Redox reactivity of the binuclear iron active site of porcine purple acid phosphatase (uteroferrin)

Abstract

Redox interconversions (25 °C) of active Fe^IIFe^III purple acid phosphatase (PAP_r), and the inactive Fe^IIIFe^III form (PAP_o), have been explored at pH 5.0 (close to maximum activity), I= 0.100 M (NaCl). At this pH the PAP_o–PAP_r, couple has a reduction potential E°′ of 367 mV vs. normal hydrogen electrode. Whereas with [Co(phen)₃]³⁺ as oxidant for PAP_r first-order dependencies on both reactants are observed (k= 1.26 M^–1 s^–1), with [Fe(CN)₆]^3– saturation kinetics are obtained with association. K= 540 M^–1, occurring prior to electron transfer, k_et= 1.0 s^–1. The latter reaction undergoes competitive inhibition with redox inactive [Cr(CN)₆]^3–(K_cr= 550 M^–1) and [Mo(CN)₈]^4–(K_Mo= 1580 M^–1) consistent with a positively charged locality on the protein surface influencing reactivity. With [Ru(NH₃)₆]²⁺ reduction of PAP_o is too fast to monitor, but with the less strongly reducing [Ru(NH₃)₅(H₂O)]²⁺ a rate law first order in both reactants (k= 2.2 × 10⁵ M^–1 s^–1) is observed. Reactions of both these reductants with the less strongly oxidising phosphate bound PAP_o–PO₄ form (183 mV) were also studied. On reduction of PAP_r with S₂O₄^2– a bleaching of the colour is observed consistent with Fe^IIFe^II formation. After 30 min only 40% of the protein could be restored to one or other of the higher oxidation states, indicating loss of Fe^II.