The roles of weak light,oxygen and dithiothreitol in the photoreactivation of the oxygen evolving center of tris-washed and 2, 6-dichlorophenol indophenol-treated chloroplasts

The inactivated O₂-evolving center of Tris-washed chloroplasts was reactivated by DCPIP-treatment and photoreactivation in the presence of Mn²⁺, Ca²⁺, DTT and weak light. Many electron donors (Asc and reduced DCPIP, etc.) were found to be suitable substitutes for DTT. By studying the anaerobic inhibition of the reactivation, the electron acceptors O₂, NADP⁺, etc. were also found to be essential factors in photoreactivation. Weak light stimulated the chloroplast electron transport from the above-mentioned electron donors to the electron acceptor and effected the photoreactivation. More than 280 electrons were transported to NADP⁺ in the anaerobic photoreactivation of one unit of an O₂-evolving center with 400 Chl. Electron transport in the reactivation was inhibited by omitting DTT or Mn²⁺ ion, and by adding DCMU. The photoreactivated chloroplasts incorporated about 2 Mn by 400 Chl. Omission of DTT in the reactivation caused chloroplasts in the weak light to bind large amounts of excess Mn.Abbreviations Asc ascorbate - Chl chlorophyll - DCPIP 2, 6-dichlorophenol indophenol - DPC diphenyl carbazide - DTT dithiothreitol - Fd ferredoxin - STN a chloroplast preparation medium, containing 0.4 M sucrose, 0.05 M Tris-Cl and 0.01 M NaCl (pH 7.8 and 8.0) - TMPD tetramethyl-p-phenylenediamine     

Improvement of four sigma analysis for the investigation of oxygen evolution by Photosystem II

We present here an improvement to the analysis of oxygen evolution with four sigma coefficients (4-S) by computing z, the sum of the S-state probabilities, which was introduced earlier (Delrieu and Rosengard 1987, Biochim Biophys Acta 892: 163–171). We demonstrate that z is equal to the ratio of two consecutive Mean Y (the estimation of the steady state oxygen production based on local properties) found by three sigma analysis. The quantity z is useful for computing double-hits, and for showing the inactivation/activation processes of PS II complexes. Three sigma analysis assumes z=1 exactly; since this is not verified, it is argued that four sigma analysis is closer to the real workings of the water oxidizing complex. Oxygen evolution can then be interpreted in the frame of a modified Kok's model where the sum of the probabilities equals z. We therefore suggest that the closer fitting of four sigma analysis to oxygen production data is not simply due to an extra, unnecessary variable, but to the fact that PS II complexes can be inactivated and reactivated under flashing light. Finally, in order to facilitate the use of four sigma analysis, a computer program is made available upon request.     

The effect of exercise intensity and duration on the oxygen deficit and excess post-exercise oxygen consumption

Nine males with mean maximal oxygen consumption (VO2max) = 63.0 ml.kg-1.min-1, SD 5.7 and mean body fat = 10.6%, SD 3.1 each completed nine counterbalanced treatments comprising 20, 50 and 80 min of treadmill exercise at 30, 50 and 70% VO2max. The O2 deficit, 8 h excess post-exercise oxygen consumption (EPOC) and EPOC:O2 deficit ratio were calculated for all subjects relative to mean values obtained from 2 control days each lasting 9.3 h. The O2 deficit, which was essentially independent of exercise duration, increased significantly (P less than 0.05) with intensity such that the overall mean values for the three 30%, 50% and 70% VO2max workloads were 0.83, 1.89 and 3.09 l, respectively. While there were no significant differences (P greater than 0.05) between the three EPOCs after walking at 30% VO2max for 20 (1.01 l), 50 (1.43 l) and 80 min (1.04 l), respectively, the EPOC thereafter increased (P less than 0.05) with both intensity and duration such that the increments were much greater for the three 70% VO2max workloads (EPOC: 20 min = 5.68 l; 50 min = 10.04 l; 80 min = 14.59 l) than for the three 50% VO2max workloads (EPOC: 20 min = 3.14 l; 50 min = 5.19 l; 80 min = 6.10 l). An analysis of variance indicated that exercise intensity was the major determinant of the EPOC since it explained five times more of the EPOC variance than either exercise duration or the intensity times duration interaction. The mean EPOC:O2 deficit ratio ranged from 0.8 to 4.5 and generally increased with both exercise intensity and duration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Computer-controlled pulse modulation system for analysis of photoacoustic signals in the time domain

