Identification of the iron entry channels in apoferritin. Chemical modification and spectroscopic studies.
The knowledge of the route through which iron can enter and leave the apoferritin shell is a prerequisite for the understanding of ferritin's function. The involvement of the hydrophilic 3-fold channels in the iron uptake process has been studied by taking advantage of the reactivity of specific residues that line such channels, i.e., glutamic acid-127 and aspartic acid-130, the major Cd(II) binding sites, and cysteine-126. 113Cd NMR experiments have provided direct evidence for the competition between Fe(II) and Cd(II) binding to major Cd(II) binding sites on the protein and or a higher affinity of Fe(II) for these sites, in line with the well-known inhibitory effect of Cd(II) on iron uptake. Further evidence for the use of the 3-fold channels in the iron entry process has been obtained by means of chemical modification of Cys-126 with different mercurials. In particular, the introduction of the additional carboxylate carried by p-(chloromercuri)benzoate near Asp-127 and Glu-130 increases the initial rate of iron uptake and affects the coordination geometry of the metal in the Fe(III)-apoferritin complex as indicated by optical absorption and EPR data. The assignment of these effects to the carboxylate moiety of p-(chloromercuri)benzoate is brought out by the observation that the introduction in the 3-fold channel of the benzene ring only by means of phenylmercuric acetate has no effect on the initial iron uptake kinetics and on the spectroscopic properties of the Fe(III)-apoferritin complex.
Stefanini
S
S
Department of Biochemical Sciences, CNR Center of Molecular Biology, University of Rome, La Sapienza, Italy.
Desideri
A
A
Vecchini
P
P
Drakenberg
T
T
Chiancone
E
E
D000818:Animals; D001052:Apoferritins / Q000378:metabolism*; D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism; D002729:Chloromercuribenzoates; D003545:Cysteine; D004578:Electron Spin Resonance Spectroscopy; D005293:Ferritins / Q000031:analogs & derivatives*; D066298:In Vitro Techniques; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D010662:Phenylmercuric Acetate; D020245:p-Chloromercuribenzoic Acid
D002729:Chloromercuribenzoates; D002104:Cadmium; D020245:p-Chloromercuribenzoic Acid; D005293:Ferritins; D001052:Apoferritins; D007501:Iron; D003545:Cysteine; D010662:Phenylmercuric Acetate