Kinetics of the Oxyhydrogen Reaction in the Presence and Absence of Carbon Dioxide in Scenedesmus obliquus

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The hydrogenase-dependent processes, photoreduction and the dark oxyhydrogen reaction, both of which can support CO2 assimilation, were compared with aerobic photosynthesis and respiration for their sensitivity to electron transport inhibitors in cells and intact chloroplasts of Chiamydomomas reiahardii 11-32/6. Photoreduction but not photosynthesis was inhibited in chloroplasts and the oxyhydrogen reaction detected only in cells was inhibited up to 75 and 90%, respectively, by 150 micromolar rotenone, indicating the involvement of a NAD(P)H-plastoquinone oxidoreductase in the hydrogen utilizing pathways. The oxyhydrogen reaction coupled to CO2 fixation was inhibited more than 95% by 10 micromolar 2,5 -dibromo -3 -methyl -6 -isopropyl -p -benzoquinone (DBMIB), a concentration which did not affect respiratory activity. In cells, both photoreduction and the oxyhydrogen reaction exhibited a similar sensitivity to salicylhydroxamic acid (SHAM) Adapted C. reinhardii can also catalyze the uptake of H2 coupled to the consumption of 02 in the dark (6). This oxyhydrogen reaction can be further coupled to CO2 fixation through the Calvin cycle (16) indicating that this system may be closely associated with the chloroplast. In our studies with intact C. reinhardii chloroplasts, which were capable of catalyzing hydrogen-dependent photoreduction (15), we were unable to detect the oxyhydrogen reaction coupled to CO2 reduction. Studies with Scenedesmus cells indicated that the oxyhydrogen reaction had properties in common with both photosynthesis and respiration (5, 16), suggesting that possibly both mitochondrial and chloroplastic reactions were required for the C02-coupled oxyhydrogen reaction to occur.Recent studies on thylakoid fractions of C. reinhardii revealed the presence of an NAD(P)H-PQ3 oxidoreductase similar to that associated with NADH oxidation in mitochondria (1 1, 12). An uncharacterized NADPH oxidizing system associated with Chiamydomonas thylakoids had been previously reported (2) and was interpreted to function in the metabolic pathway for carbohydrate degradation in these algae (8, 9).The oxidation of reduced PQ by 02 has also been shown in Chlamydomonas thylakoids (3) indicating that the components necessary for a minimal respiratory pathway may be present in the algal chloroplast. Hiyama et al. (13)

Section: Discussionmentioning

confidence: 99%

“…After adaptation (if required), 5 (Fig. 2) at concentrations inhibitory of photosynthetic electron transport but having no effect on respiration.…”

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confidence: 99%

Association of the Chloroplastic Respiratory and Photosynthetic Electron Transport Chains of Chlamydomonas reinhardii with Photoreduction and the Oxyhydrogen Reaction

Maione

Gibbs²

1986

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“…This oxyhydrogen reaction can be further coupled to CO2 fixation through the photosynthetic carbon reduction cycle indicating a close association with the chloroplast (20). Studies with Scenedemus cells indicated that the oxyhydrogen reaction had properties in common with both photosynthesis and carbohydrate respiration, suggesting that possibly both chloroplastic and extra-chloroplastic reactions were required for the CO2-coupled oxyhydrogen reaction to occur (8,9,13).…”

Section: Cellular Fractionationmentioning

confidence: 98%

“…The procedure for isolating chloroplasts from the F-60 mutant method followed Klein et al (16) (8,9,13).…”

Section: Cellular Fractionationmentioning

confidence: 99%

Glucose Respiration in the Intact Chloroplast of Chlamydomonas reinhardtii

Chen

Gibbs²

1991

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Chloroplastic respiration was monitored by measuring 14CO2 from 14C glucose in the darkened Chiamydomonas reinhardtii F-60 chloroplast. The pattems of 14CO2 evolution from labeled glucose in the absence and presence of the inhibitors iodoacetamide, glycolate-2-phosphate, and phosphoenolpyruvate were those expected from the oxidative pentose phosphate cycle and glycolysis. The Km for glucose was 56 micromolar and for MgATP was 200 micromolar. Release of 14CO2 was inhibited by phloretin and inorganic phosphate. Comparing the inhibition of CO2 evolution generated by pH 7.5 with respect to pH 8.2 (optimum) in chloroplasts given C-1, C-2, and C-6 labeled glucose indicated that a suboptimum pH affects the recycling of the pentose phosphate intermediates to a greater extent than CO2 evolution from C-1 of glucose. Respiratory inhibition by pH 7.5 in the darkened chloroplast was alleviated by NH4CI and KCI (stromal alkalating agents), iodoacetamide (an inhibitor of glyceraldehyde 3-phosphate dehydrogenase), or phosphoenolpyruvate (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiration in the darkened Chlamydomonas chloroplast is the fructose-1,6-bisphosphatase/phosphofructokinase junction. The respiratory pathways described here can account for the total oxidation of a hexose to CO2 and for interactions between carbohydrate metabolism and the oxyhydrogen reaction in algal cells adapted to a hydrogen metabolism.profile results and the earlier finding that glucose respiration was competitive with the oxyhydrogen reaction (9, 13) prompted us to investigate carbohydrate oxidation in the Chiamydomonas reinhardtii chloroplast. We have monitored chloroplastic respiration in the intact algal chloroplast by measuring the release of "'CO2 from darkened preparations externally supplied with ['4C]glucose. This procedure has been applied to pea (23) and spinach (1) chloroplasts. MATERIALS AND METHODS Strains and Culture ConditionsChlamydomonas reinhardtii wt 137c(+) and 137c(-) and the mutant strain F-60 (obtained from R. K. Togasaki) were grown under fluorescent lights on an acetate-supplemented medium as described previously (26). Gametogenesis was induced in the wt(±) strains by excluding nitrogen from the growth media so that autolysine could be isolated through the mating of the two strains (5). Cellular FractionationThe procedure for isolating chloroplasts from the F-60 mutant method followed Klein et al. (16) (8,9,13).In contrast to higher plants (7), little is known about carbohydrate respiratory pathways and its compartmentation in the unicellular green algae. To investigate metabolism in these algae, a method for the isolation ofchloroplasts was developed (15,17). Using this method, Klein et al. (16) reported that the initial portion of glycolysis (glucose 6-P to glycerate 3-P) was located completely, and the oxidative pentose-P pathway partially (70-80%), within the algal plastid. The enzyme 14CO2 MeasurementsRates of CO2 evolution were determined by adding int...