A newly developed photoacoustic system for measurement of photosynthetic reactions in intact leaves is described. The system is based on pulsed light-emitting diodes, the pulse program and pulse response analysis being computer controlled. Separation of various components in the overall photoacoustic signal is achieved by curve fitting analysis of the responses following individual measuring light pulses in the millisecond time domain. This procedure is in distinction to the conventionally used analysis in the frequency domain, with the advantage that various signal components are obtained by on-line deconvolution, yielding simultaneous recordings of photothermal (complement of energy storage) and photobaric (evolution and uptake) signals. The basic components of the new system are described by block diagrams and the principal steps for deconvolution of the overall photoacoustic response are outlined. An example of application with simultaneous recording of chlorophyll fluorescence is given. It is apparent that the photobaric uptake component represents a significant part of the overall signal, particularly during induction of photosynthesis after dark-adaptation. This component probably contains not only O₂-uptake but uptake of CO₂ as well.Abbreviations PA photoacoustic - LED light-emitting-diode - RAM random access memory     

Control of misses in oxygen evolution by the oxido-reduction state of plastoquinone in Dunaliella tertiolecta

The way misses happen in oxygen evolution is subject to debate (Govindjee et al. 1985). We recently observed a linear lowering of the miss probability with the flash number (Meunier and Popovic 1989). Therefore, we investigated in Dunaliella tertiolecta the link between the average miss probability and the redox state of plastoquinone after n flashes. The effect of flashes was to oxidize the plastoquinone pool; we found that the oxidation of plastoquinone highly correlated (linear regression: R ²=0.996) with the lowering of the miss probability. The flash frequency was found to affect both the miss probability and the redox state of plastoquinone. When pre-flashes were given using a high flash frequency (10 Hz), the plastoquinone pool was oxidized and misses were low; however, if long dark intervals between flashes were used, the oxidizing effect of flashes was lost and the misses were high. We could not explain our results by assuming equal misses over all S-states; but unequal misses, caused by deactivations, were coherent with our results. We deduced that chlororespiration was responsible for the reduction of plastoquinone in the dark interval between flashes. We compared oxygen evolution with and without benzoquinone, using a low flash frequency (0.5 Hz) for maximum misses. Benzoquinone lowered the misses from 34% to 3%, and raised the amplitude of oxygen evolution by more than a factor of two (2). From this we deduced that the charge carrier C postulated to explain misses (Lavorel and Maison-Peteri 1983) did not account for more than 3% of miss probability in Dunaliella tertiolecta. These results indicate that the misses in oxygen evolution are controlled by the redox state of plastoquinone, through deactivations.     

Artificial inhibitory effects of sulfite on photosystem II activity measured by oxygen evolution in chloroplasts

In this report we demonstrate sulfite interaction with oxygen and PSII electron acceptors (ferricyanide and para-benzoquinone) during measurement of oxygen evolution in chloroplasts. Redox potentials of oxygen, ferricyanide and para-benzoquinone allow them to compete for sulfite. Without taking this into account, sulfite inhibition of oxygen evolution can be overestimated, since sulfite consumes oxygen and reduces ferricyanide or para-benzoquinone during the measurement. In order to correctly measure the rate of oxygen evolution in chloroplasts, it is necessary to avoid presence of sulfite during the measurement. After overcoming the artifact, mentioned above, we confirm the sulfite inhibition of oxygen evolution in chloroplasts but at a lesser extent than earlier reported. This, however, is a pretreatment effect.Abbreviations Chl Chlorophyll - EDTA Ethylenediamine Tetraacetic Acid - FeCN Potassium Ferricyanide - Hepes N-2-Hydroxyethylpiperazine-N¹-2-ethanesulfonic acid - pBQ Para-benzoquinone - PSII photosystem II     

