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Carboxysomes are protein microcompartments found in cyanobacteria, whose shell encapsulates rubisco at the heart of carbon fixation in the Calvin cycle. Carboxysomes are thought to locally concentrate CO₂in the shell interior to improve rubisco efficiency through selective metabolite permeability, creating a concentrated catalytic center. However, permeability coefficients have not previously been determined for these gases, or for Calvin-cycle intermediates such as bicarbonate ( ${\text{HCO}}_3^{-}$), 3-phosphoglycerate, or ribulose-1,5-bisphosphate. Starting from a high-resolution cryogenic electron microscopy structure of a synthetic $\beta$-carboxysome shell, we perform unbiased all-atom molecular dynamics to track metabolite permeability across the shell. The synthetic carboxysome shell structure, lacking the bacterial microcompartment trimer proteins and encapsulation peptides, is found to have similar permeability coefficients for multiple metabolites, and is not selectively permeable to ${\text{HCO}}_3^{-}$relative to CO₂. To resolve how these comparable permeabilities can be reconciled with the clear role of the carboxysome in the CO₂-concentrating mechanism in cyanobacteria, complementary atomic-resolution Brownian Dynamics simulations estimate the mean first passage time for CO₂assimilation in a crowded model carboxysome. Despite a relatively high CO₂permeability of approximately 10⁻²cm/s across the carboxysome shell, the shell proteins reflect enough CO₂back toward rubisco that 2,650 CO₂molecules can be fixed by rubisco for every 1 CO₂molecule that escapes under typical conditions. The permeabilities determined from all-atom molecular simulation are key inputs into flux modeling, and the insight gained into carbon fixation can facilitate the engineering of carboxysomes and other bacterial microcompartments for multiple applications. 

The orange carotenoid protein (OCP) functions as a sensor of the ambient light intensity and as a quencher of bilin excitons when it binds to the core of the cyanobacterial phycobilisome. We show herein that the photoactivation mechanism that converts the resting, orange-colored state, OCPO, to the active red-colored state, OCPR, requires a sequence of two reactions, each requiring absorption of a single photon by an intrinsic ketocarotenoid chromophore. Global analysis of absorption spectra recorded during continuous illumination of OCPO preparations from Synechocystis sp. PCC 6803 detects the reversible formation of a metastable intermediate, OCPI, in which the ketocarotenoid canthaxanthin exhibits an absorption spectrum with a partial red shift and a broadened vibronic structure compared to that of the OCPO state. While the dark recovery from OCPR to OCPI is a first- order, unimolecular reaction, the subsequent conversion of OCPI to the resting OCPO state is bimolecular, involving association of two OCPO monomers to form the dark-stable OCPO dimer aggregate. These results indicate that photodissociation of the OCPO dimer to form the monomeric OCPO intermediate is the first step in the photoactivation mechanism. Formation of the OCPO monomer from the dimer increases the mean value and broadens the distribution of the solvent-accessible surface area of the canthaxanthin chromophore measured in molecular dynamics trajectories at 300 K. The second step in the photoactivation mechanism is initiated by absorption of a second photon, by canthaxanthin in the OCPO monomer, which obtains the fully red-shifted and broadened absorption spectrum detected in the OCPR product state owing to displacement of the C-terminal domain and the translocation of canthaxanthin more than 12 Å into the N-terminal domain. Both steps in the photoactivation reaction of OCP are likely to involve changes in the structure of the C-terminal domain elicited by excited-state conformational motions of the ketocarotenoid. 

Carboxysomes, responsible for a substantial fraction of CO 2 fixation on Earth, are proteinaceous microcompartments found in many autotrophic members of domain Bacteria , primarily from the phyla Proteobacteria and Cyanobacteria . Carboxysomes facilitate CO 2 fixation by the Calvin-Benson-Bassham (CBB) cycle, particularly under conditions where the CO 2 concentration is variable or low, or O 2 is abundant. These microcompartments are composed of an icosahedral shell containing the enzymes ribulose 1,5-carboxylase/oxygenase (RubisCO) and carbonic anhydrase. They function as part of a CO 2 concentrating mechanism, in which cells accumulate HCO 3 − in the cytoplasm via active transport, HCO 3 − enters the carboxysomes through pores in the carboxysomal shell proteins, and carboxysomal carbonic anhydrase facilitates the conversion of HCO 3 − to CO 2 , which RubisCO fixes. Two forms of carboxysomes have been described: α-carboxysomes and β-carboxysomes, which arose independently from ancestral microcompartments. The α-carboxysomes present in Proteobacteria and some Cyanobacteria have shells comprised of four types of proteins [CsoS1 hexamers, CsoS4 pentamers, CsoS2 assembly proteins, and α-carboxysomal carbonic anhydrase (CsoSCA)], and contain form IA RubisCO (CbbL and CbbS). In the majority of cases, these components are encoded in the genome near each other in a gene locus, and transcribed together as an operon. Interestingly, genome sequencing has revealed some α-carboxysome loci that are missing genes encoding one or more of these components. Some loci lack the genes encoding RubisCO, others lack a gene encoding carbonic anhydrase, some loci are missing shell protein genes, and in some organisms, genes homologous to those encoding the carboxysome-associated carbonic anhydrase are the only carboxysome-related genes present in the genome. Given that RubisCO, assembly factors, carbonic anhydrase, and shell proteins are all essential for carboxysome function, these absences are quite intriguing. In this review, we provide an overview of the most recent studies of the structural components of carboxysomes, describe the genomic context and taxonomic distribution of atypical carboxysome loci, and propose functions for these variants. We suggest that these atypical loci are JEEPs, which have modified functions based on the presence of Just Enough Essential Parts. 

ABSTRACT Autotrophic microorganisms catalyze the entry of dissolved inorganic carbon (DIC; = CO2 + HCO3− + CO32−) into the biological component of the global carbon cycle, despite dramatic differences in DIC abundance and composition in their sometimes extreme environments. “Cyanobacteria” are known to have CO2 concentrating mechanisms (CCMs) to facilitate growth under low CO2 conditions. These CCMs consist of carboxysomes, containing enzymes ribulose 1,5-bisphosphate oxygenase and carbonic anhydrase, partnered to DIC transporters. CCMs and their DIC transporters have been studied in a handful of other prokaryotes, but it was not known how common CCMs were beyond “Cyanobacteria”. Since it had previously been noted that genes encoding potential transporters were found neighboring carboxysome loci, α-carboxysome loci were gathered from bacterial genomes, and potential transporter genes neighboring these loci are described here. Members of transporter families whose members all transport DIC (CHC, MDT and Sbt) were common in these neighborhoods, as were members of the SulP transporter family, many of which transport DIC. 109 of 115 taxa with carboxysome loci have some form of DIC transporter encoded in their genomes, suggesting that CCMs consisting of carboxysomes and DIC transporters are widespread not only among “Cyanobacteria”, but also among members of “Proteobacteria” and “Actinobacteria”.