Intrinsic tryptophan fluorescence of Schizosaccharomyces pombe mitochondrial F1-ATPase. A powerful probe for phosphate and nucleotide interactions

Abstract

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.

The Absence of the Mitochondrial ATP Synthase  Subunit Promotes a Slow Growth Phenotype of Rho− Yeast Cells by a Lack of Assembly of the Catalytic Sector F1

“…As a consequence, the presence of the 6 subunit depends on the presence of the E subunit in a rho-context. This points to an interaction between the two subunits, which was proposed previously for pig heart F, where such a 68 complex was purified from F,-ATPase as a molecular entity (Penin et al, 1990;Divita et al, 1991) and confirmed from overproduced bovine subunits (Orriss et al, 1996).…”

Fluorescence quenching of spectrin and other red cell membrane cytoskeletal proteins. Relation to hydrophobic binding sites

“…This can thus in principle be expressed (Bukulova-Orlova et al, 1974) as (KQ,protein/KQ,tryptophan) *(qtryptophan/qprotein), where the q values are the quantum yields for the protein and free tryptophan derivatives. In practice, the quenching rate constants for exposed tryptophans in denatured proteins are usually much smaller than for free tryptophan derivatives or small peptides (Ivkova et al, 1971;Eftink & Ghiron, 1976;Divita et al, 1991). Results for spectrin are given in Table 2, from which it can be seen that the tryptophan side-chains are moderately accessible to acrylamide, i.e.…”

“…For comparison, the following are literature data for acrylamide quenching constants: N-acetyltryptophanamide, 17.5; glucagon, 10.5; globular proteins, [1][2][3][4][5][6][7][8][9] (Eftink & Ghiron, 1976). The quenching constant for N-acetyltryptophanamide by iodide is 6.0 (Divita et al, 1991); and the quenching constant for tryptophan by caesium ion is 2.1 (Ivkova et al, 1971). Shows upward curvature * Determined from slope of Stern-Volmer plot at the origin.…”

The HPr Kinase from Bacillus subtilis Is a Homo-oligomeric Enzyme Which Exhibits Strong Positive Cooperativity for Nucleotide and Fructose 1,6-Bisphosphate Binding

“…The potassium iodide stock solution also contained 0.1 mM potassium thiosulfate in order to prevent I 3 Ϫ formation, which would quench tryptophan fluorescence emission (32). A control experiment conducted with similar concentrations of KCl indicated that ionic strength did not significantly modify the fluorescence emission of HprK.…”