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

Abstract

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...

Fermentation metabolism and its evolution in algae

“…Hydrogenases also function in H 2 uptake, with two distinct uptake pathways described in Chlamydomonas (Gaffron, 1944; Kessler, 1974; Maione and Gibbs, 1986a,b; Chen and Gibbs, 1992a; Figure 3 ). In the first pathway, H 2 oxidation and cyclic PSI activity in the light are linked to RuBisCO-mediated anaerobic CO 2 fixation.…”

“…This pathway requires the absence of O 2 evolution from PSII. In the second pathway, termed the oxyhydrogen reaction, H 2 oxidation occurs concomitantly with the uptake of low levels of O 2 in a process that can be coupled to CO 2 fixation (Gaffron and Rubin, 1942; Gaffron, 1944; Russell and Gibbs, 1968; Kessler, 1974; Chen and Gibbs, 1992a). Although not well characterized, it is posited that H 2 oxidation provides the reducing equivalents for CO 2 fixation and that the low levels of O 2 present are respired to provide ATP (Gaffron and Rubin, 1942; Gaffron, 1944; Maione and Gibbs, 1986a; Chen and Gibbs, 1992a).…”

“…Chen and Gibbs (1992b) detected ATP-citrate lyase, as well as PFR and α-ketoglutarate synthase activities in Chlamydomonas cell extracts, speculating that the existence of these three key enzyme activities indicated that the reverse TCA cycle could operate in Chlamydomonas . These authors showed that a Chlamydomonas mutant with a compromised Calvin–Benson cycle takes up CO 2 in the dark under minimal aerobic conditions (1% O 2 ), and that the CO 2 uptake is coupled to H 2 oxidation (Chen and Gibbs, 1992a), suggesting that the reverse TCA cycle could be a significant pathway for CO 2 assimilation when the Calvin–Benson cycle is compromised (Chen and Gibbs, 1992b). Hence, under these conditions H 2 oxidation would provide the reducing equivalents to drive the reverse TCA cycle and to allow PFR to synthesize pyruvate, leading to the accumulation of an array of biosynthetic precursors.…”

7 Hydrogenase evolution and function in eukaryotic algae

“…Hydrogen-uptake activity has also been described in several eukaryotic algae, including C. reinhardtii [48,[58][59][60][61][62]. One pathway couples H 2 oxidation in the light to CO 2 fixation.…”

A New Perspective on Hydrogen Production by Photosynthetic Water Splitting

“…1). This fermentative metabolic process has been well studied (10,11). Since the substrate of the fermentative pathway is generated photosynthetically by reduction of CO,, the net result of the sequential process, photosynthesis and fermentation, is still splitting water to H, and O,, with CO, as an intermediate.…”