MECHANISM OF IRON RELEASE FROM HUMAN SERUM C-TERMINAL MONOFERRIC TRANSFERRIN TO PYROPHOSPHATE - KINETIC DISCRIMINATION BETWEEN ALTERNATIVE MECHANISMS

The kinetics of iron release from C-terminal monoferric transferrin (FeCTf) to pyrophosphate (PP) under pseudo-first-order conditions show an apparent saturation linear dependence of k(obs) on [PP]. The variation of k(obs) with [PP] was studied under conditions of variable temperature and added anion (X) concentration (Cl-, ClO4-, NO3-, SO4(2-), HPO4(2-)). The results conform globally to a two-path mechanism (mechanism 1) involving iron removal either from a species with added anion bound to a kinetically significant anion binding (KISAB) site on the protein or with PP bound to the KISAB site, these two species being linked by a rapid equilibrium. Microscopic rate and equilibrium constants were evaluated by nonlinear regression of kinetic data to the equation k(obs) = (k1[PP] + k2K(eq)'[PP]2)/(1 + K(eq)'[PP]). The conditional constant K(eq)' (= K(eq)/[X]) obtained in the numerical regression procedure varied with [Cl-] as required by mechanism 1. Two plausible simple alternative mechanisms were also considered. Both involved a saturation pathway involving attainment of an open conformation of the protein and a nonsaturation pathway involving direct removal of iron from the transferrin. Both mechanisms led to an anion dependence of the microscopic rate and equilibrium constants on [Cl-] different from that observed experimentally. Furthermore, plots of ln k1 and ln k2 vs T-1 for mechanism 1 adhere closely to the Arrhenius model, with no curvature or breaks in the plots apparent, as required for true microscopic rate constants, thus arguing further in favor of the correctness of this mechanism.