Cofactor and substrate binding to vanadium chloroperoxidase determined by UV-VIS spectroscopy and evidence for high affinity for pervanadate

The vanadate cofactor in vanadium chloroperoxidase has been studied using W -VIS absorption spectroscopy, A band is present in the near-UV that is red- shifted as compared to free vanadate and shifts in both position and intens ity upon change in pH. Mutation of vanadate binding residues has a clear ef fect on the spectrum. Substrate-induced spectral effects allow direct measu rement of separate kinetics steps for the first time for vanadium haloperox idases. A peroxo intermediate is formed upon addition of H2O2, which causes a decrease in the absorption spectrum at 315 nm, as well as an increase at 384 nm. This peroxo form is very stable at pH 8.3, whereas it is less stab le at pH 5.0, which is the optimal pH for activity. Upon addition of halide s to the peroxo form. the native spectrum is re-formed as a result of halid e oxidation. Stopped-flow experiments show that H2O2 binding and Cl- oxidat ion occur on the millisecond to second time scale. These data suggest that the oxidation of Cl- to HOCl occurs in at least two steps. In the presence of H2O2, the affinity for the vanadate cofactor was found to be much higher than previously reported for vanadate in the absence of H2O2. This is attr ibuted to the uptake of pervanadate by the ape-enzyme. Human glucose-6-phos phatase, which is evolutionarily related to vanadium chlorsperoxidase, is a lso likely to have a higher affinity for pervanadate than vanadate. This co uld explain the enhanced insulin mimetic effect of pervanadate as compared to vanadate.