PURIFICATION AND CHARACTERIZATION OF A 1,4-BENZOQUINONE REDUCTASE FROM THE BASIDIOMYCETE PHANEROCHAETE-CHRYSOSPORIUM

An intracellular, soluble 1,4-benzoquinone reductase was purified from agitated cultures of Phanerochaete chrysosporium and characterized. The quinone reductase was expressed in cultures grown under both nitrogen-sufficient and nitrogen-limiting (12 and 1.2 mM ammonium tartrate) conditions. The protein was purified to homogeneity by using ammonium sulfate fractionation, hydrophobic interaction, and ion exchange and blue-agarose affinity chromatographies. The native flavin mononucleotide-containing protein, pI 4.3, has a molecular mass of 44 kDa as determined by gel filtration. The protein has a subunit molecular mass of similar to 22 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The quinone reductase exhibits a broad pH optimum between 5.0 and 6.5 and a temperature optimum of 30 degrees C. The enzyme catalyzes the two-electron reduction of several quinones and other electron accepters utilizing either NADH or NADPH as an electron donor. The apparent K-m for 2-methoxy-1,4-benzoquinone is 2,4 mu M, and the apparent k(cat) is 4.4 x 10(5) s(-1). Enzyme activity is strongly inhibited by Cibacron blue 3GA and by dicumarol.