Influences of energization and nucleotide binding on the reaction of Lucifer Yellow vinyl sulfone with the alpha subunits of the chloroplast ATP synthase

The catalytic portion of the chloroplast ATP synthase (CF1) consists of fiv e different polypeptides in the stoichiometry alpha(3)beta(3)gamma delta ep silon and is structurally asymmetric. Asymmetry is readily apparent in the properties of the six nucleotide binding sites and the single-copy, smaller subunits. Asymmetry is also detected in the a subunits by the rapid and co valent binding of Lucifer Yellow vinyl sulfone (LY) to one of the three che mically identical alpha subunits. The binding of LY to alpha single a subun it has allowed the investigation of whether asymmetry in the alpha subunits is a permanent feature of CF1. The development of an electrochemical proto n gradient across illuminated thylakoid membranes and the preincubation of CF1 in solution with Mg2+-ATP were found to alter the LY distribution such that multiple alpha subunits were labeled with LY, Illumination of thylakoi d membranes doubled the extent of LY labeling, and fluorescence resonance e nergy transfer measurements indicated that LY was bound to more than one al pha subunit, Since the change in LY distribution was inhibited by proton io nophores (uncouplers), alteration of a conformation by illumination is a re sult of the generation of a proton gradient. Preincubation of CF1 in soluti on with Mg2+-ATP had no effect on the extent of LY labeling but resulted in multiple alpha subunits binding LY as determined by fluorescence resonance energy transfer measurements. Adenine nucleotides at substrate level conce ntrations inhibit the reaction of LY with the a subunits. No increase in LY labeling was observed when thylakoids were illuminated under conditions in which CF1 was catalytically active.