Formation and properties of hybrid photosynthetic F-1-ATPases - Demonstration of different structural requirements for stimulation and inhibition by tentoxin

A hybrid ATPase composed of cloned chloroplast ATP synthase beta and gamma subunits (PC and gamma (C)) and the cloned alpha subunit from the Rhodospir illum rubrum ATP synthase (alpha (R)) was assembled using solubilized inclu sion bodies and a simple single-step folding procedure. The catalytic prope rties of the assembled alpha (R)(3)beta (C)(3)gamma (C) were compared to th ose of the core alpha (C)(3)beta (C)(3)gamma (C) complex of the native chlo roplast coupling factor 1 (CF1) and to another recently described hybrid en zyme containing R. rubrum alpha and beta subunits and the CF1 gamma subunit (alpha (R)(3)beta (R)(3)gamma (C)). All three enzymes were similarly stimu lated by dithiothreitol and inhibited by copper chloride in response to red uction and oxidation, respectively, of the disulfide bond in the chloroplas t gamma subunit. In addition, all three enzymes exhibited the same concentr ation dependence for inhibition by the CF1 epsilon subunit. Thus the CF1 ga mma subunit conferred full redox regulation and normal epsilon binding to t he two hybrid enzymes. Only the native CF1 alpha (C)(3)beta (C)(3)gamma (C) complex was inhibited by tentoxin, confirming the requirement for both CF1 alpha and beta subunits for tentoxin inhibition. However, the alpha (R)(3) beta (C)(3)gamma (C) complex, like the alpha (C)(3)beta (C)(3)gamma (C) com plex, was stimulated by tentoxin at concentrations in excess of 10 muM. In addition, replacement of the aspartate at position 83 in beta (C) with leuc ine resulted in the loss of stimulation in the alpha (R)(3)beta (C)(3)gamma (C) hybrid. The results indicate that both inhibition and stimulation by t entoxin require a similar structural contribution from the beta subunit, bu t differ in their requirements for alpha subunit structure.