Influences of Energization and Nucleotide Binding on the Reaction of Lucifer Yellow Vinyl Sulfone with the α Subunits of the Chloroplast ATP Synthase

The catalytic portion of the chloroplast ATP synthase (CF1) consists of five different polypeptides in the stoichiometry α3β3γδε 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 α subunits by the rapid and covalent binding of Lucifer Yellow vinyl sulfone (LY) to one of the three chemically identical α subunits. The binding of LY to a single α subunit has allowed the investigation of whether asymmetry in the α subunits is a permanent feature of CF1. The development of an electrochemical proton 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 α subunits were labeled with LY. Illumination of thylakoid membranes doubled the extent of LY labeling, and fluorescence resonance energy transfer measurements indicated that LY was bound to more than one α subunit. Since the change in LY distribution was inhibited by proton ionophores (uncouplers), alteration of α conformation by illumination is a result of the generation of a proton gradient. Preincubation of CF1 in solution with Mg2+-ATP had no effect on the extent of LY labeling but resulted in multiple α subunits binding LY as determined by fluorescence resonance energy transfer measurements. Adenine nucleotides at substrate level concentrations inhibit the reaction of LY with the α subunits. No increase in LY labeling was observed when thylakoids were illuminated under conditions in which CF1 was catalytically active.