Kinetic analysis of product inhibition in human manganese superoxide dismutase

Manganese superoxide dismutase (MnSOD) cycles between the Mn(ll) and Mn(III ) states during the catalyzed disproportionation Of O-2(.-), a catalysis th at is limited at micromolar levels of superoxide by a peroxide-inhibited co mplex with the metal. We have investigated the role in catalysis and inhibi tion of the conserved residue Trp161 which forms a hydrophobic side of the active site cavity of MnSOD. Crystal structures of mutants of human MnSOD i n which Trp161 was replaced with Ala or Phe showed significant conformation al changes on adjacent residues near the active site, particularly Gln143 a nd Tyr34 which in wild-type MnSOD participate in a hydrogen bond network be lieved to support proton transfer during catalysis. Using pulse radiolysis and observing the UV absorbance of superoxide, we have determined rate cons tants for the catalytic dismutation of superoxide. In addition, the rates o f formation and dissociation of the product-inhibited complex of these muta nts were determined by direct observation of the characteristic visible abs orption of the oxidized and inhibited states. Catalysis by W161A and W161F MnSOD was associated with a decrease of at least 100-fold in the catalytic rate of reduction of superoxide, which then promotes a competing pathway le ading to product inhibition. The structural changes caused by the mutations at position 161 led to small changes, at most a 6-fold decrease, in the ra te constant for formation of the inhibited complex. Solvent hydrogen isotop e effects support a mechanism in which formation of this complex, presumabl y the peroxide dianion bound to the manganese, involves no rate-contributin g proton transfer; however, the dissociation of the complex requires proton transfer to generate HO2- or H2O2.