Introduction
Once considered as merely a toxic food additive, inorganic nitrite (NO2-) is now a molecule of considerable interest as a messenger of cell signaling in health and disease1. Nitric oxide (NO) is an endogenously produced gas that functions as an essential signaling molecule2. Although most NO is synthesized from arginine and oxygen by NO synthases, nitrite functions as a stable reservoir of NO after reduction by hemoglobin and other proteins1. However, far from serving simply as an inert reservoir, NO2- also functions directly as an essential messenger in vascular biology3; thus defining the mechanisms of NO2- synthesis and metabolism is crucial. Though auto-oxidation of NO to NO2- occurs readily in aqueous solution, the kinetics of this reaction preclude a physiological role for this process in NO2- synthesis. Transition metals within metalloproteins are a rich source of facile electron transfer reactions, and in this issue of Nature Chemical Biology Shiva et al.4 show that ceruloplasmin functions as an NO oxidase essential for maintaining plasma NO2-.
Ceruloplasmin is a multicopper oxidase characterized by three types of spectroscopically distinct copper ions. Six copper ions are incorporated into ceruloplasmin during synthesis in hepatocytes as a secreted plasma protein and in glia and macrophages as a glycosylphosphatidylinositol (GPI)-linked plasma-membrane protein5. Aceruloplasminemia is an inherited neurodegenerative disease characterized by tissue iron accumulation secondary to loss-of-function mutations in the ceruloplasmin gene. Recognition of this disease revealed an essential role for ceruloplasmin in the oxidation of ferrous iron that is necessary for iron efflux from cells6. Interestingly, restoration of systemic iron homeostasis in aceruloplasminemia requires only 10% of the ceruloplasmin concentration, suggesting that the substantial increase in serum ceruloplasmin observed in inflammation and infection may be necessary for oxidation of plasma substrates other than ferrous iron.
Shiva et al. hypothesized that NO is a physiological substrate of ceruloplasmin that results in the generation of plasma NO2-. Observing that plasma can catalyze NO2- formation at physiologic hemoglobin concentrations, the authors showed that a redox-active plasma protein of high molecular weight functions as an NO oxidase. Removal of ceruloplasmin from plasma using specific antibodies decreased plasma nitrite content by 50%, whereas addition of ceruloplasmin to plasma increased NO2- production in the presence of an NO donor. The plasma nitrite concentrations and NO oxidase activity of mice and people with aceruloplasminemia were significantly lower than those of controls. Experiments with metal chelators and erythrocytes confirmed that it is ceruloplasmin that accounts for this plasma NO oxidase activity. Given the lower plasma nitrite concentration seen in aceruloplasminemia and the known role of NO2- in oxygen-dependent vascular responsiveness1, the authors next examined the physiologic relevance of these observations using a mouse model of liver injury that occurs after impaired liver blood flow. Notably, as compared to control littermates, aceruloplasminemic mice sustained significantly greater liver injury in this ischemia model, and the extent of liver damage was substantially reversed by administration of exogenous NO2-.
Copper is no stranger to NO2- biology. In denitrifying organisms, NO2- reduction to NO is accomplished by copper-containing nitrite reductases, and in mammalian mitochondria cytochrome c oxidase can generate NO in a copper- and nitrite-dependent reaction critical to hypoxic gene induction7. Nevertheless, the current findings address an important gap in our rapidly evolving knowledge of NO2- biology by providing convincing evidence of an enzymatic mechanism for NO2- production in the plasma. The authors did not address NO2- production outside the plasma, but given the abundance of GPI-linked ceruloplasmin in glial cells and macrophages, analysis of the role of ceruloplasmin and NO2- in neuronal function and host defense will surely follow. Indeed, ceruloplasmin is synthesized in a wide variety of tissues and cell types not directly implicated in iron homeostasis8, which raises the possibility that they are sites of NO2- production.
This discovery reveals an intersection of the pathways of copper and NO homeostasis and raises questions relevant to the study of both pathways (Fig. 1). Could the pathogenesis of tissue injury in iron-overload syndromes be dependent on NO2-? Is copper homeostasis regulated by NO2- pathways in tissues? What is the relationship between the microbiomes of the oral and stomach cavities, in which nitrite is produced by nitrate-reducing bacteria, and how is this relationship affected by alterations in copper and NO2- content in the diet? The studies of Shiva et al. remind us that the pathways comprising the inorganic chemistry of living organisms do not exist in isolation, having evolved through opportunity and selection over millions of years to permit the complex and precise interplay necessary to regulate our physiology.
Figure 1: Ceruloplasmin is a multicopper oxidase that is synthesized and secreted by the liver into the plasma.
Copper is incorporated into ceruloplasmin during synthesis and is essential for oxidase activity. Within the plasma, ceruloplasmin catalyzes the oxidation of the signaling molecule NO concomitantly with cupric (Cu2+) to cuprous (Cu1+) reduction. Nitrite (NO2-) ions can therefore be used as a sink for NO production through reduction by deoxyhemoglobin, which allows for the mobilization of NO as a signaling molecule involved in hypoxic vasodilation and ischemia-reperfusion cytoprotection. In addition, nitrite acts independently as a signaling molecule necessary for cytoprotection and post-translational modifications such as iron nitrosylation and N-and S-nitrosation.
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