EVIDENCE THAT RESIDUES EXPOSED ON THE 3-FOLD CHANNELS HAVE ACTIVE ROLES IN THE MECHANISM OF FERRITIN IRON INCORPORATION

Iron is thought to enter the ferritin cavity via the three-fold channe l, which is lined in its narrowest part by the residues Asp-131 and Gl u-134. We describe here variants of human ferritins with active and in active ferroxidase centres having Asp-131 and Glu-134 substituted with Ala and Ala or with Ile and Phe respectively. The two types of substi tution had similar effects on ferritin functionality: (i) they decreas ed the amount of iron incorporated from Fe(II) solutions and decreased ferroxidase activity by about 50%; (ii) they inhibited iron incorpora tion from Fe(III) citrate in the presence of ascorbate; (iii) they res ulted in loss of Fe and Tb binding sites; and (iv) they resulted in a marked decrease in the inhibition of iron oxidation by Tb (but not by Zn). In addition, it was found that substitution with Ala of Cys-130 a nd His-118, both of which face the three-fold channel, decreased the c apacity of H-ferritin to bind terbium and to incorporate iron from Fe( III) citrate in the presence of ascorbate. The results indicate that: (i) the three-fold channels are the major sites of iron transfer into the cavity of H- and L-ferritins; (ii) at least two metal binding site s are located on the channels which play an active role in capturing a nd transferring iron into the cavity; and (iii) the permeability of th e channel is apparently not affected by the hydrophilicity of its narr owest part. In addition, it is proposed that iron incorporation from F e(III) citrate complexes in the presence of ascorbate is a reliable, a nd possibly more physiological, approach to the study of ferritin func tionality.