Catalytic and EPR studies of the βE204Q mutant of the chloroplast F1- ATPase from Chlamydomonas reinhardtii

Chia Yuan Hu, Andrew L P Houseman, Lola Morgan, Andrew Webber, Wayne Frasch

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14 Scopus citations

Abstract

The mutation E204Q in the β subunit of the chloroplast F1-ATPase was made by biolistic transformation of Chlamydomonas reinhardtii. The yield of chloroplast F1-ATPase (CF1) purified from thylakoids was unaltered, suggesting that the mutation did not affect protein assembly. However, photoautotrophic growth of Chlamydomonas strains containing βE204Q was virtually abolished, and the effect of the mutation on the light-driven ATPsynthase activity catalyzed by purified thylakoids was comparable to the change in the photoautotrophic growth rate. The loss of ATPsynthase activity in the mutant was not the result of uncoupling. Addition of wild-type CF1 to mutant thylakoids depleted of CF1 reconstituted ATPsynthase activity indicating that the mutation did not affect assembly of F0. Furthermore, the mutant CF1F0 was capable of catalyzing ATPase-dependent photon pumping as measured by fluorescence quenching of 9-amino acridine. Although the mutation significantly affected the apparent k(cat)/K(M) of the Mg2+ -ATPase activity of the purified CF1-ATPase, no significant effect on the apparent k(cat) was observed with the mutant compared to wild-type. No significant changes in the ability of Mg2+ or Mn2+ to serve either as a cofactor or as an inhibitor of ATPase activity were observed in the mutants relative to the wild-type CF1-ATPase. EPR spectra were also taken of VO2+ bound at catalytic site 3 in its latent form. In a large fraction of the latent enzyme, a carboxyl group has displaced the nucleotide-phosphate coordination to the metal which results in the free metal inhibited Form (M3). No significant effects on the g(is parallel with) and A(is parallel with) 51V hyperfine parameters were observed between wild-type and mutant. However, the mutation increased the abundance of the M3 form relative to the M3-N3 form (metal-nucleotide-coordinated form). On the basis of these results, βE204 is not the carboxyl group that displaces the nucleotide phosphate as a ligand to form the free-metal inhibited enzyme form which predominates in site 3 in the latent state. Instead, the data are consistent with it role in which βE204 is essential to protonate an inorganic phosphate oxygen to make that oxygen a good leaving group to facilitate ATP synthesis and, via this role in H- bonding, increases the abundance of the functional metal-nucleotide complex bound to the catalytic site.

Original languageEnglish (US)
Pages (from-to)12201-12211
Number of pages11
JournalBiochemistry
Volume35
Issue number37
DOIs
StatePublished - 1996

ASJC Scopus subject areas

  • Biochemistry

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