Date Available


Year of Publication


Degree Name

Doctor of Philosophy (PhD)

Document Type



Arts and Sciences



First Advisor

Dr. Anne-Frances Miller


The enzyme nitroreductase (NR) catalyzes two-electron reduction of high explosives such as trinitrotoluene (TNT), a wide variety of other toxic nitroaromatic compounds, as well as riboflavin derivatives, using a tightly-bound flavin mononucleotide (FMN) cofactor. It has important environmental and clinical implications. Previous studies have focused on elucidating NRs catalytic mechanism and solving its crystal structure.

In this dissertation work, we first develop and implement new strategies for labeling NR with stable isotopes, and report a completely re-designed protocol for NRs purification. Then we report the successful assignment of over half of NRs backbone resonances using 3d-NMR methods. The most significant observation is that we find a well-resolved 2d 1H-15N HSQC NMR spectrum is obtained at 37°C for NR, while the HSQC at 4°C is poorly-dispersed and comprised of sharp overlapped peaks. Thus, it would appear that NR denatures at 4°C. However, as we demonstrate, the non-covalently-bound FMN cofactor is not released at the lower temperature, based on retention of the native flavin visible-CD spectrum. Similarly, far-UV CD spectroscopy shows that the protein retains full secondary structural content at 4C. In addition, near-UV CD and Fluorine-19 NMR experiments demonstrate that under completely native conditions (neutral pH, no additives) NR maintains a high degree of tertiary structure and well-defined hydrophobic side-chain packing, ruling out the possibility of a molten-globule state.

Thus, our studies report strong evidence that the dramatic low-temperature (low-T) transition near 20°C observed by NMR is not the result of protein structural changes, but rather, we propose that NR exists as an ensemble of rapidly inter-converting structures, at lower temperature, only adopting a well-defined unique structure above 20°C. Both saturation-transfer from water and solvent proton-exchange measurements support our proposed model in which the unique high-T structure is favored entropically, by release of water molecules; on the other hand, the fluxional low-T state incorporates greater water solvation at 4°C.

In the latter part of this study, we present preliminary data suggesting that the flexibility implied by easy water-access to the loosely-structured state plays a role in accommodating binding of diverse substrates. Therefore, the fluxional low-T state may be functionally important. A possible direct link between the enzyme dynamics and its catalytic activity will be an area of future investigation.

Included in

Chemistry Commons



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