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Spinach chloroplast coupling factor CF1-alpha 3 beta 3 core complex: structure, stability, and catalytic properties

M Sokolov1, Z Gromet-Elhanan

  • 1Department of Biochemistry, Weizmann Institute of Science, Rehovot, Israel.

Biochemistry
|January 30, 1996
PubMed
Summary

Tentoxin stabilizes a minimal chloroplast coupling factor CF1(alpha beta) complex, enhancing its MgATPase activity and hexameric structure. This stabilization reveals the crucial role of the CF1 gamma subunit in the functional properties of CF1-ATPase.

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Area of Science:

  • Chloroplast ATP synthase research
  • Enzyme kinetics and stabilization
  • Photosynthetic energy conversion mechanisms

Background:

  • Minimal chloroplast coupling factor CF1 core complex (CF1(alpha beta)) isolated from spinach thylakoids exhibits low MgATPase activity.
  • The CF1(alpha beta) complex is unstable, hindering structural determination and detailed functional analysis.
  • Tentoxin, a species-specific effector, influences CF1 activity but its stabilizing effect on the minimal complex was not fully understood.

Purpose of the Study:

  • To investigate the effect of tentoxin on the stability and MgATPase activity of the minimal chloroplast coupling factor CF1(alpha beta) complex.
  • To characterize the MgATPase activity of a tentoxin-stabilized CF1-alpha 3 beta 3 hexamer and compare it to a CF1 complex lacking the epsilon subunit.
  • To elucidate the role of the CF1 gamma subunit in the functional properties and catalytic cooperativity of the CF1-ATPase.

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Main Methods:

  • Isolation of a minimal chloroplast coupling factor CF1 core complex (CF1(alpha beta)) from spinach thylakoids.
  • Pretreatment of CF1(alpha beta) with high concentrations of tentoxin to induce stabilization and stimulate MgATPase activity.
  • Size-exclusion chromatography for isolation of the tentoxin-stabilized CF1-alpha 3 beta 3 hexamer.
  • Detailed characterization of MgATPase activity, including kinetic parameters, temperature profiles, and responses to inhibitors/stimulators.

Main Results:

  • High tentoxin concentrations remarkably stimulated MgATPase activity (50-fold) and stabilized the hexameric structure of CF1(alpha beta).
  • A stable CF1-alpha 3 beta 3 hexamer was isolated, exhibiting MgATPase properties similar to the parent unstable CF1(alpha beta) but differing significantly from CF1 lacking the epsilon subunit.
  • The tentoxin-stabilized CF1-alpha 3 beta 3 hexamer showed similar apparent Km values and tentoxin stimulation as CF1 lacking epsilon, but distinct temperature profiles and responses to inhibitors/stimulators.
  • Tentoxin inhibited multisite but not unisite catalysis, suggesting CF1 gamma's role in cooperative interactions between catalytic sites.

Conclusions:

  • The CF1 gamma subunit is not essential for the low MgATPase activity of CF1(alpha beta) or the enhanced activity of tentoxin-stabilized CF1-alpha 3 beta 3.
  • The CF1 gamma subunit plays a central role in conferring the typical functional properties of the CF1-ATPase, particularly in cooperative interactions.
  • Tentoxin stabilization of CF1(alpha beta) provides a tool to study the role of subunits in CF1-ATPase function and cooperativity.