Structure-Function Studies Of Human Hemoglobin With Organo-Nitrogen Oxide Compounds

Hemoglobin (Hb) is a crucial oxygen-transport metalloprotein found in the erythrocytes of all vertebrates and some invertebrates. A tetrameric Hb molecule consists of two alpha and two beta subunits, and each subunit contains a heme macrocycle for oxygen binding and transport in the body. It is well known that some organo-nitrogen oxide compounds may inhibit the Hb function via different metabolic mechanisms. In this study, we chose phenylhydroxylamine, trinitrotoluene (TNT) and n-butyl nitrite to represent three different types of biologically relevant nitrogen-containing species. We employed UV-vis spectroscopy and X-ray crystallography techniques to investigate the structure-function effects of Hb upon the reactions with those organo-nitrogen oxide compounds.In Chapter Two, we reported that phenylhydroxylamine oxidized oxyhemoglobin (oxyHb) to methemoglobin (metHb), and then induced the hemichrome formation in which both the proximal and the distal histidines were covalently bound with the heme iron in the (3 subunit. In order to generate this hemichrome geometry, the (3 heme had to shift towards the protein surface with a 5A slippage. In addition, the βCys93 was chemically modified to form S-nitrosothiol. In Chapter Three, we demonstrated that TNT could not only oxidize oxyHb under anaerobic conditions in the presence of sodium dithionite, but also induce the heme slippage to the protein surface of the β subunit, resulting in the conformational changes of the protein mainly in the β subunit. In Chapter Four, we confirmed that the n-butyl nitrite was able to react with Hb in the presence of sodium dithionite to form the Hb(NO) derivative which could be oxidized by oxygen easily.Our results demonstrated that different organo-nitrogen oxide compounds can react with Hb via different mechanisms, furthermore, inhibit the Hb function by oxidizing it to the ferric form which is no longer able to bind oxygen.