Phosphate is an inhibitor of copper, zinc superoxide dismutase

Abstract

Bovine etyrhrocyte Cu, Zn superoxide dismutase (SOD) in its oxidized form has been shown by X-ray crystallography [1] to be a dimer of two equivalent subunits, with one Cu(II) and one Zn(II) ion per subunit. The Cu ion is bound to four histidyl imidazole ligands and a water molecule making the overall geometry five-coordinate. One of these imidazoles is deprotonated and acts as a bridging ligand between Cu and Zn. The remaining ligands to Zn are two additional histidyl imidazoles and aspartyl carboxylate, forming a distorted tetrahedral geometry. Cu, Zn SOD's are found to be extremely efficient catalysts of the disproportionation of superoxide (2O 2 + 2H + → O 2 + H 2O 2) has been proposed that this activity is their primary biological function in vivo [2]. A number of anions have been observed to bind to Cu, Zn SOD e.g. CN −, N in2 3 , SC in2N , OC N , and halides. Each of these anions binds to copper(II) in the enzyme, as indicated by the pronounced spectral shifts observed upon binding [3]. Inhibitory effects of these anions have been attributed to this binding [3]. There have been several observations reported in the literature that suggest that phosphate also interacts with Cu, Zn SOD. For example, it has been mentioned in passing that phosphate interferes with the binding of C N to the enzyme [4] and that it has inhibitory effect on the SOD activity [5]. In addition, it has been reported that phosphate influences the mode of binding of cobalt to the apoprotein [6] and that it interacts with the four-copper derivative (where Cu has been substituted for Zn in the native protein) [7]. We have no visible or ESR spectral changes in solutions of Cu, Zn SOD at high concentrations of phosphate, suggesting strongly that any interaction of phosphate with the enzyme does not occur by binding of that anion to Cu(II). Contrary to the reports of McAdam [8] and Cudd and Fridovich [9], who assumed that the inhibitory effect of phosphate on the SOD activity could be entirely attributed to ionic strength effects, we found that the SOD activity of native bovine Cu, Zn SOD measured at constant ionic strength decreased significantly in the presence of increasing concentrations of phosphate. In addition, we have titrated the native enzyme with NaN 3 in differing concentrations of phosphate at constant ionic strength and thereby demonstrated that the presence of phosphate decreased the affinity of the enzyme for the azide anion. This result is reminiscent of the decrease in the binding affinity of this protein when arginine-141 is chemically modified with phenylglyoxal [10]. Arginine-141 may well be the site phosphate binding to this protein, since the binding of phosphate to the arginine-modified protein is apparently not affected by the presence of phosphate. Spectroscopic evidence concerning the nature of the interaction between phosphate and Cu, Zn SOD has also been obtained.