EPR spectroscopy of VO2+-ATP bound to catalytic site 3 of chloroplast F1-ATPase from Chlamydomonas reveals changes in metal ligation resulting from mutations to the phosphate-binding loop threonine (βT168)

Wei Chen, Russell LoBrutto, Wayne Frasch

Research output: Contribution to journalArticle

10 Scopus citations

Abstract

Site-directed mutations were made to the phosphate-binding loop threonine in the β-subunit of the chloroplast F1-ATPase in Chlamydomonas (βT168). Rates of photophosphorylation and ATPase-driven proton translocation measured in coupled thylakoids purified from βT168D, βT168C, and βT168L mutants had < 10% of the wild type rates, as did rates of Mg2+- ATPase activity of purified chloroplast F1-ATPase (CF1). The EPR spectra of VO2+-ATP bound to Site 3 of CF1 from wild type and mutants showed that EPR species C, formed exclusively upon activation, was altered in CF1 from each mutant in both signal intensity and in 51V hyperfine parameters that depend on the equatorial VO2+ ligands. These data provide the first direct evidence that Site 3 is a catalytic site. No significant differences between wild type and mutants were observed in EPR species B, the predominant form of the latent enzyme. Thus, the phosphate-binding loop threonine is an equatorial metal ligand in the activated conformation but not in the latent conformation of Site 3. The metal-nucleotide conformation that gives rise to species B is consistent with the Mg2+-ADP complex that becomes entrapped in a catalytic site in a manner that regulates enzymatic activity. The lack of catalytic function of CF1 with entrapped Mg2+-ADP may be explained in part by the absence of the phosphate-binding loop threonine as a metal ligand.

Original languageEnglish (US)
Pages (from-to)7089-7094
Number of pages6
JournalJournal of Biological Chemistry
Volume274
Issue number11
DOIs
StatePublished - Mar 12 1999

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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