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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 C[O.sub.2] by carbonic anhydrase (CA) prior to fixation. Paradoxically, many [beta]-cyanobacteria, including Thermosynechococcus elongatus BP-1, lack the conventional carboxysomal [beta]-CA, ccaA. The N-terminal domain of the carboxysomal protein CcmM is homologous to y-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 [gamma]-CA reported. The 2.0 [angstrom] structure of CcmM209 reveals a trimeric, left-handed [beta]-helix structure that closely resembles Cam, except that residues 198-207 form a third s-helix stabilized by an essential Cys194-Cys200 disulfide bond. Deleting residues 194-209 (CcmM193) results in an inactive protein whose 1.1 [Angstrom] 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 theenzyme. Thus, like membrane-bound eukaryotic cellular compartments, the [beta]-carboxysome appears to be able to maintain an oxidizing interior by precluding the entry of thioredoxin and other endogenous reducing agents. CO2 concentrating mechanism | cyanobacteria | disulfide bond www.pnas.org/cgi/doi/10.1073/pnas.0910866107