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

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The role of an electron transport pathway associated with aerobic carbohydrate degradation in isolated, intact chloroplasts was evaluated. This was accomplished by monitoring the evolution of 14CO2 from darkened spinach (Spinacia oleracea) and Chiamydo- Sugars added to and starch within the chloroplast are degraded through a series of reactions resulting initially in the formation of glucose and fructose phosphates (14,25,31). The conversion of these hexose phosphates to CO2, dihydroxyacetone-P, and 3-phosphoglycerate, the major spe-'

Section: Purity Of the Chloroplast Preparationssupporting

confidence: 82%

mentioning

confidence: 92%

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Characterization of an Electron Transport Pathway Associated with Glucose and Fructose Respiration in the Intact Chloroplasts of Chlamydomonas reinhardtii and Spinach

Singh¹,

Chen²,

Gibbs³

1992

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“…4 W m-'. One possible explanation for the ineffectiveness of DCMU is the presence of an NADPH-plastoquinone oxidoreductase in the Chlamydomonas thylakoids, which allows the functioning of a PSI-driven cyclic electron transport through NADPH, resulting in a fairly stable NADPH:NADP ratio (Maione and Gibbs, 1986).…”

Section: Results and Dlscusslonmentioning

confidence: 99%

Photoregulation of Fructose and Glucose Respiration in the Intact Chloroplasts of Chlamydomonas reinhardtii F-60 and Spinach

Singh

Chen²,

Gibbs³

1993

The photoregulation of chloroplastic respiration was studied by monitoring in darkness and in light the release of l4COz from whole chloroplasts of Chlamydomonas reinhardtii F-60 and spinach (Spinacia oleracea L.) supplied externally with ['4C]glucose and ["Clfructose, respectively. CO, release was inhibited more than 90% in both chloroplasts by a light intensity of 4 W m-'. Oxidants, oxaloacetate in Chlamydomonas, nitrite in spinach, and phenazine methosulfate in both chloroplasts, reversed the inhibition. l h e onset of the photoinhibitory effect on COz release was relatively rapid compared to the restoration of COz release following illumination. In both darkened chloroplasts, dithiothreitol inhibited release. Of the four enzymes (fructokinase, phosphoglucose isomerase, glucose-6-P dehydrogenase, and gluconate-6-P dehydrogenase) in the pathway catalyzing the release of C 0 2 from fructose, only glucose-6-P dehydrogenase was deactivated by light and by dithiothreitol.

“…For the oxyhydrogen reaction to occur, a small and tolerable amount of 02 must be introduced into the gas phase. It is known that 1% 02 is suitable for the oxyhydrogen reaction in C. reinhardtii cells (17). If chloroplastic respiratory metabolism is an integral part of the oxyhydrogen reaction, it became essential to determine whether respiration of an externally supplied carbohydrate can function in this microaerobic condition.…”

Section: Resultsmentioning

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...