Characterisation of inorganic carbon fluxes, carbonic anhydrase(s) and ribulose-1,5-biphosphate carboxylase-oxygenase in the green unicellular alga Coccomyxa

Abstract

Processes involved in the uptake and fixation of dissolved inorganic carbon (DIC) were characterised for Coccomyxa, the green algal primary photobiont of the lichen Peltigera aphthosa and compared with the freeliving alga Chlamydomonas reinhardtii Dangeard (WT cc 125+). A mass-spectrometer disequilibrium technique was used to quantify fluxes of both HCO inf3 sup− and CO2 in the two algae, while activities of carbonic anhydrases (CAs) were examined in intact cells by measuring 18O exchange from doubly labelled CO2 (13C18O18O) to water and by using CA inhibitors. The CO2-fixation kinetics of intact Coccomyxa cells were also compared with the carboxylation efficiency of its isolated and purified primary carboxylating enzyme, ribulose-1,5-bisphosphate carboxylaseoxygenase (Rubisco). The two algae were found to be significantly different in their modes of acquiring CO2 for photosynthesis. Chlamydomonas was able to actively transport both HCO inf3 sup− and CO2 from the external medium, while Coccomyxa clearly favoured CO2 as a substrate. Both algae were found to possess external as well as internal CAs, but the relative amounts of these as well as their overall significance for the functioning of photosynthesis differed. In Coccomyxa, the internal CA activity was significantly higher than in Chlamydomonas and also predominated over the external activity. In Chlamydomonas, both transport and fixation of DIC were severely inhibited by ethoxyzolamide, an inhibitor of external and internal CAs as well as the DIC-transporting system, while this inhibitor only caused a minor inhibition of photosynthesis in Coccomyxa. These results thus give strong support for earlier indirect observations of the absence of a CO2concentrating mechanism in Coccomyxa. In addition, Coccomyxa was found to possess a Rubisco with a higher carboxylation efficiency than Chlamydomonas, having a K m (CO2) of 12 +3 μM CO2 and a CO2/O2 specificity factor (S c/o) of 83 +2, and it may hence be concluded that the absence of the CO2-concentrating mechanism is positively correlated with a more efficient Rubisco in this alga.