title: A characterization of copper/zinc superoxide dismutase mutants at position 124. Zinc-deficient proteins.
abstract: Substitution of the completely conserved aspartic acid residue at position 124 of Cu,Zn superoxide dismutase with asparagine and glycine has been performed through site-directed mutagenesis on the human enzyme. Asp124 is H-bonded to the NH of two histidines, one of which is bound to copper and the other to zinc. The mutant proteins, as expressed in Escherichia coli, result in an essential zinc-free enzyme which is similar to that obtained from the wild-type derivative through chemical manipulation. Only by extensive dialysis against 0.5 M ZnCl2 or CoCl2 at pH 5.4 was it possible to reconstitute approximately 50% of the molecules in the Cu2Zn2 or Cu2Co2 form. The new derivatives have been characterized through EPR, CD and nuclear magnetic relaxation dispersion techniques. The Cu2Cox derivatives (x approximately 1) were used to monitor, through electronic and 1H-NMR spectroscopies, the metal sites which are found to be similar to those of the wild type. In addition, a double substitution with asparagine has been made, replacing the invariant aspartate at position 124 and the highly conserved aspartate at position 125. The behavior is similar to that of the other mutants in most respects. The Cu2E2 (E = empty) derivatives of the mutants are stable, even in the pH range 8-10, whereas in the case of the Cu2E2 derivative of the wild type, copper migration occurs at high pH, producing both Cu2Cu2 and apo derivatives. The activity measurements indicate that the various Cu2E2 derivatives have the same activity at low pH and similar to that of the holoenzyme. A full profile up to pH 10.5 was obtained for the mutants.
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: D E
authors.initials: DE
authors.lastname: Cabelli
authors.forename: R A
authors.initials: RA
authors.lastname: Hallewell
authors.forename: J W
authors.initials: JW
authors.lastname: Tung
authors.forename: M S
authors.initials: MS
authors.lastname: Viezzoli
mesh_terms: D003035:Cobalt / Q000032:analysis; D003300:Copper / Q000032:analysis; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D013482:Superoxide Dismutase / Q000032:analysis / Q000737:chemistry*; D015032:Zinc / Q000032:analysis*
chemical_list: D003035:Cobalt; D003300:Copper; D013482:Superoxide Dismutase; D015032:Zinc