This proposal seeks support for a program of spectroscopic and mechanistic studies of heme iron enzymes. Three important goals will be pursued. First, a fundamental aspect of protein structure will be tested: the Fe-S bond is retained in all oxidation states of cytochrome P450 and chloroperoxidase but only in ferric states of thiolate-ligated myoglobin (Mb) and cytochrome c peroxidase (CCP) mutants. The factors that lead to loss of thiolate ligation may be the same as in the active sites of the heme proteins that naturally lose thiolate ligation upon reduction. Guided by molecular modeling, Mb and CCP double/triple thiolate-ligated mutants will be prepared with H-bond donor amino acids positioned to stabilize the thiolate ligand toward reduction and oxoferryl formation. Formation of thiolate-ligated oxoferryl adducts would provide simple models for these important states. Second, use of UV-visible/near-IR magnetic circular dichroism (MCD) spectroscopy will be extended for axial ligand identification in heme and chlorin iron proteins. Heme enzymes are ubiquitous biomolecules; the function of each is significantly influenced by its axial ligands. Axial ligand identification in a new heme protein is always one of the first lines of study. MCD spectroscopy has already found great application for this purpose, but there is the potential to significantly extend its utility. Toward this end, a large number of axial ligand adducts will be prepared that involve ligand combinations not previously scrutinized by MCD. Next, MCD will be used to address key coordination structure issues for cystathione beta synthase, heme oxygenase, soluble guanylyl cyclase and iron chlorin-containing systems. The third goal is to study the mechanism of molecular oxygen activation by nitric oxide synthase (NOS) and P450. Oxyferrous NOS, stabilized at low temperatures, will be used for the first time as the starting point to address specific mechanistic questions. With P450, the putative peroxyferric intermediate reported with the D251N mutant will be characterized. Finally, spectroscopic experiments on oxyferrous states of thiolate-ligated heme proteins and their one-electron reduced products will significantly increase our knowledge of these important, but poorly understood heme states.
Showing the most recent 10 out of 76 publications
