ATP-driven stepwise rotation of FoF1-ATP synthase

Hiroshi Ueno, Toshiharu Suzuki, Kazuhiko Kinosita, Masasuke Yoshida

Research output: Contribution to journalArticle

122 Citations (Scopus)

Abstract

FoF1-ATP synthase (FoF1) is a motor enzyme that couples ATP synthesis/hydrolysis with a transmembrane proton translocation. F1, a water-soluble ATPase portion of F oF1, rotates by repeating ATP-waiting dwell, 80° substep rotation, catalytic dwell, and 40°-substep rotation. Compared with F1, rotation of FoF1 has yet been poorly understood, and, here, we analyzed ATP-driven rotations of FoF 1. Rotation was probed with an 80-nm bead attached to the ring of c subunits in the immobilized FoF1 and recorded with a submillisecond fast camera. The rotation rates at various ATP concentrations obeyed the curve defined by a Km of ≈30 μM and a V max of ≈350 revolutions per second (at 37°C). At low ATP, ATP-waiting dwell was seen and the kon-ATP was estimated to be 3.6 × 107 M-1·s-1. At high ATP, fast, poorly defined stepwise motions were observed that probably reflect the catalytic dwells. When a slowly hydrolyzable substrate, adenosine 5′-[γ-thio]triphosphate, was used, the catalytic dwells consisting of two events were seen more clearly at the angular position of ≈80°. The rotational behavior of FoF1 resembles that of F 1. This finding indicates that "friction" in Fo motor is negligible during the ATP-driven rotation. Tributyltin chloride, a specific inhibitor of proton translocation, slowed the rotation rate by 96%. However, dwells at clearly defined angular positions were not observed under these conditions, indicating that inhibition by tributyltin chloride is complex.

Original languageEnglish
Pages (from-to)1333-1338
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume102
Issue number5
DOIs
Publication statusPublished - 2005 Feb 1
Externally publishedYes

Keywords

  • ATP hydrolysis
  • Binding change mechanism
  • Membrane protein
  • Single-molecule imaging

ASJC Scopus subject areas

  • Genetics
  • General

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