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Volume 272, Number 9, Issue of February 28, 1997 pp. 5457-5463
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Catalytic Properties and Sensitivity to Tentoxin of Chlamydomonas reinhardtii ATP Synthases Changed in Codon 83 of atpB by Site-directed Mutagenesis

(Received for publication, October 4, 1996, and in revised form, November 26, 1996)

Dongli Hu , Heike R. Fiedler ^§ , Talila Golan ^¶ , Marvin Edelman ^¶ , Heinrich Strotmann ^§ , Noun Shavit and Stefan Leu

From the  Doris and Bertie Black Center for Bioenergetics in Life Sciences, Ben Gurion University of the Negev, Beer Sheva 84105, Israel, ^§ Institut für Biochemie der Pflanzen, Heinrich Heine Universität Düsseldorf, Düsseldorf, Federal Republic of Germany, and ^¶ Department of Plant Genetics, Weizmann Institute of Science, Rehovot 76100, Israel

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 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 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 cw15atpB 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 CF₁ subunits were detected by immunostaining of Western blots using CF₁ 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 µM. 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 CF₁s 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 CF₁ from the E83D mutant was sensitive to tentoxin. A lower alcohol concentration was required for optimal stimulation of the ATPase of the E83K-CF₁ than that of CF₁ from the other two strains. Moreover, the optimal activity of the E83K-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.

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