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 catal ytic mechanism of CuZn SOD are thought to be the breakage of the imida zolate bridge between copper and zinc upon reduction of the copper sit e 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 i n the reduced enzyme was provided. In contrast, a crystallographic stu dy of yeast CuZn SOD at 1.7 Angstrom has confirmed the breaking of the bridging histidine in reduced crystals. We have examined the nature o f the changes in metal coordination which result upon reduction of the enzyme using the X-ray absorption fine structure (XAFS) technique. Re sults: The copper and zinc K-edge XAFS data of bovine SOD, recorded in the buffer systems used in the two crystallographic studies, were ana lyzed by constrained refinement using fast curved wave theory, taking full account of multiple-scattering effects. The study confirms that i n 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 mole cule 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 inn er coordination sphere of the copper, with the three remaining histidi nes ligated at 1.97 +/- 0.02 Angstrom. The X-ray absorption near edge structure (XANES) of the reduced enzyme is consistent with an approxim ate trigonal planar geometry at the copper site, The XAFS at the zinc K-edge is essentially identical in both the oxidized and reduced enzym e and is accounted for by three histidines coordinated at 2.01 +/- 0.0 2 Angstrom and an aspartate ligand at 1.96 +/- 0.03 Angstrom. Conclusi ons: The existence of a three-coordinate cuprous ion in bovine CuZn SO D is demonstrated and is a key feature of catalytic degradation of sup eroxide substrate by SOD involving alternate Cull) and Cu(ll) states o f the enzyme, Only subtle changes in the zinc K-edge XAFS take place u pon reduction, Thus the reaction mechanism which involves breakage of the bridging histidine is unambiguously supported by the XAFS data.