DEFINING THE ROLES OF THE THREEFOLD CHANNELS IN IRON UPTAKE, IRON OXIDATION AND IRON-CORE FORMATION IN FERRITIN - A STUDY AIDED BY SITE-DIRECTED MUTAGENESIS

This paper aims to define the role of the threefold intersubunit chann els in iron uptake and sequestration processes in the iron-storage pro tein, ferritin. Iron uptake, measured as loss of availability of Fe(II ) to ferrozine (due to oxidation), has been studied in recombinant hum an H-chain ferritins bearing amino acid substitutions in the threefold channels or ferroxidase centres. Similar measurements with recombinan t horse L-chain ferritin are compared. It is concluded that significan t Fe(II) oxidation occurs only at the H-chain ferroxidase centres and not in the threefold channels, although this route is used by Fe(II) f or entry. Investigations by Mossbauer and u.v.-difference spectroscopy show that part of the iron oxidized by H-chain ferritin returns to th e threefold channels as Fe(III). This monomeric Fe(III) can be displac ed by addition of Tb(III). Fe(III) also moves into the cavity for form ation of the iron-core mineral, ferrihydrite. Iron incorporated into f errihydrite becomes kinetically inert.