Intrinsic tryptophan fluorescence of Schizosaccharomyces pombe mitochondrial F1-ATPase. A powerful probe for phosphate and nucleotide interactions.
Mitochondrial F1 from the yeast Schizosaccharomyces pombe, in contrast to the mammalian enzyme, exhibits a characteristic intrinsic tryptophan fluorescence with a maximal excitation at 291 nm and a maximal emission at 332 nm. Low values of Stern-Volmer quenching constants, 4.0 M-1 or 1.8 M-1, respectively, in the presence of either acrylamide or iodide, indicate that tryptophans are mainly buried inside the native enzyme. Upon subunit dissociation and unfolding by 6 M guanidine hydrochloride (Gdn.HCl), the maximal emission is shifted to 354 nm, a value very similar to that obtained with N-acetyltryptophanamide, a solute-tryptophan model compound. The tryptophan content of each isolated subunit has been estimated by fluorescence titration in the presence of Gdn.HCl with free tryptophan as a standard. Two tryptophans and one tryptophan are found respectively in the alpha and epsilon subunits, whereas none is detected in the beta, gamma, and delta subunits. These subunit contents are consistent with the total of seven tryptophans estimated for native F1 with alpha 3 beta 3 gamma 1 delta 1 epsilon 1 stoichiometry. The maximal emission of the isolated epsilon subunit is markedly blue-shifted to 310-312 nm by interaction with the isolated delta subunit, which suggests that the epsilon subunit tryptophan might be a very minor contributor to the native F1 fluorescence measured at 332 nm. This fluorescence is very sensitive to phosphate, which produces a marked blue shift indicative of tryptophans in a more hydrophobic environment. On the other hand, ADP and ATP quench the maximal emission at 332 nm, lower tryptophan accessibility to acrylamide, and reveal tryptophan heterogeneity.
Divita
G
G
Laboratoire de Biologie et Technologie des Membranes et des Systèmes Intégrés, UMR 24 du CNRS, Université Claude, Bernard de Lyon, Villeurbanne, France.
Di Pietro
A
A
Deléage
G
G
Roux
B
B
Gautheron
D C
DC
D020106:Acrylamide; D000178:Acrylamides; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines; D007454:Iodides; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology*; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000201:enzymology*; D013050:Spectrometry, Fluorescence / Q000379:methods; D014364:Tryptophan / Q000031:analogs & derivatives / Q000032:analysis / Q000737:chemistry
D000178:Acrylamides; D006146:Guanidines; D007454:Iodides; D046911:Macromolecular Substances; D020106:Acrylamide; C016952:N-acetyltryptophanamide; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D019791:Guanidine