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Structure-function studies of the CO2 uptake protein CupA of the NDH-13 complex in cyanobacteria

Structure-function studies of the CO2 uptake protein CupA of the NDH-13 complex in cyanobacteria

Juliana Artier

Microbiology and Molecular Genetics

Increase of CO2 atmospheric level has led to an intense search for solutions to mitigate the problem. A natural pathway exists in photosynthesis where CO2 is fixed into biomass. Cyanobacteria, which constitute a large phylum of natural oxygenic photosynthetic bacteria, have a huge potential for bioengineering. This includes candidates for use in diverse CO2 capture and storage projects and the potential to redirect its energy for the production of valuable compounds. Our aim in this project is to understand the structure-function of Cup (CO2 uptake proteins) in the NDH-1/CUP complex, part of the cyanobacterial CO2 concentrating mechanism (CCM). Cyanobacteria have five CCM systems responsible for increasing inorganic carbon (Ci) concentration inside of the cell, an effort to raise CO2 level close to RuBisCO, the enzyme responsible for carbon fixation. Among them, two CO2 uptake systems are specialized NDH-1 complexes, NDH-13 (NdhF3/NdhD3/CupA/CupS) and NDH-14 (NdhF4/NdhD4/CupB), which have very little known about their mechanism. CO2 is usually regulated in living organisms by carbonic anhydrases (CAs), enzymes that catalyzes the interconversion of CO2 and HCO3-. The hypothesis is that Cup, in the NDH-13,4 complex, is involved in this (non)reversible reaction, possibly coupled with release of a proton across the membrane. We constructed a double knockout mutant, where no NDH-13,4 is produced by the bacteria, and complemented with the whole cupA operon under RuBisCO promoter control in an ectopic genomic site. Physiological analysis of Ci depleted mutants by growth curves, chlorophyll fluorescence traces and O2 evolution dependent on bicarbonate, show a high Ci requirement feature on the double knockout mutant. However, the complementation strain seems to have a phenotype more like wild type, restoring cell CO2 uptake. Further studies will be directed to analyze mutants with point mutations on conserved His/Cys of CupA protein and evaluate its possible role on CA activity. Also, purification of the complex will allow additional test such as exploration of zinc presence, common on CAs.