Evidence for a linear variation of the miss and single hit S-state probabilities with the flash number,measured by oxygen evolution in Dunaliella tertiolecta

Oxygen evolution in Dunaliella tertiolecta under flashing light was measured with a bare electrode, at a 10 Hz frquency. The sigma coefficients of the oxygen evolution recurrence law (Thibault (1978) J Theor Biol 73, 271) were determined using groups of nine consecutive points. The S-state transition probabilities were computed from the sigma coefficients and plotted as a function of the flash number of the first of the points used. Low standard deviations over the sigma coefficients resulted from the use of our system (Meunier & Popovic (1988) Rev Sci Instr 59, 486). We observed a linear lowering of the miss probability with time, and a linear rise of the single-hit probability with the same absolute value of the slope. The hypothesis that the slopes were zero was statistically tested and was rejected with a 99.9% confidence interval. Our work demonstrates that, to be accurate, an oxygen evolution model has to take the variations in the properties of S-states into account.International Journal of Fracture 76 (1995) R37     

Interactions between carbon dioxide and oxygen in the photosynthesis of three species of marine red macroalgae

Summary

Red algae have the highest known selectivity factor (S_rel) for CO₂ over O₂ of ribulose bisphosphate carboxylase-oxygenase (RUBISCO). This allows the prediction that a red alga relying on diffusive supply of CO₂ to RUBISCO from air-equilibrated solution should have less O₂ inhibition of photosynthesis than would an otherwise similar non-red alga with a lower S_rel of RUBISCO. Furthermore, RUBISCO shows an increased S_rel values at low temperatures. The prediction that O ₂inhibition of photosynthesis should be small for marine red algae relying on diffusive CO₂ entry growing in the North Sea with an annual temperature range of 4–16°C was tested in O₂ electrode experiments at 12°C. Phycodrys rubens and Plocamium cartilagineum, which rely on diffusive CO₂ entry showed, as predicted, only a small inhibition at lower inorganic C concentrations. Palmaria palmata, which has a CO₂ concentrating mechanism, had the expected negligible O ₂ inhibition of photosynthesis at any inorganic C concentration except (non-significantly) for saturating inorganic C.     

Optimization of the bare platinum electrode as an oxygen measurement system in photosynthesis

This paper is concerned with the definition of the standard conditions required for optimum operation of the bare platinum electrode with photosynthetic samples. Experimental evidence shows the following: 1) Polarization circuits should have zero resistance; 2) The electrolyte layer between the electrodes should have a conductance higher than 54×10^{–6 –1} per mm² of platinum electrode area; 3) The electrodes should be polarized just before taking the measurements. All these facts can be interpreted in terms of phenomena occurring on the electrode: The adsorption of hydrogen on the electrode imposes the need for low resistances in the system, and oxygen consumption by the electrode is minimized by polarizing the electrodes as late as possible. This investigation increases the reliability of the bare platinum electrode and gives a basis for a comparison of the results from different experiments. Demonstrations of the pertinence of these conditions are made in our lab with the algae Dunaliella Tertiolecta.     

Analysis of oxygen evolution during photosynthetic induction and in multiple-turnover flashes in sunflower leaves

