Spectral properties of fluorescent derivatives of the oligomycin sensitivity conferring protein and analysis of their interaction with the F1 and Fo sectors of the mitochondrial ATPase complex

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Abstract
In order to study the kinetics and the nature of the interactions between the oligomycin sensitivity conferring protein (OSCP) and the Fo and F1 sectors of the mitochondrial ATPase complex, fluorescent derivatives of OSCP, which are fully biologically active, have been prepared by reaction of OSCP with the following fluorescent thiol reagents: 6-acryloyl-2-(dimethylamino)naphthalene (acrylodan), 2-(4-maleim-idylanilino)naphthalene-6-sulfonic acid (Mal-ANS), N-(1-pyrenyl)maleimide (Mal-pyrene), 7-(diethyl-amino)-3-(4-maleimidylphenyl)-4-methylcoumarin (Mal-coumarin), and fluorescein 5-maleimide (Mal-fluorescein). The preparation of these derivatives was based on the previous finding that the single cysteinyl residue of OSCP, Cys 118, can be covalently modified by alkylating reagents without loss of biological activity [Dupuis, A., Issartel, J. P., Lunardi, J., Satre, M., and Vignais, P. V. (1985) Biochemistry 24, 728-733]. For all fluorescent probes used, except Mal-pyrene and Mal-fluorescein, the emission spectra of conjugated OSCP were blue-shifted relative to those of the corresponding mercaptoethanol adducts, indicating that the fluorophores attached to Cys 118 were located in a hydrophobic pocket. These results were consistent with the high quantum yields and the increased fluorescence lifetimes of conjugated OSCP compared to mercaptoethanol adducts in aqueous buffer. They also fit with quenching data obtained with potassium iodide which showed that the fluorophore is shielded from the aqueous medium when it is attached to Cys 118 of OSCP. Especially noticeable was the wide half-width of the OSCP-acrylodan emission peak compared to that of mercaptoethanol-acrylodan. Denaturation of OSCP conjugated to Mal-ANS or acrylodan resulted in a red shift of the emission spectrum; in other words, the Cys 118 environment which was sequestered in a hydrophobic pocket in native OSCP became exposed to the aqueous medium in denatured OSCP. Binding of F1 to OSCP fluorescent derivatives led to the following modifications of the fluorescence parameters: (1) a red shift of the emission peak of OSCP-acrylodan and OSCP-Mal-ANS, (2) a narrowing of the emission peak of OSCP-acrylodan, (3) a decrease of the lifetime of OSCP-Mal-pyrene, and (4) a decrease of the quantum yield of OSCP-Mal-coumarin, OSCP-Mal-pyrene, and OSCP-Mal-ANS. These modifications were interpreted by exposure of Cys 118 to a more hydrophilic environment when OSCP binds of F1- Polarization assays carried out with OSCP-Mal-pyrene in both the absence and presence of F1 pointed to the existence of some rotational freedom of OSCP-Mal-pyrene bound to F1. The fast fluorescence changes reflecting the reaction of OSCP-acrylodan with F1 (less than 1 s at 25.degree.C) contrasted the slow fluorescence changes (several minutes) occurring upon binding of OSCP-acrylodan or F1-OSCP-acrylodan to Fo in AUA particles (submitochondrial particles depleted of F1 and OSCP). The Cys 118 residue of OSCP-acrylodan, which had become exposed to the aqueous medium upon binding of F1, was shielded again upon addition of AUA particles, as a result of the formation of an Fo-F1-OSCP complex.

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