spectroscopy for Rb. sphaeroides reaction centers and for Rsp. rubrum and Rp. viridis chromatophores. The samples were treated both with redox compounds, which rapidly reduce the photooxidized primary electron P+, and with inhibitors of electron transfer from QA- to the secondary quinone QB. This approach yields spectra free from P and P+ contributions which makes possible the study of the microenvironment of QA and QA-.     

Photosystem II and the unique role of bicarbonate: A historical perspective

In photosynthesis, cyanobacteria, algae and plants fix carbon dioxide (CO(2)) into carbohydrates; this is necessary to support life on Earth. Over 50years ago, Otto Heinrich Warburg discovered a unique stimulatory role of CO(2) in the Hill reaction (i.e., O(2) evolution accompanied by reduction of an artificial electron acceptor), which, obviously, does not include any carbon fixation pathway; Warburg used this discovery to support his idea that O(2) in photosynthesis originates in CO(2). During the 1960s, a large number of researchers attempted to decipher this unique phenomenon, with limited success. In the 1970s, Alan Stemler, in Govindjee's lab, perfected methods to get highly reproducible results, and observed, among other things, that the turnover of Photosystem II (PSII) was stimulated by bicarbonate ions (hydrogen carbonate): the effect would be on the donor or the acceptor, or both sides of PSII. In 1975, Thomas Wydrzynski, also in Govindjee's lab, discovered that there was a definite bicarbonate effect on the electron acceptor (the plastoquinone) side of PSII. The most recent 1.9? crystal structure of PSII, unequivocally shows HCO(3)(-) bound to the non-heme iron that sits in-between the bound primary quinone electron acceptor, Q(A), and the secondary quinone electron acceptor Q(B). In this review, we focus on the historical development of our understanding of this unique bicarbonate effect on the electron acceptor side of PSII, and its mechanism as obtained by biochemical, biophysical and molecular biological approaches in many laboratories around the World. We suggest an atomic level model in which HCO(3)(-)/CO(3)(2-) plays a key role in the protonation of the reduced Q(B). In addition, we make comments on the role of bicarbonate on the donor side of PSII, as has been extensively studied in the labs of Alan Stemler (USA) and Vyacheslav Klimov (Russia). We end this review by discussing the uniqueness of bicarbonate's role in oxygenic photosynthesis and its role in the evolutionary development of O(2)-evolving PSII. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Inhibition of photosystem II of spinach by the respiration inhibitors piericidin A and thenoyltrifluoroacetone

The effects of several respiration inhibitors on photosystem II (PS II) were investigated. Among the agents tested, piericidin A and thenoyltrifluoroacetone (TTFA) inhibited the photosynthetic electron transport of spinach as measured from chlorophyll (Chl) fluorescence parameters (Fm'-F)/Fm' and Fv/Fm. Using specific donors and acceptors of electrons, we identified the sites of inhibition in and around the PS II complex; the site of inhibition by TTFA was between QA, primary quinone acceptor in PS II, and QB, secondary quinone acceptor, in the acceptor side of P680, the reaction center Chl of PS II, while inhibition by piericidin A of the acceptor side was downstream of Q(B), out of the PS II complex. Both agents also inhibited the donor side of P680, probably between tyrosine-161 of the reaction center protein of PS II and P680.     

A new classification of the amino acid side chains based on doublet acceptor energy levels

We describe a new classification of the amino acid side chains based on the potential energy level at which each will accept an extra (doublet) electron. The doublet acceptor energy level, and the doublet acceptor orbital were calculated using semiempirical INDO/2-UHF molecular orbital theory. The results of these calculations show that the side chains fall into four groups. We have termed these groups repulsive, insulating, semiconducting, and attractive in accordance with where each lies on the relative energy scale. We use this classification to examine the role of residues between the donor and acceptor in modulating the rate and mechanism of electron transfer in proteins. With the calculated acceptor levels, we construct a potential barrier for those residues between the donor and acceptor. It is the area beneath this barrier that determines the decay of electronic coupling between donor and acceptor, and thus the transfer rate. We have used this schematic approach to characterize the four electron transfer pathways in myoglobin recently studied by Mayo et al. (Mayo, S.L., W.R. Ellis, R.J. Crutchley, and H.B. Gray. 1986. Science [Wash. DC]. 233:948-952).     