Exchange of CO₂ and O₂ and chlorophyll fluorescence were measured in the presence of 360 1 · 1^–1 CO₂ in nitrogen in Helianthus annuss L. leaves which had been preconditioned in the dark or at a photon flux density (PFD) of 24 mol · m^–2 · s^–1 either in 21 or 0% O₂. An initial light-dependent O₂ outburst of 6 mol · m^–2 was measured after aerobic dark incubation. It was attributed to the reduction of electron carriers, predominantly plastoquinone. The maximum initial rate of O₂ evolution at PFD 8000 mol · m^–2 · s^–1 was 170 mol · m^–2 · s^–2 or about four times the steady CO₂-and light-saturated rate of photosynthesis. Fluorescence measurements showed that the rate was still acceptor-limited. Fast O₂ evolution ceased after electron carriers were reduced in the dark-adapted leaf, but continued for a short time at the lower rate of 62 mol · m^–2 · s^–1 in the light-adapted leaf. The data are interpreted to show that enzymes involved in 3-phosphoglycerate reduction are dark-inhibited, but were fully active in low light. In a dark-adapted leaf, respiratory CO₂ evolution continued under nitrogen; it was partially inhibited by illumination. Prolonged exposure of a leaf to anaerobic conditions caused reducing equivalents to accumulate. This was shown by a slowly increasing chlorophyll fluorescence yield which indicated the reduction of the PSII acceptor Q_A in the dark. When the leaf was illuminated, no O₂ evolution was detected from short light pulses, although transient O₂ production was appreciable during longer light pulses. This indicates that an electron donor (pool size about 2–3 e/PSII reaction center) became reduced in the dark and the first photons were used to oxidise this donor instead of water.Abbreviations Chl chlorophyll - CRC carbon reduction cycle - GAPDH NADP-glyceraldehyde-phosphate dehydrogenase - PFD photon flux density - PGA 3-phosphoglycerate - RuBP ribulose bisphosphate - TCA tricarboxylic acid cycle To whom correspondence should be addressedThis work received support by the Estonian Academy of Sciences, the Gottfried-Wilhelm-Leibniz Program of the Deutsche For-schungsgemeinschaft and the Sonderforschungsbereich 251 of the University of Würzburg.     

Oxygen evolving membranes and particles from the transformable cyanobacterium Synechocystis sp. PCC6803

Membranes and PS II particles retaining high rates of O₂-evolving activity have been isolated from the transformable cyanobacterium, Synechocystis sp. PCC6803. Membranes from cells grown under red light exhibit rates of O₂-evolution ranging from 500–700 mole O₂/mg chl/h. PS II particles are prepared by a simple procedure involving DEAE column chromatography of detergent extracts obtained by simultaneous treatment of membranes with octylglucoside and dodecylmaltoside. The isolated PS II fraction is enriched in polypeptides immunologically cross-reactive with polypeptides present in core reaction center preparations of spinach, exhibits 77 K fluorescence emission maxima at 685 and 696 nm, but not emission and absorption due to phycobilines and is capable of rates of O₂-evolution exceeding 1000 mole O₂/mg chl/h.Abbreviations DM dodecyl--D-maltoside - OG octyl--D-glucoside     

Evidence for H2O2 as the product of reduction of oxygen by alternative oxidase in mitochondria from potato tubers

Oxygen consumption by alternative oxidase (AOX), present in mitochondria of many angiosperms, is known to be cyanide-resistant in contrast to cytochrome oxidase. Its activity in potato tuber (Solanum tuberosum L.) was induced following chilling treatment at 4 °C. About half of the total O₂ consumption of succinate oxidation in such mitochondria was found to be sensitive to SHAM, a known inhibitor of AOX activity. Addition of catalase to the reaction mixture of AOX during the reaction decreased the rate of SHAM-sensitive oxygen consumption by nearly half, and addition at the end of the reaction released nearly half of the consumed oxygen by AOX, both typical of catalase action on H₂O₂. These findings with catalase suggest that the product of reduction of AOX is H₂O₂ and not H₂O, as previously surmised. In potatoes subjected to chill stress (4 °C) for periods of 3, 5 and ?8 days the activity of AOX in mitochondria increased progressively with a corresponding increase in the AOX protein detected by immunoblot of the protein.     

