Cryogenic Spectroscopic Characterization of Nitric Oxide Synthase Catalytic Intermediates

Saturday, February 13, 2016
Abraham King Cada, San Francisco State University, San Francisco, CA
Nitric oxide synthases (NOS) catalyzes the two-step conversion of L-arginine to nitric oxide (NO) and L-citrulline through a stable intermediate NG-hydroxy-L-arginine.  Although many details of the reaction mechanism have been revealed, identifying the activated oxygen heme complexes involved in each of the two stages of the catalytic cycle remains elusive.  In order to identify these intermediates, we aim to characterize NOS heme intermediates using magnetic circular dichrosim at low temperature after the photorelease of dioxygen from a cobalt peroxo complex (μ-peroxo)(μ-hydroxo)bis[bis(bipyridyl)cobalt(III)]nitrate (HPBC). We prove the methodological feasibility of this approach by examining the reaction of hemoglobin with HPBC.  Photolysis of HPBC at 265 nm shows the formation of oxyhemoglobin from deoxyhemoglobin upon cryophotolysis confirming the release of molecular oxygen in situ. In this study, spectral comparison of ligand complexes with recombinant NOS protein  has been characterized using low-temperature magnetic circular dichroism (MCD) and UV-Vis absorption spectroscopic techniques. Subsequent cryophotolysis experiments with NOS are presented, showing that HPBC can initiate NOS catalysis to produce NO and react at cryogenic temperatures. After initiating low temperature photolysis of HPBC the subtle structural differences in the transition metal complex can be seen in the MCD spectra. We show that the oxyferrous intermediate depends upon the substrate binding of L-Arginine and the intermediate N-hydroxoyarginine.  In addition, to answering fundamental questions about NOS catalytic intermediates, we verify a universal method for investigating the reaction of oxygen to MCD active enzymes. Using HPBC to deliver dioxygen in situ under cryotemperatures, combined with temperature-controlled spectroscopy, we examine reaction protein intermediates involved during NO biosynthesis by NOS.