MEASUREMENT OF THE AMOUNT AND ISOTOPIC COMPOSITION OF THE CO2 RELEASED FROM THE CYANOBACTERIUM SYNECHOCOCCUS UTEX-625 AFTER RAPID QUENCHINGOF THE ACTIVE CO2 TRANSPORT-SYSTEM

Air-grown cells of the cyanobacterium Synechococcus UTEX 625 were susp ended in a cuvette connected to a mass spectrometer and supplied with (HCO3-)-C-13-O-18 to investigate the intracellular interconversion bet ween CO2 and HCO3- as determined from the isotopic composition of CO2 appearing in the extracellular medium under a wide variety of experime ntal conditions. Upon injection of (HCO3-)-C-13-O-18 to the cell suspe nsion in the light, the extracellular [(CO2)-C-13-O-16] increased. As the CO2 species were C-13 labelled, this demonstrated that the O-18-de pleted CO2 was originating from the added (HCO3-)-C-13-O-18. A compari son of the rates of (COO)-C-13-O-16-O-16 appearance in the medium with the formation of (COO)-C-13-O-16-O-16 from spontaneous dehydration-hy dration in the extracellular medium in the presence of cells demonstra ted that most of it had to originate from a series of intracellular de hydration - hydration cycles of (HCO3-)-C-13-O-18 that had been recent ly transported into the cells. During the time course of the experimen ts both the m/z (mass to charge) = 49 (i.e., (COO)-C-13-O-18-O-18) and 47 (i.e., (COO)-C-13-O-18-O-16) signals decreased constantly, whereas the m/z = 45 signal (i.e., (CO2)-C-13-O-16) always increased. Inhibit ing CO2 fixation enhanced the amount of CO2 arising in the medium but did not change its isotopic composition, and the CO2 was always fully depleted of O-18. When the CO2 transport system was inhibited by darke ning the cells, adding inhibitors such as Na2S or COS, or quenching th e uptake of inorganic C-13 with an excess of inorganic C-12, the magni tude of the extracellular [(CO2)-C-13-O-16] was increased but the CO2 species were still always depleted of O-18. Various incubation times o f the illuminated cells in the presence of (HCO3-)-C-13-O-18 were used to obtain a variety of internal C-i pool sizes. When the inhibitor (C OS) was added, the amount of (CO2)-C-13-O-16 arising during the respon se time of the mass spectrometer was equivalent to the amount of CO2 t hat would have been present in the whole cell if CO2 and HCO3- were in equilibrium throughout the entire cell volume, but it was at least 40 times higher than the amount of CO2 that would have been present in t he cell if the CO2 was confined to the carboxysomes. Experiments were also conducted at pH 9.0 where the spontaneous rate of (CO2)-C-13-O-16 production from (HCO3-)-C-13-O-18 dehydration-hydration would be negl igible, and again the same features were observed. Results show that i ntracellular HCO3- and CO2 are in rapid equilibrium throughout the ent ire cell volume.