Role of Tyrosine-34 in the Anion Binding Equilibria in Manganese(II) Superoxide Dismutases

Abstract

Superoxide dismutases (SODs) are proteins specialized in the depletion of superoxide from the cell through disproportionation of this anion into oxygen and hydrogen peroxide. We have used high-field electron paramagnetic resonance (HFEPR) to test a two-site binding model for the interaction of manganese-SODs with small anions. Because tyrosine-34 was thought to act as a gate between these two sites in this model, a tyrosine to phenylalanine mutant of the superoxide dismutase from R. capsulatus was constructed. Although the replacement slightly reduced activity, HFEPR measurements demonstrated that the electronic structure of the Mn(II) center was unaffected by the mutation. In contrast, the mutation had a profound effect on the interactions of fluoride and azide with the Mn(II) center. It was concluded that the absence of tyrosine-34 prevented the close approach of these anions to the metal ion. This mutation also enhanced the formation of a hexacoordinated water-Mn(II)SOD complex at low temperatures. Together, these results showed that the role of Y34 is unlikely to involve redox tuning but rather is important in regulating the equilibria between the anionic substrate in solution and the two binding sites near the metal. These observations further supported the originally proposed mutually exclusive two-binding-site model.

Peroxynitrite Mediates Active Site Tyrosine Nitration in Manganese Superoxide Dismutase. Evidence of a Role for the Carbonate Radical Anion

“…Accordingly, the observed azide inhibition of Tyr34 nitration cannot be attributed entirely to azide ligation to manganese. However, a secondary anion binding site near the MnSOD active site, involving Tyr34, has been described by Tabares et al,82-83 and a similar site for azide binding in the highly homologous FeSOD has been directly observed with NMR by Miller et al84 Significantly, the same azide-induced decrease in Tyr34 nitration selectivity was observed in the presence of CO 2 . These effects could be explained by azide interference with access to the active site by PN anion and carbonate radical anion.…”

Human SOD2 Modification by Dopamine Quinones Affects Enzymatic Activity by Promoting Its Aggregation: Possible Implications for Parkinson’s Disease

“…After reaction with DAQs, the HFEPR spectrum shows no relevant changes, indicating that in the DAQ-modified protein, the Mn(II) sites have the same structure as in the wt protein. Moreover, the resemblance is such that structural changes at the substrate access channel may be ruled out since it has been shown that mutations at this position, as well as the interaction with analogues of the substrate, have clear effects on the HFEPR spectrum [24], [25]. In addition, no evidence of spurious Mn(II) was observed in the HFEPR spectra.…”

The effect of gamma-ray irradiation on the Mn(ii) speciation in Deinococcus radiodurans and the potential role of Mn(ii)-orthophosphates

“…6). Its shape, which is a sensitive marker of the electronic structure, 44,45 remained unchanged. This demonstrated that the MnSOD active site and its environment were robust against radiation and the protein was likely to be active even after such a high dose.…”