CONFORMATIONAL-CHANGES OF THE YEAST MITOCHONDRIAL ADENOSINE-DIPHOSPHATE ADENOSINE-TRIPHOSPHATE CARRIER STUDIED THROUGH ITS INTRINSIC FLUORESCENCE .2. ASSIGNMENT OF TRYPTOPHANYL RESIDUES OF THE CARRIER TO THE RESPONSES TO SPECIFIC LIGANDS

Tryptophanyl substitution of the Saccharomyces cerevisiae adenine nucl eotide carrier (Anc2p isoform) was not deleterious for the transport a ctivity or the folding of the carrier [preceding paper by Le Saute et al. (1996) Biochemistry 35, 16116-16124], Conformational changes of th e isolated wild-type and Trp-substituted Anc2p variants, induced upon binding of specific substrates [adenosine triphosphate (ATP) or diphos phate (ADP)] or inhibitors [carboxyatractyloside (CATR) or bongkrekic acid (BA)], were studied by measurement of intrinsic fluorescence, Tit ration of CATR and BA binding sites ended in the same number of sites, namely, 6-7 nmol/mg of wild-type and variant Anc2p. Isolated Anc2p in detergent presented similar emission spectra, suggesting that all try ptophanyl residues were in environments of similar hydrophobicity, Trp 87 and Trp126 contributed largely and to a similar extent to the fluor escence enhancement observed in response to ATP binding, while Trp235 contributed negatively and to a small extent to the fluorescence chang e, Both Trp126 and Trp235, and to a lower extent Trp87, participate in the CATR-induced fluorescence decrease of Anc2p. Responses to BA bind ing were observed only in the presence of ATP; they consisted of a fur ther fluorescence increase of the Anc2p . ATP complex, which was mainl y due to Trp87 and Trp126, Trp235 being much less responsive, The diff erent fluorescence responses of the three Trp residues of Anc2p varian ts to ATP, CATR, and BA are in agreement with distinct binding sites f or these ligands and distinct conformations of the carrier protein rec ognizing specifically CATR or BA, A mechanistic model is proposed to i nterpret the transitions between the different conformational states o f Anc2p.