ON THE MECHANISM OF SULFITE ACTIVATION OF CHLOROPLAST THYLAKOID ATPASE AND THE RELATION OF ADP TIGHTLY BOUND AT A CATALYTIC SITE TO THE BINDING CHANGE MECHANISM

Washed chloroplast thylakoid membranes upon exposure to [3H]ADP retain a tightly bound [3H]ADP on a catalytic site of the ATP synthase. The presence of sufficient endogenous or added Mg2+ results in an enzyme with essentially no ATPase activity. Sulfite activates the ATPase, and many molecules of ATP per synthase can be hydrolyzed before most of the bound [3H]ADP is released, a result interpreted as indicating that the ADP is not bound at a site participating in catalysis by the sulfite-activated enzyme [Larson, E. M., Umbach, A., & Jagendorf, A. T. (1989) Biochim. Biophys. Acta 973, 75–85]. We present evidence that this is not the case. The Mg2+- and ADP-inhibited enzyme when exposed to MgATP and 20–100 mM sulfite shows a lag of about 1 min at 22 °C and of about 15 s at 37 °C before reaching the same steady-state rate as attained with light-activated ATPase that has not been inhibited by Mg2+ and ADP. The lag is not eliminated if the enzyme is exposed to sulfite prior to MgATP addition, indicating that ATPase turnover is necessary for the activation. The release of most of the bound [3H]ADP parallels the onset of ATPase activity, although some [3H]ADP is not released even with prolonged catalytic turnover and may be on poorly active or inactive enzyme or at noncatalytic sites. The results are consistent with most of the tightly bound [3H]ADP being at a catalytic site and being replaced as this Mg2+- and ADP-inhibited site regains equivalent participation with other catalytic sites on the activated enzyme. The sulfite activation can be explained by sulfite combination at a Pi binding site of the enzyme-ADP-Mg2+ complex to give a form more readily activated by ATP binding at an alternative site. © 1990, American Chemical Society. All rights reserved.