Myeloperoxidase (MPO) is abundant hemoprotein present in neutrophils and monocytes which plays an essential role in immune surveillance and host defense mechanisms. It also is implicated in the pathogenesis of atherosclerosis and other inflammatory disorders. Upon phagocyte activation, MPO is secreted into both the extracellular milieu and the phagolysosome where it uses hydrogen peroxide (H2O2) produced during a respiratory burst as co-substrate. Activated intermediates, Compounds I and II, are sequentially formed which generate cytotoxic oxidants and diffusible radical species. Despite the potential significance of MPO to both human health and disease, little is known about the factors that influence MPO catalytic activity and function. In this proposal we focus on the potential role of nitric oxide (NO, nitrogen monoxide) and physiological reductants like ascorbate (Vitamin C) in the regulation of MPO activity, conformation and function. MPO and inducible nitric oxide synthase (NOS) are both stored and secreted in primary granules of activated leukocytes, and NO is known to react with the iron center of hemoproteins at near diffusion-controlled rates. However, the potential interactions between NO and the distal heme moiety of MPO are essentially unexplored. Similarly, ascorbate and other physiological reductants function in regulation the redox state of tissues. However, their role in modulating MPO catalysis through heme reduction has not been explored. The overall goal of this proposal is to identify the biochemical mechanisms through which NO and physiological reductants like ascorbate modulate MPO catalytic activity, conformation and function. We will examine the role of NO in modulating MPO activity and function and develop a comprehensive kinetic model for the interaction of nitrogen oxides with MPO. In parallel, we will examine the potential role of peroxidases in serving as a catalytic sink for NO, modulating its bioavailability and function. We will test the hypothesis that MPO-nitrosyl complexes serve as a novel mechanism for catalyzing formation of nitrosothiol adducts both in vitro and in vivo. Finally, we will explore the role of physiological reductants in reducing MPO-Fe(III) to the inactive form MPO- Fe(II), as well as characterize the role of heme reduction on MPO structure and function. Studies of MPO catalytic mechanisms and function are essential to a more fundamental understanding of the factors which govern MPO-dependent processes in human health and disease.
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