| Abstract
| - Manganese superoxide dismutase (MnSOD) cycles between the Mn(II) and Mn(III) statesduring the catalyzed disproportionation of O2•-, a catalysis that is limited at micromolar levels of superoxideby a peroxide-inhibited complex with the metal. We have investigated the role in catalysis and inhibitionof the conserved residue Trp161 which forms a hydrophobic side of the active site cavity of MnSOD.Crystal structures of mutants of human MnSOD in which Trp161 was replaced with Ala or Phe showedsignificant conformational changes on adjacent residues near the active site, particularly Gln143 and Tyr34which in wild-type MnSOD participate in a hydrogen bond network believed to support proton transferduring catalysis. Using pulse radiolysis and observing the UV absorbance of superoxide, we have determinedrate constants for the catalytic dismutation of superoxide. In addition, the rates of formation and dissociationof the product-inhibited complex of these mutants were determined by direct observation of the characteristicvisible absorption of the oxidized and inhibited states. Catalysis by W161A and W161F MnSOD wasassociated with a decrease of at least 100-fold in the catalytic rate of reduction of superoxide, which thenpromotes a competing pathway leading to product inhibition. The structural changes caused by the mutationsat position 161 led to small changes, at most a 6-fold decrease, in the rate constant for formation of theinhibited complex. Solvent hydrogen isotope effects support a mechanism in which formation of thiscomplex, presumably the peroxide dianion bound to the manganese, involves no rate-contributing protontransfer; however, the dissociation of the complex requires proton transfer to generate HO2- or H2O2.
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