CRYSTALLOGRAPHIC DETERMINATION OF REDUCED BOVINE SUPEROXIDE-DISMUTASEAT PH-5.0 AND OF ANION-BINDING TO ITS ACTIVE-SITE

The crystal structures of dithionite-reduced bovine Cu(I),Zn superoxid e dismutase and of its adducts with the inorganic anions azide and thy ocyanide have been determined in a C222(1) crystal form obtained at pH 5.0. This crystal form is characterized by a high solvent content (72 %) and by having the two Cu,ZnSOD monomers (A and B) in different crys tal environments. One of them (B) is involved in few intermolecular cr ystal contacts so that it is in a more ''solution like'' environment, as indicated by average temperature factors which are about twice thos e of the other monomer. The differences in crystal packing affect the active site structures. While in the A monomer the Cu(I) is coordinate d to all four histidine residues, in the B monomer the bridging His61 side chain is found disordered, implying partial detachment from coppe r. The same effect occurs in the structures of the anion complexes. Th e inorganic anions are found bound in the active site cavity, weakly i nteracting with copper at distances ranging from 2.5 to 2.8 Angstrom. The copper site in the A subunit of the native enzyme structure displa ys significant electron density resembling a diatomic molecule, bound side-on at about 2.8 A from the metal, which cannot be unambiguously i nterpreted. The crystallographic data suggest that the existence of th e His61 bridge between copper and zinc is dominated by steric more tha n electronic factors and that the solution state favors the His61 deta chment. These structures confirm the existence of an energetically ava ilable state for Cu(I) in Cu,ZnSOD where the histidinato bridge to zin c is maintained. This state appears to be favored by tighter crystal c ontacts. The binding of the anions in the active site cavity is differ ent from that observed in the oxidized enzyme and it appears to be dom inated by electrostatic interactions within the cavity. The anion bind ing mode observed may model the substrate interaction with the reduced enzyme during catalysis.