Kinetic and structural characterization of human manganese superoxide dismulase containing 3-fluorotyrosines

Incorporation of 3-fluorotyrosine and site-specific mutagenesis have been used with stopped-flow spectrophotometry and pulse radiolysis to investigate the catalytic properties of human manganese superoxide dismutase (MnSOD). All of the nine tyrosine residues in each of the four subunits of the homotetramer of human MnSOD were replaced with 3-fluorotyrosine. Previous studies showed that the crystal structures of the unfluorinated and fluorinated human MnSOD are nearly superimposable with the root-mean-square deviation for 198 alpha-carbon atoms at 0.3 angstrom. However, the catalytic activity k(cat)/K-m of the fluorinated MnSOD at 30 mu M-1 s(-1) was less than unfluorinated wild type at 800 mu M-1 s(-1). Comparison of the values of k(cat)/K-m for fluorinated and unfluorinated wild-type and Y34F MnSOD showed that this decrease for the fluorinated enzyme was in significant part due to 3-fluorotyrosine residues distant (> 7 angstrom) from the active-site metal, not to 3-fluorotyrosine at position 34 close (similar to 5 angstrom) to the metal. Although many rate constants for the catalysis are decreased by this fluorination, the rate of dissociation of the product-inhibited complex appears unchanged by the presence of fluorinated tyrosines. These results suggest that Tyr34 is not a proton donor in the release of the product-inhibited complex, which involves protonation of a peroxo complex of the metal with release of hydrogen peroxide. (c) 2006 Elsevier B.V. All rights reserved.