Forms of Dissolved Carbon Dioxide Required for Photosystem II Activity in Chloroplast Membranes

Abstract

It is established that PSII activity in chloroplast membranes requires catalytic amounts of dissolved CO2 (11,12,15). This substance, denoted as C',2 is bound to the reaction center complex (9). It has not been determined with certainty if CO2 or HCO3 is the active form. The ligand interacts with the reaction center in a dynamic fashion (10). It is released very briefly in light, possibly when electrons are passed from the reaction center to PC, but is bound again in a subsequent "dark" reaction. To understand this cyclic binding and release of the ligand and to formulate an explanation of the "bicarbonate effect," it is necessary to know the form of inorganic carbon which is taken up, used and released by the reaction center. Early experiments indicated that HCO3 was, in fact, the "active form" (3,6,12). This conclusion, however, remained tentative.A previous study (10) room temperature and harvested 8-14 days after planting. After harvesting, the leaves were ground for 30 s in a Waring Blendor set at maximum speed. This grinding regime will yield predominantly mesophyll grana but no attempt was made to completely exclude bundle sheath chloroplasts. The isolation medium contained 0.4 M sucrose, 0.01 M NaCi, and 0.05 M Na-phosphate (pH 6.8). All operations were done at near freezing temperatures. After grinding, the chloroplast slurry was filtered through four layers of cheesecloth and the filtrate was centrifuged for 1 min at 200g. The supernatant suspension was poured into clean tubes and centrifuged for 5 min at 1,500g. The resulting supernatant fluid was discarded. The chloroplasts comprising the pellet were then osmotically shocked by suspension in sucrose-free isolation medium. The broken chloroplasts were collected by centrifugation at 1,500g for 5 min. The resulting pellet was resuspended in a small amount of isolation medium, divided into portions, placed in a freezer and kept at -80 C. The chloroplasts were thawed just before use.To deplete chloroplasts of endogenous C', thawed grana were suspended in depletion medium which contained 0.175 M NaCl, 0.1 M sodium formate, and 0.05 M Na-phosphate (pH 5.0). TheChl concentration was about 50 jg/ml. The room-temperature suspension was allowed to stand for 10 min, then it was centrifuged. The thylakoid membranes, now depleted of C', were resuspended in a small amount of depletion medium and kept on ice until used (see ref. 9).Ferricyanide-supported 02 evolution was monitored with a Rank Brothers, Clark-type electrode. The light source was a Sylvania 150-w projector lamp and the light was focused by a Prado slide projector. The incident light intensity of white light was 500 w/m2.The rate of binding of radioactive C' to PSII reaction centers was measured as follows. Broken chloroplasts were depleted of endogenous C' by the procedure given above. They were then collected by centrifugation and resuspended in a small amount of depletion medium. In some experiments the chloroplasts were suspended in other media as noted in the figure legends. Twenty

Characteristics of Light-Dependent Inorganic Carbon Uptake by Isolated Spinach Chloroplasts

“…3); and (d) studies with either isolated mesophyll protoplasts or with wheat leafextracts suggest that RuBP carboxylase is mostly in the activated form in vivo (19,23). In addition to protein, inorganic carbon has been reported to bind directly to chloroplast membranes (25). The slow release of Ci in the dark is further evidence for a pool of bound CO2 in the stroma.…”

Enhanced Mass Transfer in Microbubble Driven Airlift Bioreactor for Microalgal Culture

“…This theory is also applicable to the mass transfer within a medium without microalgae, but such alkaline pH values were not encountered in the experiments conducted. Stemler (1980) also noted the effect of pH on the amount of dissolved CO 2 and discusses the effect of pH on the relative levels of CO 2 and 3 HCO solved  present within a solution [19]. He found that in from pH 8.0 to 7.3, the amount of 3 …”

Bicarbonate, not CO2, is the species required for the stimulation of Photosystem II electron transport

“…However, the pH optimum of the requirement falls closely at the pK~ of HCO~/CO 2 [21,22], and it has been suggested that both CO 2 and HCO 3 are required [22]. From the effect of pH on the rate of HCO 3 binding, it was suggested that CO 2 is involved [23]. Similarly, measurements of the lag time between CO 2 vs. HCOj addition and restimulation of the Hill reaction suggest that CO 2 is required, at least for diffusion to the active site [24,25].…”