Spectral characterization and chemical modification of FMN-containing ascorbyl free-radical reductase from Pleurotus ostreatus.

Ascorbyl free-radical reductase was purified 1143-fold with an overall yield of 9.9% from the cytosolic fraction of Pleurotus ostreatus. The native enzyme had a molecular mass of 127 kDa and SDS/PAGE revealed that the enzyme consists of two subunits, each with a molecular mass of 62 kDa. The enzyme utilized only NADH as an electron donor. The enzyme was highly specific for ascorbyl free radical as an electron acceptor and capable of catalysing the reduction of ferricyanide and 2,6-dichloroindophenol as artificial electron acceptors. The apparent K(m) values of the enzyme towards NADH and ascorbyl free radical were 35+/-0.22 and 2.1+/-0.03 microM, respectively. The catalytic mechanism of this enzyme is of Ping Pong type. The enzyme contained FMN as a prosthetic group and showed the characteristic absorption spectrum ascribed to the charge-transfer interaction of thiolate anion with FMN. The enzyme contained eight cysteine residues per monomer and was inactivated more rapidly by mercurials than by thiol-alkylating reagents. Kinetic analysis of the inactivation process revealed that the enzyme had 1 mol of thiol group/mol of subunit in the active site with a pK(a) of 6.9. The modification of the thiol group of the enzyme caused the loss of charge-transfer absorbance centred at 540 nm and blocked the electron-transfer process from NADH to FMN. The modification of lysine, arginine and histidine residues led to the loss of its activity. Unlike the active enzyme, the fluorescence quenching of NADH was not observed in the lysine-modified enzyme, which implies that lysine residues can participate in the interaction with NADH.