Formate as an NMR probe of anion binding to Cu,Zn and Cu,Co bovine erythrocyte superoxide dismutases.
The binding of formate to bovine Cu,Zn superoxide dismutase has been studied by NMR spectroscopy. The distance between the copper ion and the proton covalently bound to formate has been evaluated from the broadening of the resonance of such proton. The effect on the copper-coordinated water molecule was evaluated from the bulk water relaxation effect by pulsed low-resolution NMR. The broadening of the resonance due to the formate carboxyl in the 13C NMR spectrum gave further indications about the carbon-copper distance thus providing information about the orientation of the formate ion. Changes of isotropically shifted resonances of the Cu,Co enzyme, where cobalt substitutes the native zinc, indicate that rearrangements of imidazoles of the liganding histidines occur upon binding. Transient NOE experiments gave indication of the proximity of the formate proton to resonance H of the NMR spectrum assigned to the imidazole proton of the copper-liganding His 118 of the active site. 2D NMR NOESY experiments made clear that no important rearrangement of the liganding histidines occurred in the presence of a saturating amount of formate. The absence of relevant changes of the intensity of NOE cross-peaks which are sensitive to interatomic distances in the active site revealed that only slight changes have occurred. Molecular graphics representation on the basis of all the information obtained allowed us to locate the formate in the proximity of the active site. The formate binding occurs via hydrogen bonds through the carboxylate ion and the NH groups of the side chains of Arg 141 which is external to the copper coordination sphere and faces the active site of the enzyme.
Sette
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Department of Biology, University of Rome Tor Vergata, Italy.
Paci
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M
Desideri
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A
Rotilio
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D000818:Animals; D000838:Anions / Q000097:blood*; D001665:Binding Sites; D002417:Cattle; D004912:Erythrocytes / Q000201:enzymology*; D005561:Formates / Q000097:blood*; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011485:Protein Binding; D013482:Superoxide Dismutase / Q000097:blood*
D000838:Anions; D005561:Formates; D013482:Superoxide Dismutase