Abstract
Toluene dioxygenase, from Pseudomonas putida , oxidizes toluene to (+)- cis -1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene. The oxygenase-component of this multienzyme system was purified to homogeneity by a two-step procedure that utilized affinity and ion exchange chromatography. The purified enzyme would oxidize toluene only in the presence of NADH, ferrous iron and partially purified preparations of NADH cytochrome c reductase and an iron-sulfur protein (ferredoxin TOL). Spinach NADPH cytochrome c reductase and NADPH could substitute for the Pseudomonas reductase and NADH. The molecular weight of the oxygenase-component was determined to be 151,000 and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the enzyme is composed of two subunits with molecular weights of 52,500 and 20,800. The absorption spectrum showed maxima at 550 (Shoulder), 450, 326 and 278 nm and preliminary experiments have indicated the presence of 2 gram atoms of iron and 2 gram atoms of acid-labile sulfur per mole of protein. The results indicate that the oxygenase-component of the toluene dioxygenase enzyme system is an iron-sulfur protein that has been designated ISP TOL.
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