The crystal structure of the monomeric human SOD mutant F50E/G51E/E133Q atatomic resolution. The enzyme mechanism revisited

The crystal structure of the engineered monomeric human Cu,ZnSOD triple mut ant F50E/G51E/E133Q (Q133M2SOD) is reported at atomic resolution (1.02 Angs trom). This derivative has about 20% of the wild-type activity. Crystals of Q133M2SOD have been obtained in the presence of CdCl2. The metal binding s ite is disordered, with both cadmium and copper ions simultaneously binding to the copper site. The cadmium (II) ions occupy about 45 % of the copper sites by binding the four histidine residues which Ligate copper in the nat ive enzyme, and two further water molecules to complete octahedral coordina tion. The copper ion is tri-coordinate, and the fourth histidine (His63),is detached from copper and bridges cadmium and zinc. X-ray absorption spectr oscopy performed on the crystals suggests that the copper ion has undergone partial photoreduction upon exposure to the synchrotron Light. The structu re is also disordered in the disulfide bridge region of loop TV that is loc ated at the subunit/subunit interface in the native SOD dimer. As a consequ ence, the catalytically relevant Arg143 residue is disordered. The present structure has been compared to other X-ray structures on various isoenzymes and to the solution structure of the same monomeric form. The structural r esults suggest that the low activity of monomeric SOD is due to the disorde r in the conformation of the side-chain of Arg143 as well as of loop TV. It is proposed that the subunit-subunit interactions in the multimeric forms of the enzyme are needed to stabilize the correct geometry of the cavity an d the optimal orientation of the charged residues in the active channel. Fu rthermore, the different coordination of cadmium and copper ions, contempor aneously present in the same site, are taken as models for the oxidized and reduced copper species, respectively. These proper ties of the structure h ave allowed us to revisit the enzymatic mechanism. (C) 1999 Academic Press.