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Degree

Doctor of Philosophy, Ohio State University, Ohio State Biochemistry Program, 2004.

Abstract

The interaction between trimethylamine dehydrogenase (TMADH) and electron-transferring flavoprotein (ETF), both from bacterium Methylophilus methylotrophus W3A1, has been examined. Our results indicate that the absorbance change previously attributed to changes in the environment of the FAD of ETF upon binding to TMADH is instead due to electron transfer from partially reduced as-isolated TMADH to ETF. Further, when the semiquinone form of ETF is used instead of the oxidized form, the mirror-image of the spectral change seen with as-isolated TMADH and oxidized ETF is observed, attributable to a small amount of electron transfer in the reverse of the physiological direction. Kinetic determination of the dissociation constant and limiting rate constant for electron transfer within the complex of (reduced) TMADH with (oxidized) ETF is reconfirmed and discussed in the context of a recently proposed model involving “structural imprinting” of ETF. The reaction of TMADH with two homologous series of substituted substrates: N,N-dimethylanilines and benzylamines is studied by rapid-reaction kinetics. The four possible mechanisms- direct nucleophilic attack, one-electron oxidation, proton abstraction and hydrogen atom abstraction- previously proposed for the oxidative demethylation of flavoenzymes are thus evaluated. Hammett plots of log (klim/Kd) versus óI do not yield linear correlations for either series of substrates, indicating the most likely mechanism is one involving hydrogen atom abstraction. A linear correlation is observed between the free energy and Charton’s size values in the case of the benzylamine series, reflecting a significant spatial effect for these substrates. The pH-dependence of flavin reduction of TMADH has been examined by using two forms of enzymes (wild-type TMADH and an H172Q mutant) as well as two forms of substrate (protiated and perdeuterated). Careful comparisons indicate that the substrate binds as the protonated cation rather than the neutral form. The observation that there is only one kinetically influential ionization for the profile of the reaction-limiting rate (klim) against pH re-confirms our previous result and contrasts with a recent report in which two ionizations were observed. This single ionization within the enzyme-substrate complex is attributed to neither substrate nor the residue His-172. No obvious kinetically influential ionization is due to this residue His-172 and it appears not to function as a general base in the reaction.

Keywords

trimethylamine dehydrogenase electron transfer flavin reduction imprinted mechanism kinetics ETF

Advisor

Russ Hille

Document number: osu1085107954
Permalink: http://rave.ohiolink.edu/etdc/view?acc_num=osu1085107954

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