Evaluation of bacterial biosensors to determine chromate bioavailability and to assess ecotoxicity of soils.

Chromate can be considered a potent environmental contaminant and consequently, an understanding of chromate availability and toxicity to soil biology is essential for effective ecological assessment of metal impact in soils. This study shows the response of two bacterial bioreporters, pCHRGFP1 Escherichiacoli and pCHRGFP2 Ochrobactrumtritici, to increasing concentrations of chromate in two different soils. The bioreporters, carrying the regulatory gene chrB transcriptionally fused to the gfp reporter system, exhibited different features. In both, the fluorescence signal and the chromate concentration could be linearly correlated but E. coli biosensor functioned within the range of 0.5-2 µM and O. tritici biosensor within 2-10 µM chromate. The bioreporters were validated through comparative measurements using the chemical chromate methods of diphenylcarbazide and ionic chromatography. The bacterial sensors were used for the estimation of bioavailable fraction of chromate in a natural soil and OECD artificial soil, both spiked with chromate in increasing concentrations of 0-120 mg Cr(VI) kg(-1) of soil. OECD soil showed a faster chromate decrease comparing to the natural soil. The toxicity of soils amended with chromate was also evaluated by ecotoxicological tests through collembolan reproduction tests using Folsomia candida as test organism. Significant correlations were found between collembolans reproduction and chromate concentration in soil (lower at high chromate concentrations) measured by biosensors. Data obtained showed that the biosensors tested are sensitive to chromate presence in soil and may constitute a rapid and efficient method to measure chromate availability in soils.

Requirement for Asn298 on D1 protein for oxygen evolution: analyses by exhaustive amino acid substitution in the green alga Chlamydomonas reinhardtii.

PSII generates strong oxidants used for water oxidation. The secondary electron donor, Y(Z), is Tyr161 on PSII reaction center D1 protein and mediates electron transfer from the oxygen-evolving Mn(4)CaO(5) cluster to the primary electron donor, P680. The latest PSII crystal structure revealed the presence of a hydrogen bond network around Y(Z), which is anticipated to play important roles in the electron and proton transfer reactions. Y(Z) forms a hydrogen bond with His190 which in turn forms a hydrogen bond with Asn298 on D1 protein. Although functional roles of Y(Z) and His190 have already been characterized, little is known about the functional role of Asn298. Here we have generated 19 mutants from a green alga Chlamydomonas reinhardtii, in which the Asn298 has been substituted by each of the other 19 amino acid residues. All mutants showed significantly impaired or no photosynthetic growth. Seven mutants capable of photosynthetic growth showed oxygen-evolving activity although at a significantly reduced rate. Interestingly the oxygen-evolving activity of these mutants was markedly photosensitive. The 19 mutants accumulated PSII at variable levels and showed a light-induced electron transfer reaction from 1,5-diphenylcarbazide (DPC) to 2,6-dichlorophenolindophenol (DCIP), suggesting that Asn298 is important for the function and photoprotection of the Mn(4)CaO(5) cluster.

Development of a new chromium reducing antioxidant capacity (CHROMAC) assay for plants and fruits.

A chromium reducing antioxidant capacity (CHROMAC) assay was presented to measure antioxidant capacity of selected plants and fruits and compared its performance with other commonly used antioxidant capacity methods of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and cupric reducing antioxidant capacity (CUPRAC). The assay is based on the spectrophotometric measurement of colored a chelate complex of Cr(III) and diphenylcarbazone formed by the reaction of Cr(VI) and 1,5-diphenylcarbazid in acidic medium. Phenolic compounds react with excessive amounts of Cr(VI) at low pH values, causing reduction of Cr(VI) to Cr(III) and conversion of phenols to oxidized products. The assay comprises of the antioxidant with a chromium(VI) solution, a 1,5-diphenylcarbazid in acidic medium and subsequent measurement of the developed absorbance at 540 nm after 50 min. The color development is stable for phenolic compounds in plant and fruit. The selectivity of the assay for phenolic compounds was improved by adjusting pH to 2.8 and reduction potential between 0.2 and 0.9 V. The developed assay was successfully applied to the measurement of antioxidant capacity of three plants and one fruit (Prunus divaricata Ledeb.subsp. divaricata) samples and comparable results were obtained by ABTS and CUPRAC assays.

Glycinebetaine protects the D1/D2/Cytb559 complex of photosystem II against photo-induced and heat-induced inactivation.

