Abstract
The NO dioxygenation reaction catalyzed by heme-containing globin proteins is a crucial aerobic detoxification pathway. Accordingly, the second order reaction of NO with oxymyoglobin and oxyhemoglobin has been the focus of a large number of kinetic and spectroscopic studies. Stopped-flow and rapid-freeze-quench (RFQ) measurements have provided evidence for the formation of a Fe(III)-nitrato complex with millisecond lifetime prior to release of the nitrate product, but the temporal resolution of these techniques is insufficient for the characterization of precursor species. Most mechanistic models assume the formation of an initial Fe(III)-peroxynitrite species prior to homolytic cleavage of the OO bond and recombination of the resulting NO(2) and Fe(IV)=O species. Here we report vibrational spectroscopy measurements for the reaction of oxymyoglobin with a photolabile caged NO donor at cryogenic temperatures. We show that this approach offers efficient formation and trapping of the Fe(III)-nitrato, enzyme-product, complex at 180 K. Resonance Raman spectra of the Fe(III)-nitrato complex trapped via RFQ in the liquid phase and photolabile NO release at cryogenic temperatures are indistinguishable, demonstrating the complementarity of these approaches. Caged NO is released by irradiation <180 K but diffusion into the heme pocket is fully inhibited. Our data provide no evidence for Fe(III)-peroxynitrite of Fe(IV)=O species, supporting low activation energies for the NO to nitrate conversion at the oxymyoglobin reaction site. Photorelease of NO at cryogenic temperatures allows monitoring of the reaction by transmittance FTIR which provides valuable quantitative information and promising prospects for the detection of protein sidechain reorganization events in NO-reacting metalloenzymes.