Characterization of the product-inhibited complex in catalysis by human manganese superoxide dismutase.

The reduction with excess H(2)O(2) of human Mn(III) superoxide dismutase (SOD) and the active-site mutant Y34F Mn(III)SOD was measured by scanning stopped-flow spectrophotometry and revealed the presence of an intermediate in the reduction of the manganese. The visible absorption spectrum of this intermediate closely resembled that of the enzyme in the inhibited, zero-order phase of the catalyzed disproportionation of superoxide. The decay of the visible spectrum of this intermediate was 2-fold faster for the wild-type compared with the mutant Y34F Mn-SOD. This correlates with the enhanced product inhibition of Y34F during the catalysis of O-(2) dismutation. The visible spectrum of the product-inhibited complex resembles that of the azide-Mn-SOD complex, suggesting that the inhibited complex has expanded geometry about the metal to octahedral. This study shows that the inhibited complex responsible for the zero-order phase in the catalysis by Mn-SOD of superoxide dismutation can be reached through both the forward (O-(2)) and reverse (H(2)O(2)) reactions, supporting a mechanism in which the zero-order phase results from product inhibition.