Binding of modified adenine nucleotides to isolated coupling factor from chloroplasts as measured by polarization of fluorescence

1 Fluorescent nucleotides (1,N6‐ethenoadenosine diphosphate and triphosphate, ɛADP and ɛATP) replace the natural nucleotides rather efficiently (65–85%) in several chloroplast reactions (ADP inhibition of electron transport, ATP inhibition of electron transport, ATP stimulation of proton uptake, etc.). 2 ɛAMP does not bind to coupling factor protein. 3 The latent isolated coupling factor protein, which reconstitutes phosphorylation well when added to depleted chloroplasts, binds (in less than 1 min) both ɛADP and ɛATP at two divalent cation‐sensitive sites each of similar binding affinity (dissociation constant Kd∼ 1–3 μM) although the ɛADP and ɛATP sites are not the same. 0.1 M NaCl increases Kd by 3–5‐fold. 4 Polarization of fluorescence of nucleotides and coupling factor suggest that cation‐induced changes of protein conformation exist. 5 Binding of coupling factor to nucleotides is maximal at pH 8 where phosphorylation in chloroplasts is also maximal, but changes (with isolated coupling factor) at other pH values are not directly related to the enzyme action. 6 Interaction of inorganic phosphate (Pi) with coupling factor in the presence of nucleotide occurs as monitored by a sizable increase in Kd for ɛADP‐coupling factor and ɛATP‐coupling factor binding sites, and by decrease in polarization of fluorescence of coupling factor; this suggests negative interaction of adenine nucleotides and Pi on coupling factor. 7 Interaction of coupling factor with ɛADP and ATP on the one hand, and with ɛATP and ADP on the other, occurs as indicated by sizable increases in the Kd for ɛADP‐coupling factor and ATP‐coupling factor, respectively; this suggests s negative interaction of ADP and ATP on coupling factor. 8 The kinetics of release of ɛADP and ɛATP from coupling factor when excess ADP or ATP were added in competition were relatively slow compared to the rate at which photophosphorylation proceeds in vivo; this suggests that coupling factor may have to be bound to the hydrophobic HFo protein and/or the thylakoid membrane for fully functional adenine nucleotide binding to be expressed. Already bound ɛATP was more difficult to remove, even with Dowex 2X, from coupling factor than already bound ɛADP suggesting a more ‘internal’ binding of the former.