A model for the mechanism of chloride activation of oxygen evolution in photosystem II

A hypothesis is proposed to explain the function of Cl^- in activating the oxygenevolving complex (OEC) of photosystem II (PS II), based on the results of recent ³⁵Cl-NMR studies. The putative mechanism involves Cl^- binding to two types of sites. An intrinsic site is suggested to be composed of three histidyl residues (His 332 and His 337 from D1 and His 337 D2). It is proposed that Cl^- binding to this site accelerates the abstraction of H⁺ from water by raising the pK_a's of the histidine imidazole groups. Cl^- binding also stimulates the transfer of H⁺ from this intrinsic site to a set of extrinsic sites on the 33 kD extrinsic polypeptide. The extrinsic Cl^- binding sites are suggested to involve four protein domains that are linked together by salt-bridge contacts. Chloride and H⁺ donated from the intrinsic site attack these intramolecular salt-bridges in a defined sequence, thereby exposing previously inaccessible Cl^- and H⁺ binding sites and stimulating the oxidation of water. This hypothesis also proposes a possible structure for the Mn active site within the D1/D2 complex. Specific amino-acid residues that are likely to participate as Mn lignads are identified on the lumenal portions of the D1 and D2 proteins that are different from those in the L and M subunits of photosynthetic bacteria; the choice of these residues is based on the metal coordination chemistry of these residues, their location within the polypeptide chain, the regularity of their spacing, and their conservation through evolution. The catalytic Mn-binding residues are suggested to be D-61, E-65, E-92, E-98, D-103; D-308, E-329, E-342 and E-333 in D1, and H-62, E-70, H-88, E-97, D-101; E-313, D-334, E-338 and E-345 in D2. Finally, this hypothesis identifies sites on both D2 and the 33 kD extrinsic polypeptide that might be involved in high- and low-affinity Ca²⁺ binding.To whom correspondence should be addressed     

Quantitative aspects of oxygen and carbon dioxide exchange through the lungs in Ocypode ceratophthalmus (Crustacea: Decapoda) during rest and exercise in water and air

Ghost crabs Ocypode ceratophthalmus were exercised in air and water to measure CO₂ and O₂ exchange rates using the method of instantaneous measurements of oxygen consumption rate (MO₂) where applicable. Average heart rate increased from 100 to nearly 400 pulses per minute after five minutes of exercise on a treadmill at a run rate of 0.133 m s^?1. It took less than a minute for oxygen taken up through the lung epithelium from the air inside the branchial cavity to reach the maximal oxygen consumption rate of 26.1 mmol O₂ kg^?1 h^?1. Resting MO₂ was 4.06 mmol O₂ kg^?1 h^?1 in air, but decreased to 3.37 mmol O₂ kg^?1 h^?1 in seawater. Radioactive CO₂ from injected l-lactate is released linearly by the lung. The percent accumulated 14-CO₂ in exhaled air, plotted against time, intersects zero time on the x -axis, indicating rapid gas exchange at the lung surface. The P ₅₀ values for native haemocyanin of 4.89 mm Hg before exercise, and 8.99 mm Hg after exercise, are typical of a high-affinity haemocyanin usually associated with terrestrial crabs. The current notion that Ocypode ceratophthalmus drown when submerged in seawater was not substantiated by our experiments. MO₂ in seawater increased from 3.37 mmol O₂ kg^?1 h^?1 for resting crabs to 5.72 mmol O₂ kg^?1 h^?1 during exercise. When submerged by wave-seawater in the natural environment and during exercise in respirometer-seawater O. ceratophthalmus do not swim but, having a specific density of 1.044, float nearly weightless with a minimum of body movements.     

Reactive oxygen species generation through NADH oxidation by 6-formylpterin derivatives in the dark

6-formylpterin (6FP) has been reported to produce reactive oxygen species (ROS) such as *O2- and H2O2 from O2 in the presence of NADH under light condition. In the present study, we prepared a variety of 6FP derivatives and found that 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3-pivaloylpteridin-4-one and 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3-methylpteridin-4-one, in which the 2-amino groups are modified by a dimethylaminomethylene group and the 3-positions by pivaloyl and methyl groups and 2-amino-6-formyl-3-methylpteridin-4-one in which the amino group at the 2-position is free and the 3-position is modified by a methyl group generated H2O2 from O2 on oxidation of NADH to NAD+ in the dark. However, 6FP and 2-(N,N-dimethylaminomethyleneamino)-6-formylpteridin-4-one, in which the 3-position is free did not yield H2O2. These results indicate that modification of the 3-position is essential to make the activities of 6FP available in the dark and would be suggestive for designing pharmaceutical compounds that generate appropriate and controllable amounts of ROS in vivo.     