The presence of 1.0 mol/L glycinebetaine during isolation of D1/D2/Cytb559 reaction centre (RC) complexes from photosystem II (PSII) membrane fragments preserved the photochemical activity, monitored as the light-induced reduction of pheophytin and electron transport from diphenylcarbazide to 2.6-dichlorophenol-indophenol.-Glycinebetaine also protected the D1/D2/Cytb559 complexes against strong light-induced damage to the photochemical reactions and the irreversible bleaching of beta-carotene and chlorophyll. The presence of glycinebetaine also enhanced thermotolerance of the D1/D2/Cytb559 complexes isolated in the presence of 1.0 mol/L betaine with an increase in the temperature for 50% inactivation from 29 degrees C to 35 degrees C. The results indicate an increased supramolecular structural stability in the presence of glycinebetaine.

Comparative study of effects of artificial electron donors on the AT-band of photosystem II thermoluminescence.

Extraction of the Mn-cluster from photosystem II (PS II) inhibits the main bands of thermoluminescence and induces a new AT-band at -20 degrees C. This band is attributed to the charge recombination between acceptor QA- and a redox-active histidine residue on the donor side of PS II. The effect of Mn(II) and Fe(II) cations as well as the artificial donors diphenylcarbazide and hydroxylamine on the AT-band of thermoluminescence was studied to elucidate the role of the redox-active His residue in binding to the Mn(II) and Fe(II). At the Mn/PS II reaction center (RC) ratio of 90 : 1 and Fe/PS II RC ratio of 120 : 1, treatment with Mn(II) and Fe(II) causes only 60% inhibition of the AT-band. Preliminary exposure of Mn-depleted PS II preparations to light in the presence of Mn(II) and Fe(II) causes binding of the cations to the high-affinity Mn-binding site, thereby inhibiting oxidation of the His residue involved in the AT-band formation. The efficiency of the AT-band quenching induced by diphenylcarbazide and hydroxylamine is almost an order of magnitude higher than the quenching efficiency of Mn(II) and Fe(II). Our results suggest that the redox-active His is not a ligand of the high-affinity site and does not participate in the electron transport from Mn(II) and Fe(II) to YZ. The concentration dependences of the AT-band inhibition by Mn(II) and Fe(II) coincide with each other, thereby implying specific interaction of Fe(II) with the donor side of PS II.

Effects of carboxyl amino acid modification on the properties of the high-affinity, manganese-binding site in photosystem II.

Our previous work using the "diphenylcarbazide (DPC)-inhibition assay" has identified four amino acid (two carboxyls and two histidyls) ligands to four Mn2+ bound with high affinity on Tris-washed photosystem II (PSII) membrane fragments [Preston and Seibert (1991) Biochemistry 30, 9615-9624, 9625-9633]. One of the ligands binds a photooxidizable Mn, specifically, and the others bind either nonphotooxidizable Mn2+, Zn2+, or Co2+ [Ghirardi et al. (1996) Biochemistry 35, 1820-1828]. The current paper shows the following: (a) the high-affinity photooxidizable Mn, which donates to the oxidized primary PSII donor (YZ*), is bound to a carboxyl residue with a KM = 1.5 microM or Kd = 0.94 microM in the absence of DPC, and a Ki = 1.3 microM in the presence of DPC (both steady-state and flash approaches were used); (b) if this carboxyl is chemically modified using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC), Mn2+ is photooxidized at a lower affinity (Kd = 25 microM) site that does not involve carboxyl ligands; (c) low-affinity Mn is photooxidized (possibly by YD*, the oxidized form of the alternative PSII donor) with a KM = 220 microM at a completely different site that also requires a carboxyl ligand; (d) photooxidation of high-affinity DPC by YZ* with a KM of 40-42 microM or Kd of 49-58 microM occurs at a site that does not require carboxyl residues; (e) photooxidation of low-affinity DPC with a KM = 1200 microM occurs at a site (possibly near YD) that is not affected by carboxyl modification with EDC. Due to the similarities between the binding of the high-affinity photooxidizable Mn to EDC-treated membranes and to PSII complexes from Asp170D1 mutants [Nixon and Diner (1992) Biochemistry 31, 942-948], we identify its carboxyl residue ligand as Asp170 on D1, one of the reaction-center proteins. The second carboxyl ligand identified using the DPC-inhibition assay binds Mn (but not a photooxidizable one), Zn, or Co ions. At least one of the two histidyl ligands (either His337 on D1 or another unidentified histidyl) that bind nonphotooxidizable, high-affinity Mn2+ also binds Zn2+ and Co2+.

Use of a novel histidyl modifier to probe for residues on Tris-treated photosystem II membrane fragments that may bind functional manganese.

