INVESTIGATIONS INTO KINETICS OF ENZYMES VIA NMR

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Toluene 4-monooxygenase (T4MO) catalyzes NADH- and O2-dependent conversion of toluene to p-cresol, and can also catalyze the adventitious oxidation of numerous other hydrocarbons such as indole, naphthalene, chloroform, and trichloroethylene. This four-protein complex consists of an NADH oxidoreductase, a catalytic effector protein, a diiron center-containing hydroxylase, and a small Mr soluble ferredoxin (T4MOC, 12,326Da). T4MOC is a monomeric protein which contains a Rieske-type iron sulfur center. Catalytic reconstitution studies have shown that T4MOC is obligately required for electron transfer between the oxidoreductase and diiron hydroxylase components. We will conduct research to determine 1D, 2D and 3D structures of T4MOC by using multinuclear high field NMR and isotopically labeled protein samples. These studies will provide electronic and dynamic information about correlations between redox states, proton exchange, and spin delocalization that may contribute to electron transfer reactions between Riseke and diiron centers. Moreover, by using the completely soluable T4MO enzyme system, we will study changes in T4MOC redox and protonation states, coordination geometry, and electron delocalization upon complex formation with T4MOH, in catalytically relevant electron transfer complexes.