Sulfite stimulation of chloroplast coupling factor ATPase

Abstract

The latent ATPase of spinach chloroplasts is activated by the thylakoid high-energy state, plus reduction of the disulfide bond on the γ subunit. If the high-energy state decays, activity disappears. We find that ATPase activity can be restored not only by a second illumination, but also by inorganic sulfite in the dark. With sufficient sulfite present, high concentrations of uncoupler no longer inhibit ATP hydrolysis. However, sulfite does not replace light in permitting rapidreduction of the disulfide bond on the γ subunit by dithioerythreitol. Sulfite together with saturating levels of the uncouplers, NH 4 Cl and gramicidin, stimulates very high rates of Mg 2+ -dependent ATPase. Using thylakoids treated with trypsin following reduction in the light, the consequent rates of ATPase can be as high as 3000 μmol/mg chlorophyll per h, or5-times faster than any previously reported. The system follows hyperbolic kinetics with respect to sulfite, and extensive kinetic characterization of thylakoid-bound ATPase became possible. Kinetic analyses indicated competitive relationships between ADP and both ATP and sulfite, and between azide (a high-affinity inhibitor in this system) and sulfite. There was a mutual interdependence of kinetic constants for ATP and for sulfite: as the concentration of one of these decreased, the values for both K m and V m of the other decreased.