A γ-subunit point mutation in Chlamydomonas reinhardtii chloroplast FF-ATP synthase confers tolerance to reactive oxygen species.

The chloroplast FF-ATP synthase (CFF) drives ATP synthesis and the reverse reaction of ATP hydrolysis. The enzyme evolved in a cellular environment where electron transfer processes and molecular oxygen are abundant, and thiol modulation in the γ-subunit via thioredoxin is important for its ATPase activity regulation. Especially under high light, oxygen can be reduced and forms reactive oxygen species (ROS) which can oxidize CFF among various other biomolecules. Mutation of the conserved ROS targets resulted in a tolerant enzyme, suggesting that ROS might play a regulatory role. The mutations had several side effects in vitro, including disturbance of the ATPase redox regulation [F. Buchert et al., Biochim. Biophys. Acta, 1817 (2012) 2038-2048]. This would prevent disentanglement of thiol- and ROS-specific modes of regulation. Here, we used the F catalytic core in vitro to identify a point mutant with a functional ATPase redox regulation and increased HO tolerance. In the next step, the mutation was introduced into Chlamydomonas reinhardtii CFF, thereby allowing us to study the physiological role of ROS regulation of the enzyme in vivo. We demonstrated in high light experiments that CFF ROS targets were involved in the significant inhibition of ATP synthesis rates. Molecular events upon modification of CFF by ROS will be considered.