PMID:11863465
| Citation |
Benson, DE, Haddy, AE and Hellinga, HW (2002) Converting a maltose receptor into a nascent binuclear copper oxygenase by computational design. Biochemistry 41:3262-9 |
|---|---|
| Abstract |
Computational protein design methods were used to identify mutations that are predicted to introduce a binuclear copper center coordinated by six histidines, replacing the maltose-binding site in Escherichia coli maltose-binding protein (MBP) with an oxygen-binding site. A small family of five candidate designs consisting of 9 to 10 mutations each was constructed by oligonucleotide-directed mutagenesis. These mutant proteins were expressed and purified, and their stability, copper- and cobalt-binding properties, and interactions of the resulting metalloprotein complexes with azide, hydrogen peroxide, and dioxygen were characterized. We identified one 10-fold mutant, MBP.Hc.E, that can form Cu(II)(2) and Co(II)(2) complexes that interact with H(2)O(2) and O(2). The Co(II)(2) protein reacts with H(2)O(2) to form a complex that is spectroscopically similar to a synthetic model that structurally mimics the oxy-hemocyanin core, whereas the Cu(II)(2) protein reacted with O(2) or H(2)O(2) does not. We postulate that the equilibrium between the open and closed conformations of MBP allows species with variable Cu-Cu distances to form, and that such species can bind ligands in geometries that are not observed in natural type III centers. Introduction of one additional mutation in the hinge region of MBP, I329F, known to favor formation of the closed state, results in a binuclear copper center that when reacted with low concentrations of H(2)O(2) mimics the spectroscopic signature of oxy-hemocyanin. |
| Links | |
| Keywords |
ATP-Binding Cassette Transporters; Carrier Proteins/chemistry; Carrier Proteins/genetics; Carrier Proteins/metabolism; Circular Dichroism; Cobalt/metabolism; Computational Biology; Copper/metabolism; Electron Spin Resonance Spectroscopy; Enzyme Stability; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins; Hydrogen Peroxide/metabolism; Maltose/metabolism; Maltose-Binding Proteins; Models, Molecular; Monosaccharide Transport Proteins; Mutagenesis; Oxidation-Reduction; Oxygenases/metabolism; Protein Conformation; Protein Engineering |
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