Recombinant Toluene-4-monooxygenase: Catalytic and Mössbauer Studies of the Purified Diiron and Rieske Components of a Four-Protein Complex†

摘要: Expression of the tmoA-F gene cluster from Pseudomonas mendocina KRI in Escherichia coli BL21(DE3) produces a catalytically active form toluene-4-monooxygenase (T4MO) complex. Here we report purification and characterization four soluble proteins required for vitro reconstitution T4MO catalytic activity. These are diiron hydroxylase (T4MOH), Riesketype ferredoxin (T4MOC), an effector protein (T4MOD), NADH oxidoreductase (T4MOF). The T4MOH component is composed tmoA, tmoB, tmoE products [quaternary structure (alpha beta epsilon)2, Mr approximately 220 kDa]. T4MOA polypeptide contains two copies amino acid sequence motif (D/E)X(28-37)DEXRH; same provides all protein-derived ligands to centers ribonucleotide reductase, methane monooxygenase, stearoyl-ACP delta 9 desaturase. Mossbauer, optical, EPR measurements show that suggest center hydroxo bridge(s) diferric state, as observed monooxygenase. Mossbauer also T4MOC Rieske-type iron-sulfur center. This assignment accord with presence CPHX(15-17)CX2H, which has been found bacterial, chloroplastic, mitochondrial Rieske well bacterial NADH-dependent cis-dihydrodiol-forming aromatic dioxygenases. While single-turnover studies confirm function hydroxylase, multiple-turnover hydroxylation activity increased by more than 100-fold T4MOC, mediates highly specific electron transfer between T4MOF T4MOH. T4MOD can be purified 11.6 kDa monomeric devoid cofactors or redox-active metal ions; this detected substoichiometric consitutent rate reaction mildly stimulated further addition separately T4MOH, implying formation high affinity, competent complex these components. characterizations define novel, four-component oxygenase combining elements oxidation methanotrophic bacteria complexes soil pseudomonads.