Catalytic consumption of
nitric oxide (NO) by
myeloperoxidase and related
peroxidases is implicated as playing a key role in impairing NO bioavailability during inflammatory conditions. However, there are major gaps in our understanding of how
peroxidases consume NO in physiological fluids, in which multiple reactive
enzyme substrates and
antioxidants are present. Notably, ascorbate has been proposed to enhance
myeloperoxidase-catalyzed NO consumption by forming NO-consuming substrate radicals. However, we show that in complex
biological fluids ascorbate instead plays a critical role in inhibiting NO consumption by
myeloperoxidase and related
peroxidases (
lactoperoxidase,
horseradish peroxidase) by acting as a competitive substrate for
protein-bound redox intermediates and by efficiently scavenging
peroxidase-derived radicals (e.g.,
urate radicals), yielding ascorbyl radicals that fail to consume NO. These data identify a novel mechanistic basis for how ascorbate preserves NO bioavailability during
inflammation. We show that NO consumption by
myeloperoxidase Compound I is significant in substrate-rich fluids and is resistant to competitive inhibition by ascorbate. However,
thiocyanate effectively inhibits this process and yields
hypothiocyanite at the expense of NO consumption.
Hypothiocyanite can in turn form NO-consuming radicals, but
thiols (
albumin,
glutathione) readily prevent this. Conversely, where ascorbate is absent,
glutathione enhances NO consumption by
urate radicals via pathways that yield
S-nitrosoglutathione. Theoretical kinetic analyses provide detailed insights into the mechanisms by which ascorbate and
thiocyanate exert their protective actions. We conclude that the local depletion of ascorbate and
thiocyanate in inflammatory microenvironments (e.g., due to increased metabolism or dysregulated transport) will impair NO bioavailability by exacerbating
peroxidase-catalyzed NO consumption.