IRON RELEASE FROM RECOMBINANT N-LOBE AND MUTANTS OF HUMAN TRANSFERRIN

Mutations of kinetically active residues in the recombinant N-lobe of human transferrin may accelerate or retard release of iron from the pr otein to pyrophosphate, thereby providing means for exploring the indi vidual roles of such residues in the concerted mechanisms of release. Using an established spectrofluorometric method and pyrophosphate as t he required iron-sequestering agent, we have compared release from una ltered native transferrin and recombinant N-lobe half-transferrin to r elease from six N-lobe mutants, R124S, R124K, K206R, H207E, H249Y, and Y95H. Mutation of R124, which serves as a principal anchor for the sy nergistic carbonate anion ordinarily required for iron binding by tran sferrin, accelerates release. This effect is most marked at endosomal pH, 5.6, and is also evident at extracellular pH, 7.4, pointing to a c ritical and perhaps initiating role of carbonate in the release proces s. Mutation of K206 to arginine, or of H207 to glutamine, each lying i n the interdomain cleft of the N-lobe, gives products mimicking the ar rangements in lactoferrin. Release of iron from these two mutants, as from lactoferrin, is substantially slower than from unaltered recombin ant N-lobe. Interdomain residues not directly involved in iron or anio n binding may therefore participate in the control of iron release wit hin the endosome. The H249Y mutant releases iron much more rapidly tha n its wild-type parent or any other mutant, possibly because of steric effects of the additional phenolic ring in the binding site. No simpl e explanation is available to account for a stabilizing effect of the Y95H mutation. Chloride (or another simple anion) promotes and is esse ntial for iron release from the C-lobe of human transferrin but exerts a retarding effect on release from the N-lobe in native and mutant tr ansferrins alike. A simple model, entailing binding competition betwee n pyrophosphate used to effect release and chloride, substantially acc ounts for the negative effect of chloride on the N-lobe.