Haemocyanin oxygen binding and the physiological ecology of a range of talitroidean amphipods (Crustacea)

Summary Haemolymph PO₂ and pH of two amphipod species, Apohyale pugettensis (aquatic) and Megalorchestia californiana (semi-terrestrial) in vivo were examined during immersion and emersion at 15 and 25°C, and also after activity in air at 15°C. For M. californiana arterial O₂ tensions were higher in air than in water. This situation was reversed in A. pugettensis, although all O₂ tensions measured for both species were comparatively high. No arterial-venous PO₂ difference was apparent in the haemolymph of quiescent M. californiana. Haemocyanin (Hc) was 100% saturated in vivo only in the following; A. pugettensis in water (15 and 25°C) and air (15°C), and M. californiana in air (15°C). The Hc of both species becomes important in O₂ transport during activity; under such circumstances the haemolymph of M. californiana delivered more O₂ to the tissues than did that of A. pugettensis, despite the greater O₂ content of the latter. The animals studied here may exhibit a stage (size class?) where cutaneous gas exchange is sufficient for resting aerobic metabolism while specialized respiratory carriers (and respiratory structures) are important in meeting the increased aerobic demands of activity or environmental stress.Abbreviations Hc haemocyanin - PO₂ partial pressure of oxygen     

On the role of bicarbonate in peroxidations catalyzed by Cu,Zn superoxide dismutase

The interaction of Cu,ZnSOD with H2O2 generates an oxidant at the active site that can then cause either the inactivation of this enzyme or the oxidation of a variety of exogenous substrates. We show that the rate of inactivation, imposed by 10-mM H2O2 at 25 degrees C and pH 7.2, is not influenced by 10-mM HCO3-; whereas the oxidation of 2,2'-azino-bis-[3-ethylbenzothiazoline sulfonate] (ABTS=) is virtually completely dependent upon HCO3-. The reduction of the active site Cu(II) by H2O2, which precedes inactivation of the enzyme, occurred at the same rate in phosphate buffer with or without bicarbonate added. These results indicate that HCO3- does not play a role in facilitating the interaction of H2O2 with the active site copper, but they can be accommodated by the proposal that HCO3- is oxidized to HCO3*, which then diffuses from that site and causes the oxidation of substrates, such as ABTS=, that are too large to traverse the solvent access channel to the Cu(II).     

A sensitive,simultaneous analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase efficiencies: Graphical determination of the CO2/O2 specificity factor

A simple approach to determine CO₂/O₂ specificity factor () of ribulose 1,5-bisphosphate carboxylase/oxygenase is described. The assay measures the amount of CO₂ fixation at varying [CO₂]/[O₂] ratios after complete consumption of ribulose 1,5-bisphosphate (RuBP). Carbon dioxide fixation catalyzed by the carboxylase was monitored by directly measuring the moles of ¹⁴CO₂ incorporated into 3-phosphoglycerate (PGA). This measurement at different [CO₂]/[O₂] ratios is used to determine graphically by several different linear plots the total RuBP consumed by the two activities and the CO₂/O₂ specificity factor. The assay can be used to measure the amounts of products of the carboxylase and oxygenase reactions and to determine the concentration of the substrate RuBP converted to an endpoint amount of PGA and phosphoglycolate. The assay was found to be suitable for all [CO₂]/[O₂] ratios examined, ranging from 14 to 215 micromolar CO₂ (provided as 1–16 mM NaHCO₃) and 614 micromolar O₂ provided as 50% O₂. The procedure described is extremely rapid and sensitive. Specificity factors for enzymes of highly divergent values are in good agreement with previously published data.Abbreviations HEPPS N-(2-hydroxyethyl)piperazine-N-(3-propanesulfonic acid) - L large subunit of rubisco - PGA 3-phosphoglyceric acid - rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP d-ribulose 1,5-bisphosphate - S small subunit of rubisco - XuBP d-xylulose 1,5-bisphosphate