Structural organization of chloroplast coupling factor.
Fluorescence resonance energy transfer measurements have been used to construct spatial maps for the accessible sulfhydryl of the gamma subunit (dark site) and the essential tyrosine residue of the beta subunits relative to previously mapped sites on the H+-ATPase from chloroplasts. The extent of energy transfer was measured between a coumarinylmaleimide derivative reacted covalently at the dark site and acceptor species selectively bound at the gamma-disulfide and the three nucleotide binding sites of the solubilized coupling factor complex. The nucleotide energy acceptor was 2'(3')-(trinitrophenyl)adenosine triphosphate, and the gamma-disulfide site was labeled with fluoresceinylmaleimide. The dark-site sulfhydryl also was labeled with pyrenylmaleimide which served as an energy donor for 7-chloro-4-nitro-2,1,3-benzoxadiazole reacted at the beta-tyrosine sites. Similar measurements were also made with pyrenylmaleimide covalently attached to the gamma-sulfhydryl accessible only under energized conditions on the thylakoid membrane surface (light site). The observed transfer efficiencies indicate that the dark-site sulfhydryl is approximately 45 A from all three nucleotide sites and 41-46 A from the gamma-disulfide site. The average distances separating the essential beta-tyrosines and the light- and dark-site sulfhydryls are 38 and 42 A, respectively. (In calculating these distances, random orientation of the donor-acceptor dipoles was assumed.) The results are consistent with a previously described structural model of the intact enzyme and can be used to gain insight into the overall structural organization or alpha-, beta-, and gamma-polypeptides within the coupling factor.
Snyder
B
B
Hammes
G G
GG
D009327:4-Chloro-7-nitrobenzofurazan / Q000378:metabolism; D004735:Energy Transfer; D046911:Macromolecular Substances; D008433:Mathematics; D006180:Proton-Translocating ATPases / Q000032:analysis*; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds; D014443:Tyrosine
D046911:Macromolecular Substances; D013438:Sulfhydryl Compounds; D014443:Tyrosine; D006180:Proton-Translocating ATPases; D009327:4-Chloro-7-nitrobenzofurazan