A Method For Cryogenic Magnetic Circular Dichroism Characterization of Reaction Intermediates During Nitric Oxide Synthase Catalysis

Nitric oxide (NO) plays many essential roles in physiology including vasodilation, neurotransmission, and immune response. NO is produced in mammals by the enzyme nitric oxide synthase (NOS).  Understanding the catalytic and regulatory mechanisms of NOS in mammals is of fundamental biochemical significance. Our research group is developing a method to better understand the mechanism of NOS catalysis.  Our method involves initiating NOS activity using a photoactivated caged-oxygen complex [(µ-peroxo)(µ-hydroxo)bis-[bis(bipyridyl)cobalt(III)] nitrate, cryogenically trapping the intermediates, and characterizing the heme active site of these intermediates using magnetic circular dichroism (MCD) spectroscopy.  This work demonstrates the feasibility of the method using myoglobin and presents preliminary data on the short-lived intermediates in the enzymatic reaction catalyzed by nitric oxide synthase.  This method will help clarify fundamental questions about the mechanism of NOS catalysis and provide insight into how the protein environment controls this chemistry.  This method will be a valuable resource in characterizing the mechanism of any oxygen dependent heme proteins.