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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

Carboxysomes are proteinaceous microcompartments that encapsulate carbonic anhydrase (CA) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco); carboxysomes, therefore, catalyze reversible HCO dehydration and the subsequent fixation of CO. The N- and C-terminal domains of the β-carboxysome scaffold protein CcmM participate in a network of protein-protein interactions that are essential for carboxysome biogenesis, organization, and function. The N-terminal domain of CcmM in the thermophile Thermosynechococcus elongatus BP-1 is also a catalytically active, redox regulated γ-CA. To experimentally determine if CcmM from a mesophilic cyanobacterium is active, we cloned, expressed and purified recombinant, full-length CcmM from Nostoc sp. PCC 7120 as well as the N-terminal 209 amino acid γ-CA-like domain. Both recombinant proteins displayed ethoxyzolamide-sensitive CA activity in mass spectrometric assays, as did the carboxysome-enriched TP fraction. NstCcmM209 was characterized as a moderately active and efficient γ-CA with a k of 2.0 × 10 s and k/ K of 4.1 × 10 M s at 25 °C and pH 8, a pH optimum between 8 and 9.5 and a temperature optimum spanning 25-35 °C. NstCcmM209 also catalyzed the hydrolysis of the CO analog carbonyl sulfide. Circular dichroism and intrinsic tryptophan fluorescence analysis demonstrated that NstCcmM209 was progressively and irreversibly denatured above 50 °C. NstCcmM209 activity was inhibited by the reducing agent tris(hydroxymethyl)phosphine, an effect that was fully reversed by a molar excess of diamide, a thiol oxidizing agent, consistent with oxidative activation being a universal regulatory mechanism of CcmM orthologs. Immunogold electron microscopy and Western blot analysis of TP pellets indicated that Rubisco and CcmM co-localize and are concentrated in Nostoc sp. PCC 7120 carboxysomes. [ABSTRACT FROM AUTHOR]

We studied the interactions of the CO-concentrating mechanism and variable light in the filamentous cyanobacterium Leptolyngbya sp. CPCC 696 acclimated to low light (15 μmol m s PPFD) and low inorganic carbon (50 μM Ci). Mass spectrometric and polarographic analysis revealed that mediated CO uptake along with both active Na-independent and Na-dependent HCO transport, likely through Na/HCO symport, were employed to concentrate Ci internally. Combined transport of CO and HCO required about 30 kJ mol of energy from photosynthetic electron transport to support an intracellular Ci accumulation 550-fold greater than the external Ci. Initially, Leptolyngbya rapidly induced oxygen evolution and Ci transport to reach 40-50% of maximum values by 50 μmol m s PPFD. Thereafter, photosynthesis and Ci transport increased gradually to saturation around 1,800 μmol m s PPFD. Leptolyngbya showed a low intrinsic susceptibility to photoinhibition of oxygen evolution up to PPFD of 3,000 μmol m s. Intracellular Ci accumulation showed a lag under low light but then peaked at about 500 μmol photons m s and remained high thereafter. Ci influx was accompanied by a simultaneous, light-dependent, outward flux of CO and by internal CO/HCO cycling. The high-affinity and high-capacity CCM of Leptolyngbya responded dynamically to fluctuating PPFD and used excitation energy in excess of the needs of CO fixation by increasing Ci transport, accumulation and Ci cycling. This capacity may allow Leptolyngbya to tolerate periodic exposure to excess high light by consuming electron equivalents and keeping PSII open. [ABSTRACT FROM AUTHOR]