Core formation in Escherichia coli bacterioferritin requires a functional ferroxidase center.
Bacterioferritin from Escherichia coli is able to accumulate large quantities of iron in the form of an inorganic iron(III) mineral core. Core formation in the wild-type protein and a number of ferroxidase center variants was studied to determine key features of the core formation process and, in particular, the role played by the ferroxidase center. Core formation rates were found to be iron(II)-dependent and also depended on the amount of iron already present in the core, indicating the importance of the core surface in the mineralization reaction. Core formation was also found to be pH-dependent in terms of both rate and iron-loading characteristics, occurring with maximum efficiency at pH 6.5. Even at this optimum pH, however, the effective iron capacity was approximately 2700 per molecule, i.e., well below the theoretical limit of approximately 4500, suggesting that competing oxidation/precipitation processes have a major influence on the amount of iron accumulated. Disruption of the ferroxidase center, by site-directed mutagenesis or by chemical inhibition with zinc(II), had a profound effect on core formation. Effective iron capacities were found to be linked to iron(II) oxidation rates, and in zinc(II)-inhibited wild-type and E18A bacterioferritins core formation was severely restricted. Zinc(II) was also able, even at low stoichiometries (12-60 ions/protein), to significantly inhibit further core formation in protein already containing a substantial core, indicating the importance of the ferroxidase center throughout the core formation process. A mechanism is proposed that incorporates essential roles for the core surface and the ferroxidase center. A central feature of this mechanism is that dioxygen cannot readily gain access to the core, perhaps because the channels through the bacterioferritin coat are hydrophilic and dioxygen is nonpolar.
Baaghil
Suzanne
S
Center for Metalloprotein Spectroscopy and Biology, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, United Kingdom.
Lewin
Allison
A
Moore
Geoffrey R
GR
Le Brun
Nick E
NE
D000409:Alanine / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D002570:Ceruloplasmin / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics; D003300:Copper / Q000737:chemistry; D003573:Cytochrome b Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry; D004793:Enzyme Reactivators / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005290:Ferric Compounds / Q000737:chemistry* / Q000378:metabolism; D005293:Ferritins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D013056:Spectrophotometry, Ultraviolet; D055672:Static Electricity; D015032:Zinc / Q000737:chemistry
D001426:Bacterial Proteins; D003573:Cytochrome b Group; D004791:Enzyme Inhibitors; D004793:Enzyme Reactivators; D029968:Escherichia coli Proteins; D005290:Ferric Compounds; D018698:Glutamic Acid; D003300:Copper; D005293:Ferritins; C018032:bacterioferritin; D002570:Ceruloplasmin; D015032:Zinc; D000409:Alanine