In this study, we addressed the presence and location of nucleotide-binding sites in the voltage-dependent anion channel (VDAC). VDAC bound to reactive red 120-agarose, from which it was eluted by ATP, less effectively by ADP and AMP, but not by NADH. The photoreactive ATP analog, benzoyl-benzoyl-ATP (BzATP), was used to identify and characterize the ATP-binding sites in VDAC.[␣-32 P]BzATP bound to purified VDAC at two or more binding sites with apparent high and low binding affinities. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis of BzATP-labeled VDAC confirmed the binding of at least two BzATP molecules to VDAC. The VDAC BzATP-binding sites showed higher specificity for purine than for pyrimidine nucleotides and higher affinity for negatively charged nucleotide species. VDAC treatment with the lysyl residue modifying reagent, fluorescein 5-isothiocyanate, markedly inhibited VDAC labeling with BzATP. The VDAC nucleotide-binding sites were localized using chemical and enzymatic cleavage. Digestion of [␣-32 P]BzATP-labeled VDAC with CNBr or V8 protease resulted in the appearance of ϳ17-and ϳ14-kDa labeled fragments. Further digestion, high performance liquid chromatography separation, and sequencing of the selected V8 peptides suggested that the labeled fragments originated from two different regions of the VDAC molecule. MALDI-TOF analysis of BzATP-labeled, tryptic VDAC fragments indicated and localized three nucleotide binding sites, two of which were at the N and C termini of VDAC. Thus, the presence of two or more nucleotidebinding sites in VDAC is suggested, and their possible function in the control of VDAC activity, and, thereby, of outer mitochondrial membrane permeability is discussed.It has recently been recognized that there exists a metabolic coupling between the cytosol and the mitochondria, with the outer mitochondrial membrane (OMM), 2 the boundary between these compartments, fulfilling an important function in this coupling (1-4). In the cross-talk between mitochondria and cytosol, mitochondrial ATP and Ca 2ϩ play major roles. As the primary transporter of nucleotides, Ca 2ϩ , and other ions and metabolites across the OMM (reviewed in Refs. 5 and 6), the voltage-dependent anion channel (VDAC) mediates a substantial portion of the OMM molecular traffic. It has been demonstrated that VDAC is permeable to Ca 2ϩ and possesses Ca 2ϩ -binding sites that control its activity (reviewed in Ref. 5). VDAC permeability is also regulated by associated protein(s) (reviewed in Refs. 5-7), and by different ligands that bind to VDAC, such as glutamate, ruthenium red, La 3ϩ , and NADH (reviewed in Refs. 5 and 6). Thus, by providing the pathway for anions, cations, ATP, and other metabolites into and out of the mitochondria, as well as possessing ligand-binding sites, VDAC plays an important role in the regulation of mitochondrial functions. Indeed, VDAC has been suggested to control OMM permeability, coupled respiration, and cell survival (3,4,8).It has been proposed that activati...