A significant portion of the total carbon fixed in the biosphere is attributed to the autotrophic metabolism of prokaryotes. In cyanobacteria and many chemolithoautotrophic bacteria, CO 2 fixation is catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), most if not all of which is packaged in protein microcompartments called carboxysomes. These structures play an integral role in a cellular CO 2 -concentrating mechanism and are essential components for autotrophic growth. Here we report that the carboxysomal shell protein, CsoS3, from Halothiobacillus neapolitanus is a novel carbonic anhydrase (-class CA) that has an evolutionary lineage distinct from those previously recognized in animals, plants, and other prokaryotes. Functional CAs encoded by csoS3 homologues were also identified in the cyanobacteria Prochlorococcus sp. and Synechococcus sp., which dominate the oligotrophic oceans and are major contributors to primary productivity. The location of the carboxysomal CA in the shell suggests that it could supply the active sites of RuBisCO in the carboxysome with the high concentrations of CO 2 necessary for optimal RuBisCO activity and efficient carbon fixation in these prokaryotes, which are important contributors to the global carbon cycle.

Carboxysomes are proteinaceous biochemical compartments that constitute the enzymatic "back end" of the cyanobacterial CO 2 -concentrating mechanism. These protein-bound organelles catalyze HCO 3 ؊ dehydration and photosynthetic CO 2 fixation. In Synechocystis sp. strain PCC6803 these reactions involve the ␤-class carbonic anhydrase (CA), CcaA, and Form 1B ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The surrounding shell is thought to be composed of proteins encoded by the ccmKLMN operon, although little is known about how structural and catalytic proteins integrate to form a functional carboxysome. Using biochemical activity assays and molecular approaches we have identified a catalytic, multiprotein HCO 3 ؊ dehydration complex (BDC) associated with the protein shell of Synechocystis carboxysomes. The complex was minimally composed of a CcmM73 trimer, CcaA dimer, and CcmN. Larger native complexes also contained RbcL, RbcS, and two or three immunologically identified smaller forms of CcmM (62, 52, and 36 kDa). Yeast two-hybrid analyses indicated that the BDC was associated with the carboxysome shell through CcmM73-specific protein interactions with CcmK and CcmL. Protein interactions between CcmM73 and CcaA, CcmM73 and CcmN, or CcmM73 and itself required the N-terminal ␥-CA-like domain of CcmM73. The specificity of the CcmM73-CcaA interaction provided both a mechanism to integrate CcaA into the fabric of the carboxysome shell and a means to recruit this enzyme to the BDC during carboxysome biogenesis. Functionally, CcaA was the catalytic core of the BDC. CcmM73 bound H 14 CO 3 ؊ but was unable to catalyze HCO 3 ؊ dehydration, suggesting that it may potentially regulate BDC activity.

Cyanobacterial RuBisCO is sequestered in large, icosahedral, protein-bounded microcompartments called carboxysomes. Bicarbonate is pumped into the cytosol, diffuses into the carboxysome through small pores in its shell, and is then converted to CO 2 by carbonic anhydrase (CA) prior to fixation. Paradoxically, many β-cyanobacteria, including Thermosynechococcus elongatus BP-1, lack the conventional carboxysomal β-CA, ccaA . The N-terminal domain of the carboxysomal protein CcmM is homologous to γ-CA from Methanosarcina thermophila (Cam) but recombinant CcmM derived from ccaA -containing cyanobacteria show no CA activity. We demonstrate here that either full length CcmM from T. elongatus , or a construct truncated after 209 residues (CcmM209), is active as a CA—the first catalytically active bacterial γ-CA reported. The 2.0 Å structure of CcmM209 reveals a trimeric, left-handed β-helix structure that closely resembles Cam, except that residues 198–207 form a third α-helix stabilized by an essential Cys194-Cys200 disulfide bond. Deleting residues 194–209 (CcmM193) results in an inactive protein whose 1.1 Å structure shows disordering of the N- and C-termini, and reorganization of the trimeric interface and active site. Under reducing conditions, CcmM209 is similarly partially disordered and inactive as a CA. CcmM protein in fresh E. coli cell extracts is inactive, implying that the cellular reducing machinery can reduce and inactivate CcmM, while diamide, a thiol oxidizing agent, activates the enzyme. Thus, like membrane-bound eukaryotic cellular compartments, the β-carboxysome appears to be able to maintain an oxidizing interior by precluding the entry of thioredoxin and other endogenous reducing agents.