“…By monitoring the initial rates of H2, 02, and '4CO2 uptakes Supported by U.S. Department of Energy DE.FG02-86ER 13486. 2 Present address: N-212 Agricultural Science N, University of Kentucky, Lexington, and the effect of inhibitors on these rates with gas-sensing electrodes and isotopic techniques, Erbes and Gibbs (9) supplied additional evidence supporting the participation of respiration and the reductive pentose-P cycle of photosynthesis in the oxyhydrogen reaction coupled to CO2 fixation.…”

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confidence: 99%

Coupling of Carbon Dioxide Fixation to the Oxyhydrogen Reaction in the Isolated Chloroplast of Chlamydomonas reinhardtii

Chen

Gibbs²

1992

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The oxyhydrogen reaction (the reduction of 02 to water by H2) in the presence of CO2 was studied in the isolated Chiamydomonas reinhardtii chloroplast by monitoring the rate of 14CO2 incorporation into acid-stable products in the dark. The endogenous rate of CO2 uptake (50-125 nmol/mg chlorophyll per h) was increased about 3-to 4-fold by ATP and additionally when combined with glucose, ribose-5-phosphate, and glycerate-3-phosphate. The rate was diminished 50 to 75%, respectively, when H2 was replaced by N2 or by air. Decrease in CO2 uptake by DL-glyceraldehyde was taken to indicate that the regenerative phase and complete Calvin cycle turnover were involved. Diminution of CO2 incorporation by rotenone, antimycin A, and 2,5-dibromo-3-methyl-6-isopropanolp-benzoquinone was attributed to an inhibition of the oxyhydrogen reaction, resulting in an elevated NADPH/NADP ratio. If so, then the diminished CO2 uptake could have been by "product inhibition" of the carbon metabolic network. Our data are consistent with the proposal (H. Gaffron [1942] 1 Gen Physiol 26: 241-267) that CO2 fixation coupled to the oxyhydrogen reaction is dependent to some extent on exchloroplastic metabolism. This support is primarily ATP provided by mitochondrial respiration.The oxyhydrogen reaction in green algae adapted to a hydrogen metabolism, a process first noted and studied in depth by Gaffron (10) about 50 years ago, involves simultaneous uptake of H2 and 02 in the dark. Using Warburg manometric methods, he observed that the reductive assimilation of CO2 could be coupled to the oxyhydrogen reaction. Horowitz (12) used a mass spectrometer to monitor CO2 and 02 levels and observed that both cellular respiration and the oxyhydrogen reaction coupled to CO2 reduction had the same dependence on 02 concentration. The two investigators reached the conclusion that the oxyhydrogen reaction had some properties in common with respiration. Under oxyhydrogen conditions, the chemosynthetic reduction of CO2 is known to involve the Calvin cycle (photosynthetic reductive pentose-P cycle [3,20] and the effect of inhibitors on these rates with gas-sensing electrodes and isotopic techniques, Erbes and Gibbs (9) supplied additional evidence supporting the participation of respiration and the reductive pentose-P cycle of photosynthesis in the oxyhydrogen reaction coupled to CO2 fixation.It also became quite clear that final judgment concerning the validity of this conclusion awaited separation of the algal cell into organelles, which could be investigated for their respective roles. Taking advantage of the method developed by Klein et al. (14) for isolating photosynthetically competent chloroplasts from Chlamydomonas reinhardtii, Maione and Gibbs (17) studied its hydrogenase-mediated activities. They found that the intact chloroplast after adaptation under N2 or H2 possessed the enzyme hydrogenase that could catalyze both the anaerobic evolution of H2 and the uptake of H2 in the presence of 1% 02. Their results demonstrated that the plastid not only c...