This proposal is designed to use the unique advantages of NMR spectroscopy for paramagnetic molecules, in conjunction with other complementary techniques, to examine hemes, skeletally modified hemes (verdoheme, meso-hydroxylated heme, isoporphyrins), heme enzymes (peroxidases, heme oxygenase), and reactive chemical models for these enzymes. 1D and 2D NMR studies offer the opportunity to examine geometric and electronic structure of the heme and its immediate environment (axial ligation, protein pocket) in intact, functioning enzymes and in reacting model systems to investigate critical factors that are responsible for the binding and activation of substrates, especially small molecules (dioxygen, peroxides), and in the case of heme oxygenase (HO), heme itself. One portion of the project will focus on NMR strategies for the use of 1D and 2D methods to enlarge the experimental capabilities and quantify the interpretive bases of hyperfine shifts. For proteins these will examine the second layer of protein residues near the heme (now that the first layer can be studied), evaluate methodologies for proteins larger than horseradish peroxidase (HRP), extract information from cross correlation peaks in COSY maps, and extract structural information from high-spin as well as low-spin states of heme enzymes. For models these techniques will focus on understanding the electronic effects of heme modification by introduction of functionalities on the heme periphery as occurs during heme degradation that accompanies heme enzyme operation. The methodological advances are expected to contribute not only to effective pursuit of the our goals, but to contribute significantly to the NMR investigation of a large variety of other paramagnetic metalloproteins. Studies on peroxidases will focus on horseradish peroxidase where the geometry of the active site, the substrate binding sites relevant to peroxidase vs oxygenase activity, as well as the effects of selective mutations on each of these, will be examined. Studies on HO will focus on establishing the identity, sequence origin and geometry of the components of the active site of the heme (substrate)/HO complex, with the goal of eventually understanding factors that account for the a- specificity of meso-site attack that occurs during heme catabolism. This process initiates bile pigment (especially bilirubin) and carbon monoxide (a putative neurotransmitter) formation. The complex series of events that result in heme catabolism will be elucidated at the molecular level through studies of fundamental, insoluble iron compounds that are likely intermediates in the process and through their structural characterization, examination of reactive models (coupled oxidation of heme with 02) for this process, and attempts to trap or prepare reactive intermediates with intact heme oxygenase itself. Throughout this work the goal will be to define the oxidation/ligation/spin profanation state of the heme or modified heme as it passes step-wise through the intricate process of heme catabolism in order to understand the role of the enzyme in guiding and hastening this process.
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