Catalytic properties and sensitivity to tentoxin of Chlamydomonas reinhardtii ATP synthases changed in codon 83 of atpB by site-directed mutagenesis

Abstract

The participation of the amino acid β83 in determining the sensitivity of chloroplast ATP synthases to tentoxin was reported previously. We have changed codon 83 of the Chlamydomonas reinhardtii atpB gene by site-directed mutagenesis to further examine the role of this amino acid in the response of the ATP synthase to tentoxin and in the mechanism of ATP synthesis and hydrolysis. Amino acid β83 was changed from Glu to Asp (βE83D) and to Lys (βE83K), and the highly conserved tetrapeptide βT82-E83-G84-L85 (ΔTEGL) was deleted. Mutant strains were produced by particle gun transformation of atpB deletion mutants cw15AatpB and FUD50 with the mutated atpB genes. The transformants containing the βE83D and βE83K mutant genes grew well photoautotrophically. The ΔTEGL transformant did not grow photoautotrophically, and no CF1 subunits were detected by immunostaining of Western blots using CF1 specific antibodies. The rates of ATP synthesis at clamped ΔpH with thylakoids isolated from cw15 and the two mutants, βE83D and βE83K, were similar. However, only the phosphorylation activity of the mutant βE83D was inhibited by tentoxin with 50% inhibition attained at 4 μM. These results confirm that amino acid β83 is critical in determining the response of ATP synthase to tentoxin. The rates of the latent Mg-ATPase activity of the CF1s isolated from cw15, βE83D, and βE83K were similar and could be enhanced by heat, alcohols, and octylglucoside. As in the case of the membrane-bound enzyme, only CF1 from the βE83D mutant was sensitive to tentoxin. A lower alcohol concentration was required for optimal stimulation of the ATPase of the βE83K-CF1 than that of CF1 from the other two strains. Moreover, the optimal activity of the βE83K-CF1 was also lower. These results suggest that introduction of an amino acid with a positively charged side chain in position 83 in the 'crown' domain affects the active conformation of the CF1-ATPase.