The preprotein translocase of the inner membrane of mitochondria (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. Tim50 is a major receptor for transporting the precursor protein across the mitochondrial inner membrane in TIM23 complex. However, the interaction of divalent metal ions with Tim50 and the contribution in the interaction of presequence peptide with Tim50 are still unknown. Herein, we investigated the interaction of divalent metal long with the intermembrane space domain of Tim50 (Tim50(IMS)) and the interaction of presequence peptides with Tim50(IMS) in presence of Ca2+ ion by fluorescence spectroscopy in vitro. The static fluorescence quenching indicates the existence of strong binding between divalent metal ions and Tim50(IMS). The order of the binding strength is Ca2+, Mg2+, Cu2+, Mn2+, and Co2+ (from strong to weak). Moreover, the interaction of presequence peptides with Tim50(IMS) is weakened in presence of Ca2+ ion, which implicates that Ca2+ ion may play an important role in the protein import by TIM23 complex. (C) 2012 Elsevier Inc. All rights reserved.