An Alternative Mechanism of Bicarbonate-mediated Peroxidation by Copper-Zinc Superoxide Dismutase

Abstract

Hydrogen peroxide can interact with the active site of copper-zinc superoxide dismutase (SOD1) to generate a powerful oxidant. This oxidant can either damage amino acid residues at the active site, inactivating the enzyme (the self-oxidative pathway), or oxidize substrates exogenous to the active site, preventing inactivation (the external oxidative pathway). It is well-established that the presence of bicarbonate anion dramatically enhances the rate of oxidation of exogenous substrates. Here, we show that bicarbonate also substantially enhances the rate of self-inactivation of human wild type SOD1. Together, these observations suggest that the strong oxidant formed by hydrogen peroxide and SOD1 in the presence of bicarbonate arises from a pathway that is mechanistically distinct from that producing the oxidant in its absence. Self-inactivation rates are further enhanced in a mutant SOD1 protein (L38V) linked to the fatal neurodegenerative disorder familial amyotrophic lateral sclerosis (FALS). The 1.4 resolution crystal structure of pathogenic SOD1 mutant D125H reveals the mode of oxyanion binding in the active site channel and implies that phosphate anion attenuates the bicarbonate effect by competing for binding to this site. The orientation of the enzyme-associated oxyanion suggests that both the self- and external oxidative pathways can proceed through an enzyme-associated peroxycarbonate intermediate. As the concentration of bicarbonate in biological fluids is significant (~25 mM), the outcomes of peroxycarbonate-mediated activities by SOD1 have implications for understanding oxidative cellular damage and FALS etiology.