title: Mutation of the metal-bridging proton-donor His63 residue in human Cu, Zn superoxide dismutase. Biochemical and biophysical analysis of the His63-->Cys mutant.
abstract: The bridging His63 residue in human Cu, Zn superoxide dismutase, which binds both metals, has been replaced by a Cys residue. The mutant protein has been purified from Escherichia coli and appears to be a normal dimer. Spectroscopic techniques (electronic spectroscopies, EPR, nuclear magnetic relaxation dispersion) show that Cys63 binds the zinc ion, but not the copper ion, and that the latter is probably five co-ordinated with three histidine ligands and two water molecules. The reduction potential of the copper ion in the Cu2+/Cu+ pair decreases from 0.41 V to 0.27 V at neutral pH but still remains intermediate between those of the O2/O2- and O2-/H2O2 pairs so that copper can both oxidize and reduce the O2- substrate, a requirement for dismutase activity. The enzyme binds the substrate-analogue azide (N3-), which displaces one water molecule, with near normal affinity, whereas the enzyme activity with the O2- substrate is reduced to less than 1% of wild-type levels at pH 7.8. The properties of the mutant enzyme are discussed in relation to the superoxide-copper electron transfer process and to the catalytic mechanism.
authors.affiliation: Department of Chemistry, University of Florence, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Banci
authors.forename: I
authors.initials: I
authors.lastname: Bertini
authors.forename: M
authors.initials: M
authors.lastname: Borsari
authors.forename: M S
authors.initials: MS
authors.lastname: Viezzoli
authors.forename: R A
authors.initials: RA
authors.lastname: Hallewell
mesh_terms: D003300:Copper / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D006639:Histidine / Q000378:metabolism; D006801:Humans; D009154:Mutation; D011522:Protons; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
chemical_list: D011522:Protons; D006639:Histidine; D003300:Copper; D013482:Superoxide Dismutase; D015032:Zinc; D003545:Cysteine