Myoglobin promotes nitrite-dependent mitochondrial S-nitrosation to mediate cytoprotection after hypoxia/reoxygenation

Highlights

• Myoglobin is a more efficient nitrite reductase than mitochondrial cytochromes.

• Myoglobin catalyzes nitrite-dependent S-nitrosation of mitochondria.

• Partial oxidation of myoglobin enhances nitrite-dependent S-nitrosation.

Abstract

It is well established that myoglobin supports mitochondrial respiration through the storage and transport of oxygen as well as through the scavenging of nitric oxide. However, during ischemia/reperfusion (I/R), myoglobin and mitochondria both propagate myocardial injury through the production of oxidants. Nitrite, an endogenous signaling molecule and dietary constituent, mediates potent cardioprotection after I/R and this effect relies on its interaction with both myoglobin and mitochondria. While independent mechanistic studies have demonstrated that nitrite-mediated cardioprotection requires the presence of myoglobin and the post-translational S-nitrosation of critical cysteine residues on mitochondrial complex I, it is unclear whether myoglobin directly catalyzes the S-nitrosation of complex I or whether mitochondrial-dependent nitrite reductase activity contributes to S-nitrosation. Herein, using purified myoglobin and isolated mitochondria, we characterize and directly compare the nitrite reductase activities of mitochondria and myoglobin and assess their contribution to mitochondrial S-nitrosation. We demonstrate that myoglobin is a significantly more efficient nitrite reductase than isolated mitochondria. Further, deoxygenated myoglobin catalyzes the nitrite-dependent S-nitrosation of mitochondrial proteins. This reaction is enhanced in the presence of oxidized (Fe³⁺) myoglobin and not significantly affected by inhibitors of mitochondrial respiration. Using a Chinese Hamster Ovary cell model stably transfected with human myoglobin, we show that both myoglobin and mitochondrial complex I expression are required for nitrite-dependent attenuation of cell death after anoxia/reoxygenation. These data expand the understanding of myoglobin's role both as a nitrite reductase to a mediator of S-nitrosation and as a regulator of mitochondrial function, and have implications for nitrite-mediated cardioprotection after I/R.

Introduction

Myoglobin and mitochondria are closely associated in the heart and together form a functional metabolome to sustain cardiac bioenergetics [[1], [2], [3], [4]]. Physiologically, mitochondria consume oxygen, which is coupled to the generation of ATP to supply 99% of the energy required for normal cardiac function. The monomeric heme protein, myoglobin, sustains oxidative phosphorylation through the storage and transport of oxygen to the mitochondrion within the myocyte [5,6]. However, during prolonged periods of ischemia and subsequent reperfusion (I/R), this functional relationship is disrupted and mitochondria and myoglobin both contribute to the pathogenesis of I/R injury [7]. Specifically, mitochondrial ATP generation is diminished during ischemia, leading to depleted free energy supply for cellular homeostasis. Upon reperfusion, the accumulation of reducing substrates and oxygen stimulates excessive mitochondrial reactive oxygen species (ROS) generation, leading to oxidation of mitochondrial protein complexes, the release of cytochrome c, and apoptosis [8,9]. Myoglobin potentiates this reperfusion-induced oxidative damage through its auto-oxidation and catalysis of superoxide generation [10,11].

Nitrite (NO₂⁻), the one electron oxidation product of nitric oxide (NO), is an endogenous signaling molecule that confers potent cardioprotection in numerous ex vivo and in vivo cell, isolated heart and animal models of myocardial I/R [[12], [13], [14], [15], [16]]. While the precise mechanisms underlying nitrite's cytoprotective effects after I/R are still being elucidated, two seminal observations implicate myoglobin and mitochondria as independent sub-cellular components that are central to nitrite-mediated protection: 1) nitrite covalently modifies a critical cysteine residue (cysteine 39 of the ND3 subunit) on mitochondrial electron transport chain complex I by S-nitrosation during I/R). This post-translational modification results in the inhibition of electron entry and transport in the mitochondrion, effectively attenuating reperfusion ROS generation and preventing protein oxidation and apoptosis [17,18]. 2) Myoglobin expression is required for nitrite-mediated cardioprotection as demonstrated by the inability of nitrite to decrease infarct size or protect cardiac function in myoglobin knockout mice subjected to myocardial infarction [19,20]. This necessity for myoglobin in nitrite-mediated cytoprotection is attributed to its efficient hypoxic nitrite reductase activity, whereby deoxygenated myoglobin (deoxyMb) reduces nitrite to bioavailable NO via the following reaction:

Nitrite + deoxyMb (Fe²⁺) + H⁺ → NO + metMb (Fe³⁺) + OH⁻(k = 12.4 M⁻¹s⁻¹; pH 7, 37 °C)

[21,22].

Despite the recognition that myoglobin-dependent nitrite reduction and mitochondrial S-nitrosation are both required for nitrite-induced cytoprotection, it is unknown whether myoglobin mediates mitochondrial S-nitrosation.

In the context of S-nitrosation, it is important to note that NO does not directly modify reduced protein thiols to form S-nitrosothiols but can be converted to nitrosating species in aerobic conditions [23,24]. Relevant to hypoxic S-nitrosation, nitrite is known to catalyze reductive nitrosylation, involving the reduction of metmyoglobin (Fe³⁺) by NO [25]. Further, a reductive anhydrase reaction has also been proposed as a potential mechanism in which metheme (Fe³⁺) proteins react with nitrite. Both these reactions require metheme and result in the formation of the potent nitrosating species dinitrogen trioxide (N₂O₃) [26,27]. In this regard, heme proteins such as hemoglobin and mitochondrial cytochrome c have been shown to promote S-nitrosation [[28], [29], [30]]. However, the ability of myoglobin to catalyze these reactions in a physiological milieu, with physiological levels of nitrite, and its impact on mitochondrial protein modification has previously not been assessed. Additionally, several studies demonstrate that components of the mitochondrial electron transport chain can directly reduce nitrite to NO [22,28,31], but the efficiency of this activity has never been compared to that of myoglobin.

Herein, we directly compare the nitrite reductase activity of mitochondria and myoglobin and show that myoglobin is a significantly more efficient nitrite reductase. We demonstrate that myoglobin promotes mitochondrial S-nitrosation in purified proteins and in a cell system and demonstrate that this is essential to nitrite-mediated protection from hypoxia/reoxygenation. We discuss the implications of myoglobin-dependent mitochondrial S-nitrosation for the regulation of metabolism in physiology and hypoxic/ischemic disease.