Role of bicarbonate at the acceptor side of Photosystem II

Besides being the substrate for the carboxylation reaction of photosynthesis, CO₂ (bicarbonate) is required for the activity of Photosystem II (water plastoquinone oxido-reductase). It plays a role on the electron donor side as well as the electron acceptor side. In this contribution, attention will mostly be focused on the history of research into the effects of bicarbonate on electron flow reactions on the acceptor side. Donor side reactions are discussed in this issue by Alan Stemler. This revised version was published online in June 2006 with corrections to the Cover Date.     

Probing the donor and acceptor substrate specificity of the γ-glutamyl transpeptidase

γ-Glutamyl transpeptidase (GGT) is a two-substrate enzyme that plays a central role in glutathione metabolism and is a potential target for drug design. GGT catalyzes the cleavage of γ-glutamyl donor substrates and the transfer of the γ-glutamyl moiety to an amine of an acceptor substrate or water. Although structures of bacterial GGT have revealed details of the protein-ligand interactions at the donor site, the acceptor substrate site is relatively undefined. The recent identification of a species-specific acceptor site inhibitor, OU749, suggests that these inhibitors may be less toxic than glutamine analogues. Here we investigated the donor and acceptor substrate preferences of Bacillus anthracis GGT (CapD) and applied computational approaches in combination with kinetics to probe the structural basis of the enzyme's substrate and inhibitor binding specificities and compare them with human GGT. Site-directed mutagenesis studies showed that the R432A and R520S variants exhibited 6- and 95-fold decreases in hydrolase activity, respectively, and that their activity was not stimulated by the addition of the l-Cys acceptor substrate, suggesting an additional role in acceptor binding and/or catalysis of transpeptidation. Rat GGT (and presumably HuGGT) has strict stereospecificity for L-amino acid acceptor substrates, while CapD can utilize both L- and D-acceptor substrates comparably. Modeling and kinetic analysis suggest that R520 and R432 allow two alternate acceptor substrate binding modes for L- and D-acceptors. R432 is conserved in Francisella tularensis, Yersinia pestis, Burkholderia mallei, Helicobacter pylori and Escherichia coli, but not in human GGT. Docking and MD simulations point toward key residues that contribute to inhibitor and acceptor substrate binding, providing a guide to designing novel and specific GGT inhibitors.     

Clara cell damage and inhibition of pulmonary mixed-function oxidase activity by naphthalene

The photosystem II electron acceptor 3,6-dichloro-2,5-dimethoxy-p-benzoquinone [DCDMQ] is suggested to replace the second quinone-type two electron acceptor B (or R); the DCDMQ Hill reaction is sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea, but is insensitive to dry heptane extraction of thylakoids and other photosystem II inhibitors. Addition of HCO₃^? to CO₂-depleted thylakoids in silicomolybdate, DCDMQ, diaminodurene and ferricyanide Hill reactions brought about 1,3,10 and 10 fold increase in the electron transport rates; these data confirm that HCO₃^? affects both Q^? to B and B^2? to PQ reactions.     

NMR-based exploration of the acceptor binding site of human blood group B galactosyltransferase with molecular fragments

A substantial body of work has been devoted to the design and synthesis of glycosyltransferase inhibitors. A major obstacle has always been the demanding chemistry. Therefore, only few potent and selective inhibitors are known to date. Glycosyltransferases possess two distinct binding sites, one for the donor substrate, and one for the acceptor substrate. In many cases binding to the donor site is well defined but data for acceptor binding is sparse. In particular, acceptor binding sites are often shallow, and in many cases the dimensions of the binding pocket are not well defined. One approach to glycosyltransferase inhibitors is to chemically link donor site and acceptor site ligands to generate high affinity binders. Here, we describe a novel approach to identify acceptor site ligands from a fragment library. We have chosen human blood group B galactosyltransferase (GTB) as a biologically important model target. The approach utilizes a combination of STD NMR, spin-lock filtered NMR experiments and surface plasmon resonance measurements. Following this route we have identified molecular fragments from a fragment library that bind to the acceptor site of GTB with affinities of the order of a natural acceptor substrate. Unlike natural substrates these fragments allow for straightforward chemical modifications and, therefore will serve as scaffolds for potent GTB inhibitors. In general, the approach described is applicable to any glycosyltransferase and may assist in the development of novel glycosyltransferase inhibitors.     

