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 ␤83 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 sitedirected 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 ␤83 was changed from Glu to Asp (␤E83D) and to Lys (␤E83K), and the highly conserved tetrapeptide ␤T82-E83-G84-L85 (⌬TEGL) was deleted. Mutant strains were produced by particle gun transformation of atpB deletion mutants cw15⌬atpB and FUD50 with the mutated atpB genes. The transformants containing the ␤E83D and ␤E83K mutant genes grew well photoautotrophically. The ⌬TEGL transformant did not grow photoautotrophically, and no CF 1 subunits were detected by immunostaining of Western blots using CF 1 specific antibodies. The rates of ATP synthesis at clamped ⌬pH with thylakoids isolated from cw15 and the two mutants, ␤E83D and ␤E83K, were similar. However, only the phosphorylation activity of the mutant ␤E83D was inhibited by tentoxin with 50% inhibition attained at 4 M. These results confirm that amino acid ␤83 is critical in determining the response of ATP synthase to tentoxin. The rates of the latent Mg-ATPase activity of the CF 1 s isolated from cw15, ␤E83D, and ␤E83K were similar and could be enhanced by heat, alcohols, and octylglucoside. As in the case of the membrane-bound enzyme, only CF 1 from the ␤E83D mutant was sensitive to tentoxin. A lower alcohol concentration was required for optimal stimulation of the ATPase of the ␤E83K-CF 1 than that of CF 1 from the other two strains. Moreover, the optimal activity of the ␤E83K-CF 1 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 CF 1 -ATPase.The eukaryotic unicellular green algae Chlamydomonas reinhardtii constitutes a powerful experimental model system for the study of the photosynthetic machinery. It is accessible to genetic analysis and grows photoautotrophically on minimal medium or heterotrophically with acetate as the sole carbon source. These properties have been used to isolate numerous photosynthetic mutants which have helped to examine the function of the photosynthetic apparatus (1).The chloroplast of C. reinhardtii contains approximately 80 copies of its 196-kb 1 circular genome (2). Due to recent progress in the molecular genetics of C. reinhardtii, chloroplast proteins can be altered by site-directed mutagenesis of the corresponding genes followed by transformation into the chloroplast (3-7). Chloroplast transformation was first demonstrated in 1988 by Boynton and co-workers (3), by complementation of an atpB deletion mutant with the cloned wild type gene. The transforming DNA integrates into the recipient chloroplast DNA by homologous recombination. Goldschmidt-Clermont (5) has constructed a chimeric selectable marker using ...

Hybrid Rhodospirillum rubrumF0F1 ATP Synthases Containing Spinach Chloroplast F1 β or α and β Subunits Reveal the Essential Role of the α Subunit in ATP Synthesis and Tentoxin Sensitivity

“…6, inset) did not enable the determination of the ratio of the bound CF 1 ␣ to the remaining RrF 1 ␣, the full inhibition of the hybrid containing both CF 1 ␣ and ␤ by tentoxin sets a lower limit of 1 mol CF 1 ␣ bound/mol of reconstituted RrF 0 F 1 /CF 1 . CF 1 ␤-Asp 83 was shown to be required for the inhibitory action of tentoxin and residues of different charge, such as lysine, or different spacer length such as glutamate, could not replace it (22,23). Table II illustrates that this aspartate is indeed essential for obtaining full inhibition by tentoxin in the presence of CF 1 ␣.…”

“…This result suggested that the ␤ subunit might be responsible for conferring tentoxin sensitivity to the F 1 enzyme. An aspartic acid residue at position 83 of CF 1 ␤ was indeed implicated as being essential for tentoxin sensitivity of the chloroplast CF 0 F 1 ATP synthase (22,23). However, the observation that the MgATPase activity of membranes isolated from an uncD-deleted Escherichia coli strain complemented with the chloroplast atpB gene was insensitive to tentoxin (24) suggests that additional CF 1 subunits might also be involved in conferral of this sensitivity.…”

Kinetic Analysis of Tentoxin Binding to Chloroplast F1-ATPase

“…First, a high resolution structure of CF 1 is still lacking. Second, there is only indirect evidence (24,26) that ␤-Asp-83 2 takes part in the TTX binding site. However, for illustrating the TTX effects in relation to the CF 1 structure, we can take for granted that the TTX binding site is located in this region and that the structure of this well conserved domain is equivalent in CF 1 and MF 1 , although MF 1 is TTX-insensitive.…”

The Structure of the Chloroplast F1-ATPase at 3.2 Å Resolution

“…Both structures should promote additional contacts in the N-terminal crown-region of the F 1 complex, which might increase the thermal stability of the enzyme. In the thermophilic enzyme a hydrogen bond between ␤Asn 40 and ␣Arg 90 and van der Waal's interaction of residues ␤Asn 38 -␣Ser 21 and ␤Glu 41 -␣Met 48 are proposed to cause this increased stability (10). The high resolution structure of the chloroplast enzyme suggests that a salt bridge ␤Asp 83 -␣Arg 297 enhances the stability of the enzyme.…”