RECOMBINANT TOLUENE-4-MONOOXYGENASE - CATALYTIC AND MOSSBAUER STUDIESOF THE PURIFIED DIIRON AND RIESKE COMPONENTS OF A 4-PROTEIN COMPLEX

Expression of the tmoA-F gene cluster from Pseudomonas mendocina KR1 i n Escherichia coli BL21(DE3) produces a catalytically active form of t he toluene-4-monooxygenase (T4MO) complex. Here we report the purifica tion 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 effe ctor protein (T4MOD), and an NADH oxidoreductase (T4MOF). The T4MOH co mponent is composed of the tmoA, tmoB, and tmoE gene products [quatern ary structure (alpha beta epsilon)(2), M(r) approximate to 220 kDa]. T he T4MOA polypeptide contains two copies of the amino acid sequence mo tif (D/E)X((28-37))DEXRH; the same motif provides all of the protein-d erived ligands to the diiron centers of ribonucleotide reductase, the soluble methane monooxygenase, and the stearoyl-ACP Delta(9) desaturas e. Mossbauer, optical, and EPR measurements show that the T4MOH contai ns diiron centers and suggest that the diiron center contains hydroxo bridge(s) in the diferric state, as observed for methane monooxygenase . Mossbauer and EPR measurements also show that the T4MOC contains a R ieske-type iron-sulfur center. This assignment is in accord with the p resence of the amino acid sequence motif CPHX((15-17))CX(2)H, which ha s also been found in the bacterial, chloroplastic, and mitochondrial R ieske proteins as well as the bacterial NADH-dependent cis-dihydrodiol -forming aromatic dioxygenases. While single-turnover catalytic studie s confirm the function of the T4MOH as the hydroxylase, the NADH-depen dent multiple-turnover hydroxylation activity is increased by more tha n 100-fold in the presence of the T4MOC, which mediates highly specifi c 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 substoichiome tric consitutent of the purified T4MOH. The rate of the hydroxylation reaction can be mildly stimulated by the further addition of separatel y purified T4MOD to the T4MOH, implying the formation of a high affini ty, catalytically competent complex between these two components. Thes e characterizations define a novel, four-component oxygenase combining elements from the soluble methane oxidation complex of the methanotro phic bacteria and the aromatic hydroxylation complexes of the soil pse udomonads.