2,6-Dichloro-phenol indophenol prevents switch-over of electrons between the cyanide-sensitive and -insensitive pathway of the mitochondrial electron transport chain in the presence of inhibitors

To evolve a simple oxygen electrode-based method to estimate alternative respiration, one needs to develop a procedure to prevent switch-over of electrons to either pathway upon inhibition by cyanide or salicylhydroxamic acid. It was hypothesized that the inclusion of appropriate electron acceptor, possessing redox potential close to one of the electron transport carriers in between ubiquinone (branch point) and cytochrome a-a3, should be able to stop switch-over of electrons to either pathway by working as an electron sink. To test the hypothesis, 2,6-dichloro-phenol indophenol (DCPIP; redox potential +0.217 V), an artificial electron acceptor having a redox potential quite similar to the site near cytochrome c1 (redox potential +0.22 V) on the cyanide-sensitive pathway, was used with isolated mitochondria and leaf discs in the absence and presence of inhibitors (potassium cyanide, antimycin A, and salicylhydroxamic acid). Polarographic data confirmed electron acceptance by DCPIP only from the inhibited (by cyanide or salicylhydroxamic acid) mitochondrial electron transport chain, hence preventing switch-over of electrons between the cyanide-sensitive and cyanide-insensitive pathway of respiration. Results with antimycin A and reduction status of DCPIP further confirmed electron acceptance by DCPIP from the mitochondrial electron transport chain. Possible implications of the results have been discussed.     

Identification of tenuazonic acid as a novel type of natural photosystem II inhibitor binding in Q(B)-site of Chlamydomonas reinhardtii

Tenuazonic acid (TeA) is a natural phytotoxin produced by Alternaria alternata, the causal agent of brown leaf spot disease of Eupatorium adenophorum. Results from chlorophyll fluorescence revealed TeA can block electron flow from Q(A) to Q(B) at photosystem II acceptor side. Based on studies with D1-mutants of Chlamydomonas reinhardtii, the No. 256 amino acid plays a key role in TeA binding to the Q(B)-niche. The results of competitive replacement with [(14)C]atrazine combined with JIP-test and D1-mutant showed that TeA should be considered as a new type of photosystem II inhibitor because it has a different binding behavior within Q(B)-niche from other known photosystem II inhibitors. Bioassay of TeA and its analogues indicated 3-acyl-5-alkyltetramic and even tetramic acid compounds may represent a new structural framework for photosynthetic inhibitors.     

Relationship between hydrogen consumption, dehalogenation, and the reduction of sulfur oxyanions by Desulfomonile tiedjei

