Ethoxyzolamide Inhibition of C 02-Dependent Photosynthesis in the Cyanobacterium Synechococcus PCC 79421

Cells of the cyanobacterium, Synechococcus PCC7942, grown under high inorganic carbon (C,) conditions (1% C02; pH 8) were found to be photosynthetically dependent on exogenous CO2. This was judged by the fact that they had a similar photosynthetic affinity for CO2 (Ko.5[CO2] of 3.4-5.4 micromolar) over the pH range 7 to 9 and that the low photosynthetic affinity for C, measured in dense cell suspensions was improved by the addition of exogenous carbonic anhydrase (CA). The CA inhibitor, ethoxyzolamide (EZ), was shown to reduce photosynthetic affinity for CO2 in high Ci cells. The addition of 200 micromolar EZ to high Ci cells increased Ko.5(CO2) from 4.6 micromolar to more than 155 micromolar at pH 8.0, whereas low C, cells (grown at 30 microliters CO2 per liter of air) were less sensitive to EZ. EZ inhibition in high and low C, cells was largely relieved by increasing exogenous C, up to 100 millimolar. Lipid soluble CA inhibitors such as EZ and chlorazolamide were shown to be the most effective inhibitors of CO2 usage, whereas water soluble CA inhibitors such as methazolamide and acetazolamide had little or no effect. EZ was found to cause a small drop in photosystem 11 activity, but this level of inhibition was not sufficient to explain the large effect that EZ had on CO2 usage. High C cells of Anabaena variabilis M3 and Synechocystis PCC6803 were also found to be sensitive to 200 micromolar EZ. We discuss the possibility that the inhibitory effect of EZ on CO2 usage in high Cl cells of Synechococcus PCC7942 may be due to inhibition of a 'CA-like' function associated with the CO2 utilizing C, pump or due to inhibition of an intemal CA activity, thus affecting CO2 supply to ribulose bisphosphate carboxylase-oxygenase. It is now well documented that cyanobacteria possess an inducible CO2 concentrating mechanism (CCM2) which functions to elevate internal CO2 levels around Rubisco (5, 6, 10, 13, 17). Both CO2 and HCOy can be transported and/or utilized by the CCM (6, 7, 24), but the ability to utilize HCOy is most apparent when cells are grown at very low 'This work was supported by a National Research Fellowship (to G. D. P.) awarded by the Australian Government Department of Science and Technology. 'Abbreviations: CCM, CO2 concentrating mechanism; CA, carbonic anhydrase; C,, dissolved inorganic carbon; DMQ, 2,5-dimethylp-benzoquinone; EZ, ethoxyzolamide; Rubisco, ribulose bisphosphate carboxylase-oxygenase; Ko. , concentration required for half maximal response. levels of Ci (7, 15). Although most research has centered upon characterizing the high affinity HCOuptake system, it has been known for some time that CO2 is often a more efficient substrate for the CCM than HCO3 (6,7,24) Nevertheless, the most recent model for the CCM postulates a pivotal role for a HCO3 transporter on the plasma membrane (24). In this proposal the ability to utilize CO2 is accommodated for by the addition of a 'CA-like' moiety that converts CO2 to HCO3 in close proximity to the HCO3 pump. It was assumed that CO2 acts as a better substrate due to a higher diffusion rate into the periplasmic space than the charged HCOion. The best evidence for this model is that in low CO2 grown cells ofAnabaena variabilis M3, a low concentration (1O .M) of the CA inhibitor, EZ, inhibits CO2 uptake more than HCOT uptake (24). Unfortunately this type of evidence is not generally applicable to other cyanobacteria, such as Synechococcus sp. , which are virtually unaffected by 10 to 100 ,uM EZ (9). Recent studies have verified that low CO2 grown cyanobacteria possess the ability to transport or utilize both CO2 and HCO3 (1, 7). More important, it has been shown that cells of Synechococcus PCC6301 grown at high CO2 retain the ability to take up CO2 while losing the capacity for HCO3 uptake (7). This CO2 uptake activity predominates in cells grown in media (pH 8.2) equilibrated with CO2 levels above normal air levels (>350 ,uL/L), whereas maximum induction of HCO3 uptake capacity requires CO2 levels of less than 50 ,uL/L in the gas phase (7). They suggest, in contrast to Volokita et al. (24), that a CO2 utilizing Ci pump may be the primary component of the CCM and that HCOutilization would require 'front end'conversion ofHC03 to CO2 in the vicinity of the C, pump. The studies described in this paper were aimed at extending the characterization of the ecologically important CO2 utilization system in Synechococcus PC7942. We show that CO2 utilization can be severely inhibited by high concentrations of the CA inhibitor, EZ, with high CO2 grown cells being more sensitive to EZ than low CO2 grown cells. We also show that EZ inhibits CO2 utilization in other cyanobacterial strains, namely Anabaena variabilis M3 and Synechocystis PCC6803. MATERIAL AND METHODS