The SIR provided D, L-[imidazole-""""""""N2]Histidine; lg Toluene-4-monooxygenase (T4MO) catalyzes the NADH- and 02-dependent oxidation of toluenen, indole, naphthalene, chloroform, trichlorethylene, an dother hydrocarbons. The enzyme complex consists of four components: a 12.5 kDa Rieske ferredoxin (T4MOC, the primary subject of this study) an 11.6 kDa regulatory protein (T4MOD); a 216 kDa diiron hydroxylase (T4MOH); and a 36kDa NADH oxidoreductase. We are studying T4MOC using multinuclear, high field NMR. Our plan is to make extensive use of methods developed for the study of other iron-sulfur proteins at the National Magnetic Resonance Facility at Madison. Our studies of T4MOC represent the first detailed NMR studies of a Rieske protein. As such, they will provide an experimental framework for evaluating and improving existing NMR methods, and will provide eletronic and dynamic information about correlations between redox states, proton exchange, and spin delocalization that may contribut to electron transfer between Rieske and dfiron centers. Two distinct areas will be addressed in this research effort: 1)properties of hyperfine-shifted resonances associated with the oxidized and reduced states of T4MOC; and 2) 2-D and 3-D NMR studies of the diamagnetic resonances arising from the protein backbones of T4MOC and T4MOD. The objective of this research is to understand the molecular properties of protein (T4MOD), and the diiron hydroxylase (T4MOH) that lead to the 100- to 300-fold enhancements in catalytic rate. The T4MO system offers a unique opportunity to study the energetics of electron transer between Rieske and diiron centers, and to study the protein-based control of oxygense catalysis by regulatory proteins such as T4MOD within a single experimental system. Thus, it is likely that significan new insights about thermodynamic and biological control of diiron oxygenase enzymes will be obtained from the proposed activity.
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