Implications For the Role of the Peroxoferric Intermediate In the Second Step of Nitric Oxide Synthase Derived From the Reactivity of P450-Cam With Camphor-5-Oxime and Characterization of the Heme Iron Coordination Structures of Tyrosine- and Histidine-Ligated Heme Enzymes Using Magnetic Circular Dichroism Spectroscopy
Date of Award
Campus Access Dissertation
Chemistry and Biochemistry
John H Dawson
Mammalian cytochrome P450s (P450s) are membrane-bound thiolate-ligated monooxygenases that play critical roles in steroid biosynthesis and drug metabolism. Although discovered over fifty years ago, a major remaining challenge in the study of these enzymes has been to establish the reactivity of the different active oxygen intermediates in the P450 catalytic cycle including the peroxo- and hydroperoxo-ferric states and the high-valent ferryl [Fe(IV)=O] porphyrin radical species known as compound I. Like P450, nitric oxide (NO,,G) synthase (NOS) is a thiolate-ligated heme monooxygenase. It produces L-citrulline and NO,,G from L-arginine via Nfç-hydroxy-L-arginine by a mechanism similar to that of P450. In this study 1R-(+)-camphor-5-oxime has been synthesized and its reactions with P450-CAM, a soluble P450 from Pseudomonas putida, and its T252A mutant have been investigated to probe the mechanism of the second step of the NOS reaction. Previous studies have shown that the T252A mutant is unable to form compound I. Formation of a significant amount of 1R-(+)-2,5-diketocamphor and NO,,G products from camphor-oxime with this mutant implicates involvement of the peroxo- or hydroperoxo-ferric intermediates as active oxidants in this step of the NOS catalytic cycle.
Magnetic circular dichroism (MCD) and UV-Visible (UV-Vis) absorption spectroscopy have been used to characterize the heme-iron coordination structure of (A) several tyrosinate-ligated heme enzymes and (B) the high potential (HP) and low potential (LP) heme centers of the diheme cytochrome c peroxidases (DHCCP) from Shewenella onendeisis. Even though coral (Plexaura homomalla) allene oxide synthase (cAOS) (43 kDa) and Micobacterium avium protein (MAP) (33 kDa) show some (< 20%) sequence homology to catalases, both have little catalase activity. MCD and UV-Vis spectra of cAOS and MAP resemble those of Tyr-ligated bovine liver catalase (BLC) in their native ferric (Fe(III)) and Fe(III)-CN¢w states. The spectra of the Tyr-ligated heme transport protein (PhuT) from Pseudomonas aeruginosa more closely resembles those of another heme-binding protein ShuT from Shigella dyseteriae than those of BLC or AOS. Facile formation of mono- and, especially, bis-imidazole (Im) and cyanide (CN¢w) PhuT adducts is likely related to its function in heme-acquisition/transport/release. Finally, we have analyzed MCD and UV-Vis spectral properties of DHCCP and have been able to spectrally separate and identify the individual HP and LP hemes in their various oxidation (di-ferric, ferric/ferrous and di-ferrous) and ligation states.
Bandara, D.(2010). Implications For the Role of the Peroxoferric Intermediate In the Second Step of Nitric Oxide Synthase Derived From the Reactivity of P450-Cam With Camphor-5-Oxime and Characterization of the Heme Iron Coordination Structures of Tyrosine- and Histidine-Ligated Heme Enzymes Using Magnetic Circular Dichroism Spectroscopy. (Doctoral dissertation). Retrieved from https://scholarcommons.sc.edu/etd/656