In this paper, we investigate the effects of histidyl amino acid modification on high-affinity Mn binding to photosystem II (PSII) using methods similar to those used in the preceding paper [Ghirardi et al. (1998) Biochemistry 37, 0000] for carboxyl amino acid modification. Given the rather low specificity of diethyl pyrocarbonate (DEPC) for histidine modification, we modified Tris-washed PSII membranes with a novel and more specific histidyl modifier, platinum(II) (2,2':6',2"-terpyridine) chloride (Pt-TP). Both the "diphenylcarbazide (DPC)-inhibition assay" and single-turnover flash approaches were used. The concentration dependence of Pt-TP modification on steady-state measurements shows two types of interactions, each accounting for about half of the full effect. At concentrations <50 microM, Pt-TP modifies mostly histidyls and abolishes half of the observed Mn inhibition of DPC-mediated 2,6-dichlorophenolindophenol (DCIP) photoreduction (equivalent to two high-affinity, Mn-binding ligands). This effect can be blocked by addition of Mn2+ during Pt-TP modification. Double-modification experiments with DEPC and Pt-TP demonstrate that both modifiers affect the same observable histidyl residues in PSII. Above 50 microM, Pt-TP modifies mostly cysteines (or histidines in a more hydrophobic environment) and has an additional effect on the reducing side of PSII that (a) does not involve Mn binding and (b) results in the apparent abolishment of all four of the Mn-binding ligands detected by the DPC-inhibition assay. Single-flash experiments show that histidyl modification does not eliminate the binding of the high-affinity, photooxidizable Mn2+ to Asp170 on D1 (nor does it significantly affect high-affinity DPC photooxidation), but it does decrease the binding affinity (Kd) of that Mn from 0.6 to 1.5 microM, particularly at lower (<50 microM Pt-TP) concentrations. Double-modification experiments also demonstrate that the lower affinity, photooxidizable Mn-binding site, uncovered when the high-affinity site is modified with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) [see Ghirardi et al. (1998)], is not associated with a histidyl ligand. Three nonphotooxidizable, high-affinity Mn2+ ions bind to a second carboxyl and two histidyl ligands, and these Mn are not photooxidized by a flash even when the ligand to the photooxidizable Mn is modified by EDC. Proteolytic enzyme studies indicate that the two histidyl ligands identified by the DPC-inhibition assay are probably His337 on D1 and His 339 on D2, but His 332 on D1 is not eliminated.

Photochemical reactions of photosystem II in ethylene glycol.

The behavior of photosystem II (PSII) reactions was investigated under conditions of decreasing water content by the addition of increasing concentrations of ethylene glycol (EG). The photosynthetic activities were measured for PSII samples either directly in aqueous solutions of EG or in the standard buffer medium following EG treatment. Several effects on PSII arise upon exposure to EG. Below 50% EG there are no significant irreversible changes, although there is a slowing of the QA-reoxidation kinetics in the presence of EG. At concentrations of 50-70% EG, protein structural changes occur that include the release of the 16, 23, and 33 kDa extrinsic proteins and two of the catalytic Mn ions. For these samples, the capacity for O2 evolution is considerably reduced and the formation of donor side H2O2 is enhanced. In 60% EG, the nanosecond components in the rate of P680+ reduction are converted entirely to microsecond kinetics which upon return of the sample to the standard buffer medium are partially restored, indicating that EG has a reversible, solvent effect on the PSII donor side. At concentrations of EG > 70% chlorophyll fluorescence measurements reveal reversible increases in the FO level concomitant with the generation and disappearance of a 5 microseconds decay component in the P680+ reduction kinetics. This result may indicate a solvent-induced uncoupling of the light harvesting pigment bed from the reaction center complex. As the EG concentration is increased to 80-100%, there is an irreversible loss of the primary charge separation. The use of EG as a cryoprotectant and as a water-miscible organic solvent for PSII is discussed.

Electron donation to photosystem II by diphenylcarbazide is inhibited both by the endogenous manganese complex and by exogenous manganese ions.

Diphenylcarbazide (DPC) is an efficient electron donor to the inactive oxygen-evolving complex of photosystem II (PSII). We investigated the role of manganese on the rate of electron donation from DPC to PSII in both Mn-depleted (Tris washed) and Mn-retaining (NaCl washed) PSII preparations. The rate of electron donation from DPC to PSII was significantly higher in Mn-depleted than in Mn-retaining preparations, indicating a negative role of native Mn complex on DPC electron donation. The apparent Km values for DPC were found to be 0.11 and 0.17 mM for Mn-depleted and Mn-retaining PSII preparations, respectively. This difference in the Km values also indicates an antagonistic effect of endogenous Mn cluster on electron donation from DPC, which was markedly inhibited by exogenous Mn2+. However, the magnitude of inhibition was greater in Mn-depleted than in Mn-retaining PSII preparations. This indicates a higher accessibility to DPC to PSII in the absence of native Mn complex. Our results suggest (i) that Mn, either endogenous or added, acts as an accessibility barrier for DPC to donate electrons to PSII and (ii) that the native Mn complex not only functions as an accumulator of oxidizing equivalents but may also protect PSII from exogenous reductants.

