Aurovertin, a Fluorescent Probe of Conformational Change in Beef Heart Mitochondrial Adenosine Triphosphatase

Abstract

Abstract Formation of a complex between aurovertin and soluble mitochondrial adenosine triphosphatase (F1) was accompanied by a 55-fold enhancement of fluorescence, an increase in the polarization of fluorescence from 0.278 for the free form to 0.375 for the bound form and a decrease in absorption at 366 nm. The fluorescence intensity of the complex was partially quenched by addition of ATP or Mg++ and enhanced by ADP. Two binding sites for aurovertin were found on F1 in the presence of ATP and one site in the presence of either ADP, Mg++, or dilute buffer. The dissociation constants of the fluorescent complex were 0.52, 0.07, 0.013, and 0.04 µm in the presence of ATP, ADP, Mg++, or in buffer, respectively. It is proposed that of the two binding sites for aurovertin on F1, only one site participates in inhibition of ATPase activity. Addition of succinate to a fluorescent complex of aurovertin and submitochondrial particles gave rise to an enhancement of fluorescence which depended on maintenance of an energized state. The changes in fluorescence of bound aurovertin were interpreted in terms of changes in the conformational state of the ATPase.