Observations on the oxyhydrogen reaction in Scenedesmus and its relation to respiration and photosynthesis

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...

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

Section: Cellular Fractionationmentioning

confidence: 99%

mentioning

confidence: 99%

Glucose Respiration in the Intact Chloroplast of Chlamydomonas reinhardtii

Chen

Gibbs²

1991

“…To account for these stoichiometries, perturbation of the quotient with inhibitors and the release of only traces of CO2 during the oxyhydrogen reaction, Gaffron (5) concluded that little of the 02 absorbed by the algal cell in the presence of H2 was used for normal respiratory processes. Horwitz (11) used a mass spectrometer to monitor 02 and CO2 levels continuously and observed that both cellular respiration and the oxyhydrogen reaction coupled to CO2 reduction had the same dependence on 02 tension and that rates of 02 uptake for the two reactions fell within the same range. The conclusion was reached that the oxyhydrogen reaction had some properties in common with respiration.…”

mentioning

confidence: 99%

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

Erbes¹,

Gibbs²

1981

The oxyhydrogen reaction in the presence and absence of CO2 was studied in H2-adapted Scenedesmus obliquus by monitoring the initial rates of H2, 02, and "4CO2 uptake and the effect of inhibitors on these rates with gas-sensing electrodes and isotopic techniques. In the presence of 0.02 atmosphere 02, the pH2 was varied from 0 to 1 atmosphere. Whereas the rate of 02 uptake increased by only 30%, the rate of H2 uptake increased severalfold over the range of pH2 values. At 0.1 atmosphere H2 and 0.02 atmosphere 02, rates for H2 and 02 uptake were between 15 and 25 micromoles per milligram chlorophyll per hour. As the pH2 was changed from 0 to 1 atmosphere, the quotient H2:02 changed from 0 to roughly 2. This change may reflect the competition between H2 and the endogenous respiratory electron donors. Respiration in the presence of glucose and acetate was also competitive with H2 uptake. KCN inhibited equally respiration (02 uptake in the absence of H2) and the oxyhydrogen reaction in the presence and absence of CO2. The uncoupler carbonyl cyanide ptrifluoromethoxyphenylhydrazone accelerated the rate of respiration and the oxyhydrogen reaction to a similar extent. It was concluded that the oxyhydrogen reaction both in the presence and absence of CO2 has properties in common with components of respiration and photosynthesis. Participation of these two processes in the oxyhydrogen reaction would require a closely linked shuttle between mitochondrion and chloroplast.The oxyhydrogen reaction in green algae, a process first noted and studied in depth by Gaffron (5), involves a simultaneous uptake of H2 and 02 under darkness. Using Warburg manometric techniques, he usually observed a quotient of H2:02 equal to 1. In the presence of CO2, uptake of CO2 was observed and the quotient H2:02 changed to 2. To account for these stoichiometries, perturbation of the quotient with inhibitors and the release of only traces of CO2 during the oxyhydrogen reaction, Gaffron (5) concluded that little of the 02 absorbed by the algal cell in the presence of H2 was used for normal respiratory processes. Horwitz (11) used a mass spectrometer to monitor 02 and CO2 levels continuously and observed that both cellular respiration and the oxyhydrogen reaction coupled to CO2 reduction had the same dependence on 02 tension and that rates of 02 uptake for the two reactions fell within the same range. The conclusion was reached that the oxyhydrogen reaction had some properties in common with respiration. Under oxyhydrogen reaction conditions, the reduction of CO2 is known to involve the reductive pentose-P cycle (2, 14 the oxyhydrogen reaction. The proposed mechanism involves considerable intracellular transport between mitochondrion and chloroplast.Here, we have studied the initial velocities of H2, 02, and CO2 uptake in hydrogen-adapted Scenedesmus obliquus and the effect of inhibitors on these uptake rates. The rates of H2 and 02 uptake were monitored continuously during the first few min of the oxyhydrogen reaction using gas-sensing electr...

“…Simultaneous uptake of H2 and O2 (the oxyhydrogen reaction) is inhibited when the O2 level exceeds a specific concentration (6,10). Also, the photoproduction of 02 in algae will cause cessation of H2 metabolism.…”

mentioning

confidence: 99%

“…The enzyme was assayed by MV3 reduction followed on a Gilford recording spectrophotometer at 605 nm. Hydrogenase solution was injected into a cuvette with 2.0 ml of anaerobically prepared 10 CO inhibition was observed by MV reduction after injection of hydrogenase (purified through the DEAE-cellulose step) into cuvettes containing concentrations of CO, H2, and MV specified under "Results. "…”

mentioning

confidence: 99%

Inactivation of Hydrogenase in Cell-free Extracts and Whole Cells of Chlamydomonas reinhardi by Oxygen

Erbes¹,

King²,

Gibbs³

1979

02 irreversibly inactivates hydrogenase from Chlamydomonas reinhardiThe mechanism for the inactivation involves the reaction of one molecule of hydrogenase with one molecule of 02 (or two oxygen atoms) in the transition complex of the rate-limiting step. The second order rate constant for this reaction is 190 atmospheres-' minute-1 (1.4 x 105 molar-' minute-1). At levels above 0.01 atmosphere 02, the increased numbers of 02 molecules may compete for the site of inactivation hindering the proper orientation for inactivation of any one 02 molecule and resulting in lowered rates of inactivation.CO is a reversible inhibitor of hydrogenase acting competitively against H2. The Ki for CO is 0.0010 atmosphere. CO antagonizes 02 inactivation. In a period when complete inactivation by 02 would usualHy occur, the presence of CO greatly reduces the inactivation rate.After 3 hours of adaptation in whole cells, the presence of H2 lowers the rate of deadaptation of hydrogenase. Inasmuch as H2 promotes increased 02 uptake the cellular concentration of 02 is likely to be lower. After 48 hours of adaptation 02 uptake is reduced even when H2 is present and the pattern of deadaptation under 02 with and without H2 and CO is qualitatively the same as observed for the inactivation of cell-free hydrogenase. The mechanism of inactivation of celi-free hydrogenase by 02 may be the same as the mechanism for loss of hydrogenase during deadaptation in whole algal cells.Since the initial observation of H2 metabolism in green algae the sensitivity of algal H2 metabolism to O2 has been noted (5). Simultaneous uptake of H2 and O2 (the oxyhydrogen reaction) is inhibited when the O2 level exceeds a specific concentration (6, 10). Also, the photoproduction of 02 in algae will cause cessation of H2 metabolism. Removal of photoproduced O2 reversed the inhibition of hydrogenase (9) C. moewusii was grown in minimal medium plus acetate (7).The hydrogenase was prepared as above.Assays. Protein was assayed by the biuret method (8) and Chl was assayed spectrophotometrically in 80% acetone (2). The enzyme was assayed by MV3 reduction followed on a Gilford recording spectrophotometer at 605 nm. Hydrogenase solution was injected into a cuvette with 2.0 ml of anaerobically prepared 10 mM MV in 50 mm Tris-HCl (pH 8.0) under 1.0 atm H2 at 25 C. The extinction coefficient for reduced MV was determined to be 9.6 mM-' cm-' by reduction with excess sodium dithionite at pH 8.0. One unit of hydrogenase activity is defined as the reduction of 1.0 mol MV/min.