The reactivity of ascorbate with different redox states of leghaemoglobin

Ascorbate has been previously shown to reduce the short-lived and reactive ferryl [Fe(IV)= O] states of myoglobin and haemoglobin. In this study it is shown that ascorbate is also able to reduce the otherwise long-lived and stable ferryl species [Lb(IV)] formed by the reaction of ferric or ferrous soybean leghaemoglobin with H2O2. The conversion of the ferryl species to ferric Lb, which appears to be moderately fast, is followed by a slow conversion of ferric Lb to oxyLb, by way of deoxy ferrous Lb. No reaction between ascorbate and oxyLb has been observed, in contrast to the previously reported pro-oxidant effect of ascorbate on oxymyoglobin. The addition of ascorbate prior to H2O2 to ferric Lb also prevents the formation of an additional species (which is not observed with myoglobin or haemoglobin), the optical spectra and migration on isoelectric focusing gels of this compound differ markedly from that of Lb(IV). When added after H2O2, ascorbate is able to reduce slowly this additional compound to oxyLb. These reactions are likely to occur in vivo as high ascorbate concentrations have been detected in soybean nodules. The reduction of Lb(IV) to ferric Lb is accompanied by oxidation of ascorbate, which has been detected by loss of the parent molecule in optical experiments and by the direct detection of ascorbyl radicals by electron paramagnetic resonance (EPR) spectroscopy. Analysis of the kinetics and stoichiometry of ascorbate oxidation suggests that the Lb(IV) species, the globin-derived radical(s) (which are situated on different residues and are in markedly different environments to those present in myoglobin and haemoglobin), the additional species and Lb(III) all appear to be involved in the formation of the ascorbyl radical. All of these processes are protective and may contribute to the maintenance of Lb in its functional ferrous form.