Measuring benzohydroxamate complexation with Mg2+, Mn2+, Co2+, and Ni2+ using isothermal titration calorimetry

Abstract

Isothermal titration calorimetry has been used to determine the stoichiometry and first (K1) and second (K2) conditional stability constants, where applicable, for the formation of complexes of benzohydroxamate (BzH) with Mg2+, Mn2+, Co2+, and Ni2+ ions, and benzoylphosphonate with Ni2+ ions. Binding determinations were conducted in HEPES buffer (0.1 mol L−1, pH 7.5) at 25.0 °C by titration of the ligand with metal ion. BzH and Mg2+, and benzoylphosphonate and Ni2+, formed complexes with a 1:1 stoichiometry; however, BzH formed complexes with Mn2+, Co2+, and Ni2+ ions in a step-wise binding model with a stoichiometry of 2:1 (BzH:M2+). In addition, the Gibbs energy change accompanying the binding of BzH to each of the divalent metal ions was found to follow the order of the Irving–Williams series (Mg2+ < Mn2+ < Co2+ < Ni2+) and to correlate with the electron affinities of the metal ions. Conditional stability constants, determined in this manner, may be useful for calculating the concentrations of free inhibitor when inhibition studies are conducted with metalloenzymes and enzyme inhibitors containing a hydroxamic acid group.