Insights into the effects on metal binding of the systematic substitution of five key glutamate ligands in the ferritin of Escherichia coli.
Ferritins are nearly ubiquitous iron storage proteins playing a fundamental role in iron metabolism. They are composed of 24 subunits forming a spherical protein shell encompassing a central iron storage cavity. The iron storage mechanism involves the initial binding and subsequent O2-dependent oxidation of two Fe2+ ions located at sites A and B within the highly conserved dinuclear "ferroxidase center" in individual subunits. Unlike animal ferritins and the heme-containing bacterioferritins, the Escherichia coli ferritin possesses an additional iron-binding site (site C) located on the inner surface of the protein shell close to the ferroxidase center. We report the structures of five E. coli ferritin variants and their Fe3+ and Zn2+ (a redox-stable alternative for Fe2+) derivatives. Single carboxyl ligand replacements in sites A, B, and C gave unique effects on metal binding, which explain the observed changes in Fe2+ oxidation rates. Binding of Fe2+ at both A and B sites is clearly essential for rapid Fe2+ oxidation, and the linking of FeB2+ to FeC2+ enables the oxidation of three Fe2+ ions. The transient binding of Fe2+ at one of three newly observed Zn2+ sites may allow the oxidation of four Fe2+ by one dioxygen molecule.
Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom.
Timothy J
TJ
Stillman
Paul P
PP
Connolly
Charlotte L
CL
Latimer
Andrew F
AF
Morland
Michael A
MA
Quail
Simon C
SC
Andrews
Amyra
A
Treffry
John R
JR
Guest
Peter J
PJ
Artymiuk
Pauline M
PM
Harrison
D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D018360:Crystallography, X-Ray; D004583:Electrons; D004926:Escherichia coli / Q000378:metabolism*; D005293:Ferritins / Q000737:chemistry*; D018698:Glutamic Acid / Q000737:chemistry*; D007477:Ions; D007501:Iron / Q000378:metabolism; D008024:Ligands; D008670:Metals / Q000378:metabolism / Q000494:pharmacology; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D013696:Temperature; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
D007477:Ions; D008024:Ligands; D008670:Metals; D018698:Glutamic Acid; D005293:Ferritins; D007501:Iron; D015032:Zinc; D010100:Oxygen