Date of Award

August 2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

A. Andrew Pacheco

Committee Members

Graham Moran, Dennis Bennett, Marius Schmidt, Nick Silvaggi

Keywords

Dioxygenation, Myoglobin, Nitric Oxide, Photolytic, Tuberculosis

Abstract

The previously reported NO precursor [Mn(PaPy2Q)(NO)]ClO4 (1), where (PaPy2QH) is N,N-bis(2-pyridylmethyl)-amine-N-ethyl-2-quinoline-2-carboxamide, was synthesized and proven capable of producing as much as 180 µM NO when irradiated by a single 3 mJ 500nm laser pulse, in a 0.15 cm path cell, without the need for additional sacrificial reductants or oxidants. Species 1 was first used to study the reaction of nitric oxide with oxy-myoglobin (oxyMb) to form ferric myoglobin (metMb) and nitrate. This reaction had long been assumed to proceed via the same iron-bound peroxynitrite intermediate (metMb(OONO)) as the metMb-catalyzed isomerization of peroxynitrite to nitrate. Recent research showed that the metMb-catalyzed isomerization of peroxynitrite to nitrate produces detectable amounts of nitrogen dioxide and ferryl myoglobin (ferrylMb). This suggested a “caged NO2” mechanism for peroxynitrite isomerization. The presence of free NO2 and ferrylMb products revealed that small amounts of NO2 escape from myoglobin’s interior before recombination can occur, and these should also be generated in the reaction of oxyMb with NO, if the common intermediate metMb(OONO) is formed. However, in time resolved UV/Vis spectroscopy experiments reported herein no ferrylMb was detected when oxyMb and NO reacted. The sensitivity of the methodology is such that as little as 10% of the ferrylMb predicted from the experiments with metMb and peroxynitrite should have been detectable. These results lead to the conclusion that the oxyMb + NO and metMb + ONOO− reactions do not proceed via a common intermediate as previously thought. The conclusion has significant implications for researchers that propose a possible role of oxyMb in intracellular NO regulation, because toxic NO2 and ferrylMb are not generated during NO oxidation by this species.

Nitric oxide precursor 1 was then used for investigating the interaction between NO and the protein truncated hemoglobin N (trHbN) from the pathogen Mycobacterium tuberculosis. Oxy-trHbN is exceptionally efficient at converting NO to nitrate, with a reported rate constant of 7.45108 M1 s1 compared to 4107 M1 s1 for oxyMb. This work analyzed the NO dioxygenation kinetics of wild type trHbN and a set of variants, as well as the nitrosylation kinetics for the reduced (red-trHbN) forms of these proteins. The NO dioxygenation reaction was remarkably insensitive to mutations, even within the active site, while nitrosylation was somewhat more sensitive. Curiously, the most profound change to the rate constant for nitrosylation was effected by deletion of a 12 amino acid N-terminal sequence. The deletion mutant exhibited first-order kinetics with respect to NO, but was zero-order with respect to protein concentration; by contrast all other variants exhibited second-order rate constants greater than 108 M1s1. TrHbN boasts an extensive tunnel system that connects the protein exterior with the active site, and is likely the main contributor to the protein’s impressive NO dioxygenation efficiency. The results herein suggest that N-terminal deletion abolishes a large-scale conformational motion, in the absence of which NO can still readily enter the tunnel system, but is prevented from binding to the heme for an extended period of time.

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