The ATPase activity of the alpha 3 beta 3 complex of the F1-ATPase of the thermophilic bacterium PS3 is inactivated on modification of tyrosine 307 in a single beta subunit by 7-chloro-4-nitrobenzofurazan

Abstract

The catalytically active alpha 3 beta 3 complex, assembled as described (Miwa, K., and Yoshida, M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6484-6487) from the isolated alpha and beta subunits of the F1-ATPase of the thermophilic bacterium PS3 (TF1), is inactivated by 7-chloro-4-nitrobenzofurazan (Nbf-Cl) with characteristics very similar to those observed when TF1, which has the subunit composition, alpha 3 beta 3 gamma delta epsilon, is inactivated by the reagent under the same conditions. Both native TF1 and the alpha 3 beta 3 complex are inactivated by 200 microM Nbf-Cl with a pseudo-first order rate constant of 3.7 x 10(-2) min-1 in the presence of 0.2 M Na2SO4 at pH 7.6 and 23 degrees C. The rate of increase in absorbance at 385 nm of reaction mixtures containing 200 microM [14C]Nbf-Cl and TF1, the wild-type alpha 3 beta 3 complex, or the mutant alpha 3(beta Y307----F)3 complex, each at 18 microM was also examined. Since the alpha 3(beta y307----F)3 complex is resistant to inactivation by Nbf-Cl, difference spectrophotometry revealed that inactivation of native TF1 and the wild-type alpha 3 beta 3 complex could be correlated with formation of about 1 mol of Nbf-O-Tyr/mol of enzyme or complex. Fractionation of peptic digests of the labeled enzyme and complexes by reversed-phase high performance liquid chromatography resolved a major radioactive peptide that was common to labeled TF1 and the labeled alpha 3 beta 3 complex but was absent in the digest of the labeled alpha 3(beta Y307----F)3 complex. This labeled peptide was shown to contain Tyr-beta 307 derivatized with [14C]Nbf-Cl by automatic amino acid sequence analyses. From these results, it is concluded that one-third of the sites' reactivity of Nbf-Cl with Tyr-beta 307 in TF1 or its equivalent in other F1-ATPases is not influenced by the presence of the gamma, delta, or epsilon subunits. It has also been shown that Tyr-307 is not modified to an appreciable extent when the isolated beta subunit is treated with [14C]Nbf-Cl under conditions in which this residue is nearly completely labeled in a single beta subunit when TF1 or the alpha 3 beta 3 complex is inactivated by the reagent.