Cu, Zn and Mn superoxide dismutases; Crystal structures, mutants, and function

STRUCTURE/FUNCl3ON

BOO2

Cu, Zn and Mn Superoxide Dismutases: tants, and Function

Crystal Structures,

57

Mu-

John A. Tainer ‘, Hans E. Pargea, Gloria E. 0. BorgstahY, Cindy L. Fisher”, S&an M. Redforda, Darren J. Murtar?, Michael J. Hickey”, Duncan E. McRee”, Robert A. Hallewell”, and Elizabeth D. Getzoff “Department of Molecular Biology MBd, The Scripps Research Institute, La Jolla CA 92037. bBiochemistry Department, Imperial College, London SW7 2A2, U.K. The superoxide dismutase (SOD) metalloenzymes are critical components in the physiological response to oxygen toxicity and mutations in SOD are known to cause fatal neurodegenerative disease. We are using the combination of designed mutations and x-ray crystal structures to study metal site structure and function for both the cytoplasmic Cu,Zn superoxide dismutase (Cu,Zn SOD) and the mitochondrial Mn superoxide dismutase (MnSOD). We have made and characterized over 30 Cu,Zn SOD mutants, and determined atomic structures for the wild-type human[l], bovine[2], and yeast enzymes as well as several mutants. The 10 independently fit and refined subunits in the human Cu,Zn SOD enzyme provide high accuracy, error analysis and new insights on the active site structure[l]. The Cu-ligands (His-46, His-48, His-63, His-120) form distorted square planar geometry, while the Zn-ligands (His-63, His-71, His-80, Asp-83) are tetrahedral. The Cu and Zn are linked directly by the bridging histidine 63 and indirectly by the side-chain carboxylate of buried Asp-124, which hydrogen bonds to both a Cu and a Zn-ligating histidine. A helix dipole interaction stabilizes the Zn site. Recently, we have designed and solved the structure of a mutant without the Zn site to identify the resultant effect on the remaining Cu site. Other mutant structures explore residues involved in the electrostatic recognition that allow the enzymatic rate to exceed the diffusion limit[3]. For MnSOD, the 2.2 A crystal structure of the recombinant human homotetrameric enzyme[4] shows the relationships of the secondary, tertiary, and quaternary structure with the four catalytic Mn sites. Within each subunit, both the N-terminal helical hairpin and C-terminal o//3 domains contribute ligands to the catalytic Mn site. The active site Mn ion joins the two domains and is positioned between the helical and P-sheet structural elements. Two amino acid residues from each domain, His26 in crl and His74 in CY~from the N-terminal domain, and Asp159 in ,03 and His163 in region H from the C-terminal domain, plus a water molecule, ligate the Mn(I1) with five-coordinate trigonal bipyramidal geometry. The four active sites of the MnSOD tetramer are grouped in pairs across the dimer interface with residues Glu162 and Tyr166 from one subunit contributing to the active site of the neighboring subunit. Two identical 4-helix bundles, symmetrically assembled from the N-terminal helical hairpins, form novel tetrameric interfaces and stabilize the active sites. Structures of two site-directed mutants (Tyr34 to Phe and Ile58 to Thr) help characterize the proton donor for catalysis and the role of the protein environment in metal binding affinity. 1. Parge, H. E., Hallewell, R. A. and Tainer, J. A. Proc. Natl. Acad. Sci. U.S.A., 89, 6109-6113, (1992). 2. McRee, D. E., Redford, S. M., Getzoff, E.D., Lepock, J. R., Hallewell, R. A., and Tainer, J.A. J. Biol. Chem., 265, 14234-14241, (1990). 3. Getzoff, E.D., Cabelli, D.E., Fisher, C.L., Parge, H.E., Viezzoli, M.S., Banci, L., and Hallewell, R.A. Nature, 358, 347-351, (i992). 4. Borgstahl, G. E. O., Parge, H. E., Hickey, M. J., Beyer, W. F., Hallewell, R. A. and Tainer, J. A. Cell, 71, 107-118, (1992).