Recombinant Toluene-4-monooxygenase: Catalytic and Mo ¨ssbauer Studies of the Purified Diiron and Rieske Components of a Four-Protein Complex ² Jeremie D. Pikus, ‡ Joey M. Studts, ‡ Catalina Achim, § Karl E. Kauffmann, § Eckard Mu ¨nck, § Robert J. Steffan, | Kevin McClay, | and Brian G. Fox* ,‡ The Institute for Enzyme Research, Graduate School and Department of Biochemistry, College of Agricultural and Life Sciences, UniVersity of Wisconsin, Madison, Wisconsin 53705, Department of Chemistry, Carnegie Mellon UniVersity, Pittsburgh, PennsylVania 15213, and EnVirogen, LawrenceVille, New Jersey 08648 ReceiVed February 26, 1996; ReVised Manuscript ReceiVed May 9, 1996 X ABSTRACT: Expression of the tmoA-F gene cluster from Pseudomonas mendocina KR1 in Escherichia coli BL21(DE3) produces a catalytically active form of the toluene-4-monooxygenase (T4MO) complex. Here we report the purification and characterization of four soluble proteins required for the in Vitro reconstitution of T4MO catalytic activity. These proteins are a diiron hydroxylase (T4MOH), a Rieske- type ferredoxin (T4MOC), an effector protein (T4MOD), and an NADH oxidoreductase (T4MOF). The T4MOH component is composed of the tmoA, tmoB, and tmoE gene products [quaternary structure (Rǫ) 2 , M r ≈ 220 kDa]. The T4MOA polypeptide contains two copies of the amino acid sequence motif (D/ E)X (28-37) DEXRH; the same motif provides all of the protein-derived ligands to the diiron centers of ribonucleotide reductase, the soluble methane monooxygenase, and the stearoyl-ACP Δ 9 desaturase. Mo ¨ssbauer, optical, and EPR measurements show that the T4MOH contains diiron centers and suggest that the diiron center contains hydroxo bridge(s) in the diferric state, as observed for methane monooxygenase. Mo ¨ssbauer and EPR measurements also show that the T4MOC contains a Rieske-type iron-sulfur center. This assignment is in accord with the presence of the amino acid sequence motif CPHX (15-17) CX 2 H, which has also been found in the bacterial, chloroplastic, and mitochondrial Rieske proteins as well as the bacterial NADH-dependent cis-dihydrodiol-forming aromatic dioxygenases. While single-turnover catalytic studies confirm the function of the T4MOH as the hydroxylase, the NADH- dependent multiple-turnover hydroxylation activity is increased by more than 100-fold in the presence of the T4MOC, which mediates highly specific electron transfer between the T4MOF and the T4MOH. The T4MOD can be purified as an 11.6 kDa monomeric protein devoid of cofactors or redox-active metal ions; this component is also detected as a substoichiometric consitutent of the purified T4MOH. The rate of the hydroxylation reaction can be mildly stimulated by the further addition of separately purified T4MOD to the T4MOH, implying the formation of a high affinity, catalytically competent complex between these two components. These characterizations define a novel, four-component oxygenase combining elements from the soluble methane oxidation complex of the methanotrophic bacteria and the aromatic hydroxylation complexes of the soil pseudomonads. The aerobic, bacterial oxidation of toluene is catalyzed by five distinct multicomponent complexes, whose products are shown in Figure 1. The presently available biochemical and structural properties indicate that these complexes contain three types of structurally distinct active sites. One of these complexes, the toluene dioxygenase from Pseudomonas putida F1 (TDO, 1 Figure 1A), catalyzes the NADH- and O 2 - dependent dioxygenation of toluene, forming a cis-dihy- drodiol (Gibson et al., 1970). The active site of TDO contains both a Rieske-type [2Fe-2S] center and a mono- nuclear iron center (Mason & Cammack, 1992). A second, structurally less well defined active site used for the oxidation of toluene is found in xylene monooxy- genase (Suzuki et al., 1991), which catalyzes the monooxy- genation of the methyl substituent of toluene and xylenes (Figure 1B). Xylene monooxygenase is a member of a proposed family of iron-containing integral membrane proteins (Shanklin et al., 1994) that includes the alkane hydroxylase from Pseudomonas oleoVorans and the eukary- otic fatty acid desaturases. All members of this family ² This work was supported by grants from the Institute for Enzyme Research, Graduate School, and the Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, the Petroleum Research Foundation (ACS-PRF 28405-G4), the NIH (GM-50853 to B.G.F. and GM-22701 to E.M.), and the NSF Small Business Innovation Research Program (DMI-9460076 to R.J.S.). B.G.F. is a Searle Scholar of the Chicago Community Trust (1994- 1997) and a Shaw Scientist of the Milwaukee Foundation (1994-1999). * Author to whom correspondence should be addressed. ‡ The Institute for Enzyme Research. § Carnegie Mellon University. | Envirogen. X Abstract published in AdVance ACS Abstracts, June 15, 1996. 1 Abbreviations: MOPS, 3-(N-morpholino)propanesulfonic acid; MMO, methane monooxygenase; T2MO, toluene-2-monooxygenase; T3MO; toluene-3-monooxygenase; T4MO, toluene-4-monooxygenase; T4MOH, the hydroxylase component of the T4MO; T4MOA, 55 kDa subunit of the T4MOH, the product of the tmoA gene; T4MOB, 9.6 kDa subunit of the T4MOH, the product of the tmoB gene; T4MOC, 12.5 kDa Rieske-type ferredoxin, the product of the tmoC gene; T4MOD, 11.6 kDa product of the tmoD gene; T4MOE, 35 kDa subunit of the T4MOH, the product of the tmoE gene; T4MOF, reductase component of T4MO, the product of the tmoF gene; TDO, toluene dioxygenase. 9106 Biochemistry 1996, 35, 9106-9119 S0006-2960(96)00456-4 CCC: $12.00 © 1996 American Chemical Society