The chloroperoxidase from the fungus Curvularia inaequalis; a novel vanadium enzyme.

The presence of vanadium-containing bromoperoxidases in various types of seaweed is well-documented. We now report that the terrestrial fungus Curvularia inaequalis excretes a novel chloroperoxidase which also contains vanadium as a prosthetic group. The chloroperoxidase is excreted in the medium as the only protein and is, therefore, almost purely obtained. Atomic absorption spectroscopy measurements showed that the chloroperoxidase contained vanadium, which was essential for enzymatic activity, in a stoichiometry of 1 mol vanadium per mol of enzyme. When the fungus was grown in media containing low concentrations of vanadate (VO4(3-)) or when vanadate was absent, the enzyme was excreted in an apoform. Addition of vanadate to the apoenzyme purified from the medium, dialyzed holo-enzyme or growth medium led to incorporation of the metal and to a subsequent increase in specific activity from 0.7 to about 7.5 units/mg. The reduced enzyme showed an axially symmetric EPR spectrum (g(o) = 1.971, Ao = 91.7 x 10(-4) cm-1) with 16 hyperfine lines that is essentially the same as the EPR spectrum of the vanadium-containing bromoperoxidase of the seaweed Ascophyllum nodosum. This demonstrates that the active sites in the two enzymes are very similar. The chlorinating and brominating activities of the chloroperoxidase from C. inaequalis were also studied and compared to those of the vanadium bromoperoxidase from A. nodosum. The chlorinating reaction catalyzed by the chloroperoxidase had a pH optimum around 5.5 and the Km for Cl- was small (0.25 mM at pH 4.5), but the logarithm of its value increased linearly with increasing pH. At high bromide concentrations, the pH optima of chloroperoxidase and bromoperoxidase in the brominating reaction were about the same (5.5). However, at low bromide concentrations the pH optimum of the chloroperoxidase was at higher pH values than that of the bromoperoxidase.