Evidence that residues exposed on the three-fold channels have active roles in the mechanism of ferritin iron incorporation.
Iron is thought to enter the ferritin cavity via the three-fold channel, which is lined in its narrowest part by the residues Asp-131 and Glu-134. We describe here variants of human ferritins with active and inactive ferroxidase centres having Asp-131 and Glu-134 substituted with Ala and Ala or with Ile and Phe respectively. The two types of substitution had similar effects on ferritin functionality: (i) they decreased the amount of iron incorporated from Fe(II) solutions and decreased ferroxidase activity by about 50%; (ii) they inhibited iron incorporation from Fe(III) citrate in the presence of ascorbate; (iii) they resulted 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 and His-118, both of which face the three-fold channel, decreased the capacity of H-ferritin to bind terbium and to incorporate iron from Fe(III) citrate in the presence of ascorbate. The results indicate that: (i) in three-fold channels are the major sites of iron transfer into the cavity of H- and L-ferritins; (ii) at least two metal binding sites are located on the channels which play an active role in capturing and transferring iron into the cavity; and (iii) the permeability of the channel is apparently not affected by the hydrophilicity of its narrowest part. In addition, it is proposed that iron incorporation from Fe(III) citrate complexes in the presence of ascorbate is a reliable, and possibly more physiological, approach to the study of ferritin functionality.
Levi
S
S
DIBIT Department of Biological and Technological Research, H. San Raffaele Scientific Institute, Milano, Italy.
Santambrogio
P
P
Corsi
B
B
Cozzi
A
A
Arosio
P
P
D000409:Alanine / Q000235:genetics; D001052:Apoferritins; D001224:Aspartic Acid / Q000235:genetics; D002570:Ceruloplasmin / Q000378:metabolism; D005290:Ferric Compounds / Q000378:metabolism; D005293:Ferritins / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D007501:Iron / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D013329:Structure-Activity Relationship; D013725:Terbium / Q000378:metabolism; D017186:Titrimetry
D005290:Ferric Compounds; D013725:Terbium; D001224:Aspartic Acid; D018698:Glutamic Acid; C025314:ferric citrate; D005293:Ferritins; D001052:Apoferritins; D007501:Iron; D002570:Ceruloplasmin; D000409:Alanine