The interaction of 4-chloro-7-nitrobenzofurazan with Rhodospirillum rubrum chromatophores, their soluble F1-ATPase, and the isolated purified beta-subunit.
ATP synthesis and hydrolysis by Rhodospirillum rubrum chromatophores as well as the soluble RrF1-ATPase activity are inhibited by 4-chloro-7-nitrobenzofurazan (NBD-C1) in a dithiothreitol-reversible manner. Using the method earlier developed in these chromatophores to remove specifically the beta-subunit from their membrane-bound RrF1 leaving all other subunits attached to the resulting inactive beta-less chromatophores (Philosoph, S., Binder, A., and Gromet-Elhanan, Z. (1977) J. Biol. Chem. 252, 8747-8752), we have tested the effect of NBD-Cl also on the isolated beta-subunit and on the beta-less chromatophores before and after their reconstitution with the missing beta-subunit. The isolated purified beta-subunit as well as the RrF1-ATPase bind covalently [14C]NBD-Cl with an accompanying increase in absorbance at 385 nm, indicative of a tyrosyl-O-NBD bond. But, unlike the inactive RrF1-NBD complex, the beta-NBD adduct is as capable as the native beta-subunit to reconstitute beta-less chromatophores and restore their ATP synthesis and hydrolysis activities. On the other hand, incubation of beta-less chromatophores with NBD-Cl before or after their reconstitution with either native beta or the NBD-saturated beta adduct results in complete inhibition of their restored activities. It is, therefore, concluded that there are different binding sites for NBD-Cl on the isolated beta-subunit and on the beta-less chromatophores or on chromatophores reconstituted with the beta-NBD adduct, where the beta-site is already occupied. Furthermore, the site responsible for inactivation by NBD-Cl of the coupled and reconstituted chromatophores and of the soluble RrF1 is different from the site modified by NBD-Cl on the isolated beta-subunit. Its subunit location is as yet unknown.
D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology*; D020130:Bacterial Chromatophores / Q000187:drug effects / Q000378:metabolism*; D004355:Drug Stability; D007700:Kinetics; D046911:Macromolecular Substances; D010069:Oxadiazoles / Q000494:pharmacology*; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D012247:Rhodospirillum rubrum / Q000378:metabolism*; D013053:Spectrophotometry
D046911:Macromolecular Substances; D010069:Oxadiazoles; D006180:Proton-Translocating ATPases; D009327:4-Chloro-7-nitrobenzofurazan