Toluene 4-monooxygenase (T4MO) is a member of the bacterial multicomponent monooxygenases, an enzyme family that utilizes a soluble diiron hydroxylase to oxidize a variety of hydrocarbons as the initial step in their metabolism. The hydroxylases obtain reducing equivalents from NAD(P)H via an electron transfer chain that is initiated by an oxidoreductase containing an N-terminal ferredoxin domain and C-terminal flavin- and NAD-binding domains. T4moF, the NADH oxidoreductase of T4MO, was expressed as a soluble protein in Escherichia coli BL21 (DE3) from the pUC-derived expression vector pRS205. This vector contains a lac promoter instead of a T7 promoter. A three step purification from the soluble cell lysate yielded similar to 1 mg of T4moF per gram of wet cell paste with greater than 90% purity. The purified protein contained I mol of FAD and 2 mol of Fe per mol of T4moF; quantitative EPR spectroscopy showed similar to 1 mol of the S = 1/2 signal from the reduced [2Fe-2S] cluster per mol of T4moF. Steady state kinetic analysis of p-cresol formation activity treating T4moF as the variable substrate while all other proteins and substrates were held constant gave apparent KM- and apparent k(cat)-values of 0.15 mu M and 3.0 s(-1), respectively. This expression system and purification allows for the recovery of the soluble oxidoreductase in yields that facilitate further biochemical and structural characterizations. (c) 2007 Elsevier Inc. All rights reserved.