Kinetics of photoinhibition in hydroxylamine-extracted photosystem II membranes: relevance to photoactivation and sites of electron donation.

Kinetic analyses were made of the effects of weak-light photoinhibition on the capacity of NH2OH-extracted photosystem II membranes to photooxidize the exogenous electron donors Mn2+, diphenylcarbazide, and I- or to assemble functional water-oxidizing complexes during photoactivation. The loss of capacity for photooxidation of the donors showed two first-order components (half-times of 2-3 min and 1-4 h) with relative amplitudes dependent on the donor, suggesting two photodamageable sites of electron donation (sites 1 and 2, respectively), a conclusion confirmed by analyses of velocity curves of electron donation by each donor. All of the donors appear to be oxidized preferentially by site 1 both at saturating and at limiting light intensity; however, the contribution by site 2 was nearly comparable in saturating light. Loss of photoactivation also exhibited biphasic kinetics, with components having half-times of approximately 0.8 and 3.2 min. The major component (t1/2 = 3.2 min) corresponded to loss of site 1; essentially no photoactivation was observed after its loss. From these and other analyses, we conclude (1) the relative contributions of site 1 and site 2 to the photooxidation of various exogenous electron donors is determined largely by the rates of equilibration of the donors with the two sites, and (2) only site 1 contributes to photoactivation of the water-oxidizing complex. Site 1 is attributed to tyrosine Z of the reaction center's D1 polypeptide. The molecular identity of site 2 is unknown but may be tyrosine D of the D2 polypeptide.

A requirement for Ca2+ in the extraction of O2-evolving Photosystem 2 preparations from the cyanobacterium Anacystis nidulans.

Ca2+ has been shown to be essential for the retention of maximal O2-evolving activity in Photosystem 2 particles extracted by using dodecyldimethylamine oxide from Anacystis nidulans thylakoids. The effect cannot entirely be mimicked by using Mg2+. Ca2+ stimulates electron transport from diphenylcarbazide to 2,6-dichloroindophenol catalysed by lead-inhibited cation-free preparations, showing the presence of two cation-binding sites in these particles. Photosystem 2 preparations extracted in Ca2+-containing buffer show the presence of three polypeptides at mol. wt. 30000, 33000 and 36000, which are absent or much decreased in preparations extracted in Mg2+-containing buffer. The calmodulin antagonist chlorpromazine inhibits activity of the Photosystem 2 preparation, suggesting the presence of a Ca2+-binding protein.

Photosynthetic apparatus of chilling-sensitive plants. IX. The involvement of alpha-tocopherol in the electron transport chain and the anti-oxidizing system in chloroplasts of tomato leaves.

1. The role of tocopherols in tomato chloroplasts from fresh, cold and dark-stored as well as stored and illuminated leaves was studied. 2. The cold and dark storage of leaves results in a loss of chloroplast alpha- and gamma-tocopherols of about 30-40% accompanied by an increase in chloroplast delta-tocopherol of about 40%. On illumination of stored leaves, an elevation of alpha- and gamma-tocopherol level to about 110 and 95% of the control, respectively, occurs, whilst delta-tocopherol content is not affected. 3. Experiments performed with 2,2-diphenyl-1-picrylhydrazyl-treated chloroplasts show that only about 70% of total alpha-tocopherol is functionally active in the electron transport of Photosystem II between the diphenylcarbazide (DPC) donation site and the inhibition site of DBMIB. 4. A small amount of alpha-tocopherol quinone (about 10% of alpha-tocopherol content) is found in chloroplasts from fresh, fresh and illuminated as well as cold and dark-stored tomato leaves, whereas the illumination of the latter increases the chloroplast alpha-tocopherol quinone content 3-fold. Moreover, following the illumination of chloroplasts from cold and dark-stored as well as stored and illuminated leaves, the oxidation of exogenous alpha-tocopherol to alpha-tocopherol quinone is 2-fold faster then in chloroplasts from fresh leaves. 5. The primary product ('alpha-tocopheroxide') formed during the alpha-tocopherol oxidation by illuminated chloroplasts was identified as 8a-hydroxy-alpha-tocopheron. 6. Exogenous alpha-tocopherol inhibits the lipid photoperoxidation by about 40-50% in chloroplasts from all three kinds of tomato leaf. 7. The results seem to suggest that chloroplast alpha-tocopherol is involved in both electron transport of PS II and antioxidizing system of chloroplasts.

