Contribution of Human Manganese Superoxide Dismutase Tyrosine 34 to Structure and Catalysis

Abstract

Superoxide dismutase (SOD) enzymes are critical in controlling levels of reactive oxygen species (ROS) that are linked to aging, cancer and neurodegenerative disease. Superoxide (O 2 •− ) produced during respiration is removed by the product of the SOD2 gene, the homotetrameric manganese superoxide dismutase (MnSOD). Here, we examine the structural and catalytic roles of the highly conserved active-site residue Tyr34, based upon structure-function studies of MnSOD enzymes with mutations at this site. Substitution of Tyr34 with five different amino acids retained the active site protein structure and assembly, but causes a substantial decrease in the catalytic rate constant for the reduction of superoxide. The rate constant for formation of product inhibition complex also decreases but to a much lesser extent, resulting in a net increase in the product inhibition form of the mutant enzymes. Comparisons of crystal structures and catalytic rates also suggest that one mutation, Y34V, interrupts the hydrogen-bonded network, which is associated with a rapid dissociation of the productinhibited complex. Notably, with three of the Tyr34 mutants we also observe an intermediate in catalysis, which has not been reported previously. Thus, these mutants establish a means to trap a catalytic intermediate that promises to help elucidate the mechanism of catalysis.The superoxide dismutases are found in prokaryotes, archaea and eukaryotes, where they catalyze the disproportionation of the superoxide radical anion O 2•− in cellular processes detoxifying reactive oxygen species (1). The mechanism by which all superoxide dismutases carry out the catalytic removal of superoxide involves an oxidation-reduction cycle, as shown in the reactions below.In humans three SOD enzymes exist, SOD1 is a cytoplasmic Cu,ZnSOD, SOD2 is a mitochondrial MnSOD and SOD3 is an extracellular Cu,ZnSOD. These SOD enzymes are Corresponding authors: David Silverman, Phone: 352-392-3556; Fax: 352-392-9696; e-mail: silvrmn@ufl.edu Jeff Perry, Phone: 858-784-2284; Fax 858-784-2277; email jjperry@scripps.edu. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 April 21. critical in controlling levels of reactive oxygen species (ROS) that are linked to aging, cancer and neurodegenerative disease (2). Structure-activity relationships for this class of enzymes need to be understood and distinguished, as their disease phenotypes likely occur through different mechanisms. SOD1 is a Cu,Zn dimeric enzyme with a β-barrel fold (3) with an active site channel containing a site for superoxide substrate and hydrogen peroxide product binding (4,5) adjacent to the activity important Arg143 side chain (6). The structure of human Cu,ZnSOD resembles that of bacterial Cu,ZnSOD except the dimer contact is on the opposite surface (7,8). For the human Cu,ZnSOD, dimer and framework-destabilizing mutations in SOD1 give rise to amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease (9-11), a progressive neurological dis...

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“…The resulting spectrum of Int 1 , which has an absorption maximum at 415 nm (Fig. 3B), is identical to those reported for Mn-peroxo complexes (8,10,14). Int 2 is an intermediate with broad absorption in the visible range and a maximum at 425 nm (Fig.…”

“…3A, left). Similar biphasic increases at 425 nm absorption have also been observed for several Y34 mutant human MnSODs (8). The 425 nm absorption of Y34F ScMnSOD slowly decreased at a longer time scale (Fig.…”

“…Its presence at that location supports the importance of the hydrogen-bonding chain involving Tyr34 (Gln154⋯ Tyr34⋯H 2 O⋯His30) in the WT enzyme (9). This additional water molecule is also present in Y34X (X ¼ A, N, H, or V) mutant human MnSOD (8). However, it was absent in the crystal structure of human Y34F MnSOD, which had been carried out at a resolution similar to that of our study (1.9 Å) (7).…”

“…3B). The structure of this intermediate is unknown, but species with similar spectral features have been observed in similar studies of Y34A, Y34H, and Y34N human MnSOD (8).…”

“…Wild-type (WT) and Y34F mutant human MnSOD have similar k cat ∕K m , but k cat is decreased by approximately 10-fold in the mutant protein (7). Tyr34 also plays an interesting role in the "prompt protonation" pathway, as elucidated by the changes in the kinetics of Y34 mutant human MnSOD (8).…”