Mass spectrometry has been used to confirm the presence of an active transport system for CO2 in Synechococcus UTEX 625. Cells were incubated at pH 8.0 in 100 micromolar KHCO3 in the absence of Na+ (to prevent . Upon illumination the ceUls rapidly removed almost all the free CO2 from the medium. Addition of carbonic anhydrase revealed that the CO2 depletion resulted from a selective uptake of CO2. rather than a total uptake of all inorganic carbon species. CO2 transport stopped rapidly (<3 seconds) when the light was turned off. lodoacetamide (3.3 millimolar) completely inhibited CO2 fixation but had little effect on CO2 transport. In iodoacetamide poisoned cells, transport of CO2 occurred against a concentration gradient of about 18,000 to 1. Transport of CO2 was completely inhibited by 10 micromolar diethylstilbestrol, a membrane-bound ATPase inhibitor. Studies with DCMU and PSI light indicated that CO2 transport was driven by ATP produced by cyclic or pseudocyclic photophosphorylation. Low concentrations of Na+ (<100 microequivalents per liter), but not of K+, stimulated CO2 transport as much as 2.4-fold. Unlike Na+-dependent HC03-transport, the transport of CO2 was not inhibited by high concentrations (30 milliequivalents per liter) of Li'. During illumination, the CO2 concentration in the medium remained far below its equilibrium value for periods up to 15 minutes. This could only happen if CO2 transport was continuously occurring at a rapid rate, since the continuing dehydration of HC03-to CO2 would rapidly raise the CO2 concentration to its equilibrium value if transport ceased. Measurement of the rate of dissolved inorganic carbon accumulation under these conditions indicated that at least part of the continuing CO2 transport was balanced by HCO3-efflux.Photosynthesis by cyanobacteria can occur when the CO2 concentration in the extracellular medium is so low that CO2 fixation via Rubisco2 could not occur were it not for the presence of 'CO2-concentrating' mechanisms (1,2,9,13,16,19,21,25,28 (1,2,9,18,29). For a given DIC concentration, the rate of DIC accumulation was faster under the nonequilibrium conditions (high CO,/HCO3-) than under equilibrium conditions (high HCO3-/C0.), thus indicating a lower Kmn for CO2 transport than for HCO3 transport (1. 2, 9, 29).Miller and Canvin (17) provided further evidence for a CO,-transport capacity, distinct from the HCO3-transport capacity, when they made use of the observation that HCO-transport in rapidly growing cells of Synechococcus UTEX 625 is inhibited by the absence of Na+ in the extracellular medium (8,17,22). Cells that were incubated in the absence of Na + were stimulated to accumulate normal levels of intracellular DIC by the addition of CA (17). It was postulated that, in the absence of the CA, the rate of supply of CO2 to the CO,-transport system was limited by the rate of HCO3-dehydration to CO2 in the extracellular medium. The DIC transport occurring in the presence of CA was not inhibited by the addition of Li+, whereas the Na+-dependent...

Mass spectrometric measurements of dissolved free 13CO2 were used to monitor CO2 uptake by air grown (low CO2) cells and protoplasts from the green alga Chlamydomonas reinhardtli. In the presence of 50 micromolar dissolved inorganic carbon and light, protoplasts which had been washed free of extemal carbonic anhydrase reduced the 13CO2 concentration in the medium to close to zero. Similar resuits were obtained with low CO2 cells treated with 50 micromolar acetazolamide. Addition of carbonic anhydrase to protoplasts after the period of rapid CO2 uptake revealed that the removal of CO2 from the medium in the light was due to selective and active CO2 transport rather than uptake of total dissolved inorganic carbon. demonstrated for these organisms (2, 4, 9, 15). In the case of cyanobacteria, both HCO3-and CO2 are substrates for active transport (2,3,6,7,14,15) with CO2 being selectively and preferentially used by the cells (2,6,16). In Chlamydomonas, HC03-is actively transported (4, 25, 29), but CO2 uptake has been considered to be passive (18,20). Carbon dioxide, however, is taken up from the medium faster than HCO3-by Chlamydomonas (13,28,29) and several authors (13,29) have considered the possibility of active CO2 transport.Studies on the DIC transport mechanism of green algae are complicated by their cellular compartmentation. Recently, it was shown that isolated chloroplasts of low C02 Chlamydomonas reinhardtii were able to accumulate DIC (19) and a model was presented where the only active DIC transport mechanism was located on the chloroplast envelope (18,19). In that model the plasma membrane was suggested to be only a diffusion barrier for CO2 generated by external carbonic anhydrase. In contrast, by comparison of the apparent affinities for DIC of whole cells and purified chloroplasts, Suiltemeyer et al. (26) came to the conclusion that active transport by the chloroplast alone may not be responsible for the photosynthetic characteristics of whole cells.Another difficulty in examining the DIC species taken up by whole cells is the presence of an external carbonic anhydrase (10) which catalyzes the rapid equilibrium between CO2 and HCO3-, thus making a direct discrimination between CO2 and HCO3-uptake impossible (7, 13). However, using inhibitors for external carbonic anhydrase or the cell-wall less mutant CW-15, some authors came to the conclusion that CO2 and not HCO3-(13) or that both C02 and HCO3- (29) were actively transported.Confusion about which DIC species is actively taken up from the medium may also be caused by methods which only measure total rates of transport rather than transport of CO2 or HC03-individually. Using MS, which measures free dissolved gases in liquid, several authors presented direct eviGreen algae and cyanobacteria possess a high apparent affinity for DIC3 when grown at low DIC concentrations (low CO2 cells: 2,5,9,17), and DIC accumulation has been '