Structure/Function and Reaction Mechanism
of Important Metalloproteins

Increasing number of hemeproteins have been found to play key roles in signal sensing/transduction, oxidation-reduction, energy conservation and gene regulation. Elucidation of the basic reaction mechanism of these metalloproteins will provide fundamental insights into their physiological functions, pathological roles and is also useful for development of effective therapeutic agents.

Several heme-containing proteins that control key biosynthetic pathways of eicosanoids and nitric oxide (NO) are under active investigation in my laboratory. Prostaglandin H synthase (PGHS or COX), the target of aspirin and other nonsteroidal anti-inflammatory agents (NSAIDs), catalyze the conversion of arachidonic acid to prostaglandin G2 and H2 (PGG2 and PGH2), both are active vasoconstrictor and aggregator of platelets. In addition to the substantial pharmacological interest, the enzymatic catalysis of PGHS is unique at three different aspects: one enzyme has two enzyme activities (peroxidase and cyclooxygenase), catalytic turnover is both self-propagating and self-inactivating. Our center theme is to unveil the coupling mechanism of both enzyme activities, to locate the key intermediate(s) that lead to the self-propagation of the COX activity and the self-inactivation of PGHS. Both PGHS-1 and ­2 isozymes obtained from native source or as recombinant protein are carefully studied using various spectroscopic and kinetic methods to define the structure of key intermediates and the temporal relationship among different intermediates transiently formed during catalysis. In collaboration with Dr. Lee-Ho Wang in Hematology Division, we also investigate the down stream enzyme, thromboxane synthase, which converts PGH2 to thromboxane A2, a most potent native vasoconstrictor and agonist for platelet aggregation; Prostacylin synthase, using the same substrate  PGH2 but produced a hormonal product exactly antagonizing the action of thromboxane synthase product. These two enzymes have a typical cytochrome P450 heme center but catalyze atypical peroxidase-like reaction rather than monooxygenase.

Nitric oxide synthase (NOS), the key enzyme that catalyzes the conversion from L-arginine to NO is another P450-like heme-containing protein. The catalysis involves a complex interplay of three substrates, four different enzyme cofactors and prosthetic groups to achieve a 5-electron oxidation of the nitrogen. The redox characteristics of each enzyme cofactors will be carefully evaluated and the electron-transfer sequence will be systematically defined in isolated NOS domains and the intact NOS proteins. The downstream enzyme that serves as the NO target, soluble guanylate cyclase (sGC), is another fascinating heme-containing protein to be investigated in collaboration with Drs. Ferid Murad and Emil Martin. We will focus on elucidating the structural basis for massive activation of sGC activity by NO binding to the heme center and resolving the mechanism that underlies this activation process.

Amino acid radical and substrate radical intermediates found in PGHS.
Shown in the figure are the key residues in the active site (Top panel) and the structure of the tyrosyl radical(s) (Bottom right) and the pentadienyl AA radical (Bottom left).