REDOX REACTIVITY OF THE BINUCLEAR IRON ACTIVE-SITE OF PORCINE PURPLE ACID-PHOSPHATASE (UTEROFERRIN)

Redox interconversions (25-degrees-C) of active Fe(II)Fe(III) purple acid phosphatase (PAP(r)), and the inactive Fe(III)Fe(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-degrees' of 367 mV vs. normal hydrogen electrode. Whereas with [Co(phen)3]3+ as oxidant for PAP, 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 x 10(5) 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 S2O42- a bleaching of the colour is observed consistent with Fe(II)Fe(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).