A critical assessment of the evidence from XAFS and crystallography for the breakage of the imidazolate bridge during catalysis in CuZn superoxide dismutase

Background: Copper-zinc superoxide dismutase (CuZn SOD) protects cells from the toxic effects of superoxide radicals. Key steps in the catalytic mechanism of CuZn SOD are thought to be the breakage of the imidazolate bridge between copper and zinc upon reduction of the copper site and the subsequent proton donation from the bridging histidine. This view has been recently challenged by a crystallographic study at 1.9 Angstrom resolution where evidence for a five-coordinate copper site in the reduced enzyme was provided. In contrast, a crystallographic study of yeast CuZn SOD at 1.7 Angstrom has confirmed the breaking of the bridging histidine in reduced crystals. We have examined the nature of the changes in metal coordination which result upon reduction of the enzyme using the X-ray absorption fine structure (XAFS) technique. Results: The copper and zinc K-edge XAFS data of bovine SOD, recorded in the buffer systems used in the two crystallographic studies, were analyzed by constrained refinement using fast curved wave theory, taking full account of multiple-scattering effects. The study confirms that in the oxidized form of the enzyme the copper atom is five coordinate, with four histidine ligands at 1.99 +/- 0.02 Angstrom and a water molecule at 2.18 +/- 0.03 Angstrom. In the reduced form of the enzyme, one of the histidine ligands and the water molecule are lost from the inner coordination sphere of the copper, with the three remaining histidines ligated at 1.97 +/- 0.02 Angstrom. The X-ray absorption near edge structure (XANES) of the reduced enzyme is consistent with an approximate trigonal planar geometry at the copper site, The XAFS at the zinc K-edge is essentially identical in both the oxidized and reduced enzyme and is accounted for by three histidines coordinated at 2.01 +/- 0.02 Angstrom and an aspartate ligand at 1.96 +/- 0.03 Angstrom. Conclusions: The existence of a three-coordinate cuprous ion in bovine CuZn SOD is demonstrated and is a key feature of catalytic degradation of superoxide substrate by SOD involving alternate Cull) and Cu(ll) states of the enzyme, Only subtle changes in the zinc K-edge XAFS take place upon reduction, Thus the reaction mechanism which involves breakage of the bridging histidine is unambiguously supported by the XAFS data.