Resting-cell suspensions of Desulfomonile tiedjei consumed H2 with 3-chloro-, 3-bromo-, and 3-iodobenzoate as electron acceptors with rates of 0.50, 0.44, and 0.04 mumol h-1 mg-1, respectively. However, benzoate and 3-fluorobenzoate were not metabolized by this bacterium. In addition, H2 uptake was at least fourfold faster when sulfate, sulfite, or thiosulfate was available as the electron acceptor instead of a haloaromatic substrate. When sulfite and 3-chlorobenzoate were both available for this purpose, the rate of H2 uptake by D. tiedjei was intermediate between that obtained with either electron acceptor alone. Hydrogen concentrations were reduced to comparably low levels when either 3-chlorobenzoate, sulfate, or sulfite was available as an electron acceptor, but significantly less H2 depletion was evident with benzoate or nitrate. Rates of 3-chlorobenzoate dechlorination increased from an endogenous rate of 14.5 to 17.1, 74.0, 81.1, and 82.3 nmol h-1 mg-1 with acetate, pyruvate, H2, and formate, respectively, as the electron donors. Sulfite and thiosulfate inhibited dehalogenation, but sulfate and NaCl had no effect. Dehalogenation and H2 metabolism were also inhibited by acetylene, molybdate, selenate, and metronidazole. Sulfite reduction and dehalogenation were inhibited by the same respiratory inhibitors. These results suggest that the reduction of sulfite and dehalogenation may share part of the same electron transport chain. The kinetics of H2 consumption and the direct inhibition of dehalogenation by sulfite and thiosulfate in D. tiedjei cells clearly indicate that the reduction of sulfur oxyanions is favored over aryl dehalogenation for the removal of reducing equivalents under anaerobic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization, partial purification and properties of N-methylglutamate dehydrogenase from Pseudomonas aminovorans.

1. Extracts of amine-grown Pseudomonas aminovorans contained a particle-bound N-methylglutamate dehydrogenase (EC 1.5.99.5). The enzyme was not present in succinate-grown cells, and activity appeared before growth began in succinate-grown cells which had been transferred to methylamine growth medium. 2. Membrane-containing preparations from methylamine-grown cells catalysed an N-methylglutamate-dependent uptake of O2 or reduction of cytochrome c, which was sensitive to inhibitors of the electron-transport chain. 3. N-Methylglutamate dehydrogenase activity with phenazine methosulphate or 2,6-dichlorophenol-indophenol as electron acceptor could be solubilized with 1% (w/v) Triton X-100. The solubilized enzyme was much less active with cytochrome c as electron acceptor and did not sediment in 1 h at 150000g. Solubilization was accompanied by a change in the pH optimum for activity. 4. The solubilized enzyme was partially purified by Sepharose 4B and hydroxyapatite chromatograpy to yield a preparation 22-fold increased in specific activity over the crude extract. 5. The partially-purified enzyme was active with sarcosine, N-methylalanine and N-methylaspartate as well as with N-methylglutamate. Evidence suggesting activity with N-methyl D-amino acids as well as with the L-forms was obtained. 6. The enzyme was inhibited by p-chloromercuribenzoate, iodoacetamide and by both ionic and non-ionic detergents. 2-Oxoglutarate and formaldehyde were also inhibitors. 7. Kinetic analysis confirmed previous workers' observations of a group transfer (Ping Pong) mechanism. 8. Spectral observations suggested that the partially purified preparation contained flavoprotein and a b-type cytochrome. 9. The role of the enzyme in the oxidation of methylamine is discussed.     

Role of bicarbonate in photosystem II, the water-plastoquinone oxido-reductase of plant photosynthesis

Depletion of bicarbonate (carbon dioxide) from oxygenic cells or organelles not only causes cessation of carbon dioxide fixation, but also a strong decrease in the activity of photosystem II; the photosystem II activity can be restored by readdition of bicarbonate. Effects of bicarbonate exist on both the acceptor as well as on the donor side of photosystem II. The influence on the acceptor side is located between the primary and secondary quinone electron acceptor of photosystem II, and can be demonstrated in intact cells or leaves as well as in isolated thylakoids and reaction center preparations. At physiological pH, bicarbonate ions are suggested to form hydrogen bonds to several amino acids on both D1 and D2 proteins, the reaction center subunits of photosystem II, as well as to form ligands to the non-heme iron between the D1 and D2 proteins. Bicarbonate, at physiological pH, has an important role in the water-plastoquinone oxido-reductase: on the one hand it may stabilize, by conformational means, the reaction center protein of photosystem II that allows efficient electron flow and protonation of certain amino acids near the secondary quinone electron acceptor of photosystem II; and, on the other hand, it akppears to play a significant role in the assembly or functioning of the manganese complex at the donor side. Functional roles of bicarbonate in vivo, including protection against photoinhibition, are also discussed.     