[Establishment of the site of electron transport chain disruption in mutant Chlamydomonas chloroplasts with an inactive photosystem 2]. / Ustanovlenie mesta narusheniia élektrontransportnoi tsepi khloroplastov mutantov Chlamydomonas s neaktivnoi fotosistemoi 2.

It has been shown in the studies of 6 strains of non-synthesizing Chlamydomonas reinhardi mutants with a damaged electron-transport chain (ETC) in the region of the Photosystem 2 (PS 2) that the damage is localized on the oxidizing side of PS 2. The ESR studies of the mutants have shown that signal 2 is absent in all the mutants, the width of signal 1 in some mutants is lower than in the control, which is, probably, concerned with the differences in the reaction centre structures. The ETC region from electron inlet from the donor--diphenylcarbaside to P700 is capable of functioning. It is suggested that in all the mutants studied the complex responsible for photodissociation of water is damaged.

Photooxidation reactions of diphenylcarbazide and their DCMU-sensitivity in thylakoids of the blue-green alga Oscillatoria chalybea.

Thylakoids of Oscillatoria chalybea are able to split water. The Hill reaction of these thylakoids is sensitive to DCMU. Diphenylcarbazide can substitute for water as the electron donor to photosystem II with these fully functioning thylakoids. However, the diphenylcarbazide photooxidation is completely insensitive to 3-(3,4-dichlorophenyl)-N-N'-dimethyl urea (DCMU) at high diphenylcarbazide concentrations. In with Tris-treated Oscillatoria thylakoids the water splitting capacity is lost and diphenylcarbazide restores electron transport through photosystem II as occurs with higher plant chloroplasts. However, also these photoreactions are insensitive to DCMU. If diphenylcarbazide acts in Oscillatoria as an electron donor to photosystem II the result suggests that diphenylcarbazide feeds in its electrons behind the DCMU inhibition site. This in turn indicates that in Oscillatoria the site of inhibition of DCMU is on the donor side of photosystem II.

Identification of S2 as the sensitive state to alkaline photoinactivation of photosystem II in chloroplasts.

1. Chloroplasts have been preilluminated by a sequence of n short saturating flashes immediately before alkalinization to pH 9.3, and brought back 2 min later to pH 7.8. The assay of Photosystem II activity through dichlorophenolindophenol photoreduction, oxygen evolution, fluorescence induction, shows that part of the centers is inactivated and that this part depends on the number of preilluminating flashes (maximum inhibition after one flash) in a way which suggests identification of state S2 as the target for alkaline inactivation. 2. As shown by Reimer and Trebst ((1975) Biochem. Physiol. Pflanz. 168, 225-232) the inactivation necessitates the presence of gramicidin, which shows that the sensitive site is on the internal side of the thylakoid membrane. 3. The electron flow through inactivated Photosystem II is restored by artificial donor addition (diphenylcarbazide or hydroxylamine); this suggests that the water-splitting enzyme itself is blocked. The inactivation is accompanied by a solubilization of bound Mn2+ and by the occurence of EPR Signal II "fast". 4. Glutaraldehyde fixation before the treatment does not prevent the inactivation which thus does not seem to involve a protein structural change.

Multiple-flash activation of the water-photolysis system in wheat leaves as observed by delayed emission.

The chloroplasts from wheat leaves greened under intermittent illuminations (1 ms in duration) at long intervals (5 min) are capable of photoreducing DCIP (2,6-dichlorophenolindophenol) with diphenylcarbazide as an electron donor but are incapable of photoreducing DCIP with water as the donor. On exposure of such intermittently illuminated leaves to flashes spaced at intervals of less than 10s, the delayed light emission from the leaves was greatly enhanced in parallel with the generation of Hill activity. The mechanism of this photoactivation was studied by following the changes of the delayed emission from intermittently illuminated leaves exposed to short-interval flashes programmed in various ways. Analysis of the kinetic data indicated that the photoactivation involves three consecutive photoreactions with a rate-limiting dark reaction between them; P-light leads to A0-light leads to A1-dark leads to A2-light leads to A3 in which P is a precursor convertible to A0, the first intermediate with a longer lifetime of t 1/2 approximately 100s and A3 is the final activated compound or state converted by short-interval flashes from A0 through A1 and A2, two other intermediates with shorter lifetimes of t 1/2 approximately 0.4s and 5s, respectively.