First Advisor

David H. Peyton

Date of Publication


Document Type


Degree Name

Master of Science (M.S.) in Chemistry






Hemoproteins, Myoglobin, Nuclear magnetic resonance spectroscopy



Physical Description

1 online resource (2, ix, 73 p.)


NMR studies of paramagnetic hemoproteins have improved significantly our understanding of the structure-function relationship of hemoproteins in general. Up to date most of the studies focus on low-spin ferric systems which are characterized by relatively narrow resonance peaks and concomitant better resolution. However, characterizing in detail the NMR spectra of high-spin ferric hemoproteins is important since there are several hemoproteins, such as peroxidases, catalases, oxygenases, and some ferricytochromes that contain high-spin iron (III) in their biologically active forms. Yet assigning resonances from heme peripheral protons and/or heme pocket residues in high-spin myoglobins is a daunting undertaking. Only a sparse number of active site residues are assigned in such instances, even for metaquo-myoglobin. The protons from the heme and heme pocket residues in high-spin complexes experience extremely fast relaxation and very broad linewidths, which impede the 2D methods that detect through-space and through-bond connectivities. It is the intention of this study to develop an effective strategy to gain more resonance assignments for fast-relaxing protons in hemoproteins. We have set out to use a combined strategy, using two-dimensional exchange spectroscopy (2D-EXSY) with two dimensional nuclear Overhauser effect spectroscopy I correlation spectroscopy I total correlation spectroscopy (NOESY/COSY/TOCSY). I demonstrate here that 2D EXSY experiments can be used to obtain assignment correlations for the heme protons of methydroxy-, metthiocyano-, metaquo-, and metimidazole-myoglobin forms. All these assignments are unambiguous and straightforward. Moreover, saturation-transfer experiments allow determination of ligand binding kinetics. Thus, the exchange rates between the metaquo- and metimidazole- or methyl substituted imidazole myoglobin complexes are estimated. The differences between the exchange rates reflect the differences in the hydrophobic and steric interactions between the ligands and the protein moiety. Although I only demonstrate the feasibility of2D EXSY for the myoglobin case, this assignment strategy should to be applicable to other hemoprotein systems.


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