Relationship between hydrogen consumption, dehalogenation, and the reduction of sulfur oxyanions by Desulfomonile tiedjei.

Resting-cell suspensions of Desulfomonile tiedjei consumed H2 with 3-chloro-, 3-bromo-, and 3-iodobenzoate as electron acceptors with rates of 0.50, 0.44, and 0.04 mumol h-1 mg-1, respectively. However, benzoate and 3-fluorobenzoate were not metabolized by this bacterium. In addition, H2 uptake was at least fourfold faster when sulfate, sulfite, or thiosulfate was available as the electron acceptor instead of a haloaromatic substrate. When sulfite and 3-chlorobenzoate were both available for this purpose, the rate of H2 uptake by D. tiedjei was intermediate between that obtained with either electron acceptor alone. Hydrogen concentrations were reduced to comparably low levels when either 3-chlorobenzoate, sulfate, or sulfite was available as an electron acceptor, but significantly less H2 depletion was evident with benzoate or nitrate. Rates of 3-chlorobenzoate dechlorination increased from an endogenous rate of 14.5 to 17.1, 74.0, 81.1, and 82.3 nmol h-1 mg-1 with acetate, pyruvate, H2, and formate, respectively, as the electron donors. Sulfite and thiosulfate inhibited dehalogenation, but sulfate and NaCl had no effect. Dehalogenation and H2 metabolism were also inhibited by acetylene, molybdate, selenate, and metronidazole. Sulfite reduction and dehalogenation were inhibited by the same respiratory inhibitors. These results suggest that the reduction of sulfite and dehalogenation may share part of the same electron transport chain. The kinetics of H2 consumption and the direct inhibition of dehalogenation by sulfite and thiosulfate in D. tiedjei cells clearly indicate that the reduction of sulfur oxyanions is favored over aryl dehalogenation for the removal of reducing equivalents under anaerobic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transplasma membrane electron transport in Leishmania donovani promastigotes

Leishmania donovani promastigotes are capable of reducing certain electron acceptors with redox potential at pH 7 down to -125 mV; outside the plasma membrane promastigotes can reduce ferricyanide. Ferricyanide has been used as an artificial electron acceptor probe for studying the mechanism of transplasma membrane electron transport. Transmembrane ferricyanide reduction by L. donovani promastigotes was not inhibited by such mitochondrial inhibitors as antimycin A or cyanide, but it responded to inhibitors of glycolysis. Transmembrane ferricyanide reduction by Leishmania appears to involve a plasma membrane electron transport chain dissimilar to that of hepatocyte cells. As with other cells, transmembrane electron transport is associated with proton release, which may be involved in internal pH regulation. The Leishmania transmembrane redox system differs from that of mammalian cells in being 4-fold less sensitive to chloroquine and 12-fold more sensitive to niclosamide. Sensitivities to these drugs suggest that transplasma membrane electron transport and associated proton pumping may be targets for the drugs used against leishmaniasis.     

Corrosion-enhancing potential of Shewanella putrefaciens isolated from industrial cooling waters

Microbially influenced corrosion (MIC) is catalysed by a series of metabolic activities of selected micro-organisms, notably by oxidation of cathodic hydrogen by hydrogenase, by hydrogen sulphide and by reduction of ferric iron. The sulphate-reducing bacteria are considered to be the most common catalyst of MIC, whereas the role of other bacteria has been neglected. This study examined the corrosive potential of the facultative sulphide producer, Shewanella putrefaciens , isolated from an industrial cooling water system. Shewanella putrefaciens was shown to reduce ferric iron and sulphite under anaerobic conditions and with ferric iron being the preferred electron acceptor. The isolate could utilize cathodic hydrogen as an energy source, especially when using sulphite as a terminal electron acceptor. In pure culture corrosion experiments, the highest mass loss of mild steel was observed in the presence of sulphite as sole electron acceptor, although mass loss was also detected where ferric iron was the sole electron acceptor. Our data indicate that S. putefaciens plays a role in MIC as it was able to catalyse a variety of corrosion-promoting reactions and to corrode mild steel under pure culture conditions.