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Cytoglobin is a conserved hemoprotein ubiquitously expressed in mammalian tissues, which conducts electron transfer reactions with proposed signaling functions in nitric oxide (NO) and lipid metabolism. Cytoglobin has an E7 distal histidine (His81), which unlike related globins such as myoglobin and hemoglobin, is in equilibrium between a bound, hexacoordinate state and an unbound, pentacoordinate state. The His81 binding equilibrium appears to be allosterically modulated by the presence of an intramolecular disulfide between two cysteines (Cys38 and Cys83). The formation of this disulfide bridge regulates nitrite reductase activity and lipid binding. Herein, we attempt to clarify the effects of defined thiol oxidation states on small molecule binding of cytoglobin heme, using cyanide binding to probe the ferric state. Cyanide binding kinetics to wild-type cytoglobin reveal at least two kinetically distinct subpopulations, depending on thiol oxidation states. Experiments with covalent thiol modification by NEM, glutathione, and amino acid substitutions (C38S, C83S and H81A), indicate that subpopulations ranging from fully reduced thiols, single thiol oxidation, and intramolecular disulfide formation determine heme binding properties by modulating the histidine-heme affinity and ligand binding. The redox modulation of ligand binding is sensitive to physiological levels of hydrogen peroxide, with a functional midpoint redox potential for the native cytoglobin intramolecular disulfide bond of −189 ± 4 mV, a value within the boundaries of intracellular redox potentials. These results support the hypothesis that Cys38 and Cys83 on cytoglobin serve as sensitive redox sensors that modulate the cytoglobin distal heme pocket reactivity and ligand binding.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) disease (COVID-19) has affected over 22 million patients worldwide as of August 2020. As the medical community seeks better understanding of the underlying pathophysiology of COVID-19, several theories have been proposed. One widely shared theory suggests that SARS-CoV-2 proteins directly interact with human hemoglobin (Hb) and facilitate removal of iron from the heme prosthetic group, leading to the loss of functional hemoglobin and accumulation of iron. Herein, we refute this theory. We compared clinical data from 21 critically ill COVID-19 patients to 21 non-COVID-19 acute respiratory distress syndrome (ARDS) patient controls, generating hemoglobin-oxygen dissociation curves from venous blood gases. This curve generated from the COVID-19 cohort matched the idealized oxygen-hemoglobin dissociation curve well (Pearson correlation R2=0.97, P

Cytoglobin is a heme-containing protein ubiquitous in mammalian tissues. Unlike the evolutionarily related proteins hemoglobin and myoglobin, cytoglobin shows a six-coordinated heme binding, with the heme iron coordinated by two histidine side chains. Cytoglobin is involved in cytoprotection pathways through yet undefined mechanisms and it has recently been demonstrated that cytoglobin has redox signaling properties via nitric oxide (NO) and nitrite metabolism. The reduced, ferrous cytoglobin can bind oxygen and will react with NO in a dioxygenation reaction to form nitrate, which dampens NO signaling. When deoxygenated, cytoglobin can bind nitrite and reduce it to NO. This oxidoreductase activity could be catalytic if an effective reduction system exists to regenerate the reduced heme species. The nature of the physiological cytoglobin reducing system is unknown, although it has been proposed that ascorbate and cytochrome b5 could fulfill this role. Here we describe that physiological concentrations of cytochrome b5 and cytochrome b5 reductase can reduce human and fish cytoglobins at rates up to 250-fold higher than those reported for their known physiological substrates, hemoglobin and myoglobin; and up to 100-fold faster than 5 mM ascorbate. These data suggest that the cytochrome b5/cytochrome b5 reductase system is a viable reductant for cytoglobin in vivo, allowing for catalytic oxidoreductase activity.

The bioactivation of inorganic nitrite refers to the conversion of otherwise ‘inert’ nitrite to the diatomic signaling molecule nitric oxide (NO), which plays important roles in human physiology and disease, notably in the regulation of vascular tone and blood flow. While the most well-known sources of NO are the nitric oxide synthase (NOS) enzymes, another source of NO is the nitrate-nitrite-NO pathway, whereby nitrite (obtained from reduction of dietary nitrate) is further reduced to form NO. The past few decades have seen extensive study of the mechanisms of NO generation through nitrate and nitrite bioactivation, as well as growing appreciation of the contribution of this pathway to NO signaling in vivo. This review, prepared for the volume 400 celebration issue of Biological Chemistry, summarizes some of the key reactions of the nitrate-nitrite-NO pathway such as reduction, disproportionation, dehydration, and oxidative denitrosylation, as well as current evidence for the contribution of the pathway to human cardiovascular physiology. Finally, ongoing efforts to develop novel medical therapies for multifarious conditions, especially those related to pathologic vasoconstriction and ischemia/reperfusion injury, are also explored.

Cytoglobin is a heme protein evolutionarily related to hemoglobin and myoglobin. Cytoglobin is expressed ubiquitously in mammalian tissues; however its physiological functions are yet unclear. Phylogenetic analyses indicate that the cytoglobin gene is highly conserved in vertebrate clades, from fish to reptiles, amphibians, birds, and mammals. Most proposed roles for cytoglobin require the maintenance of a pool of reduced cytoglobin (Fe(II)). We have shown previously that the human cytochrome b(5) / cytochrome b(5) reductase system, considered a quintessential hemoglobin/myoglobin reductant, can reduce human and zebrafish cytoglobins up to 250-fold faster than human hemoglobin or myoglobin. It was unclear whether this reduction of zebrafish cytoglobins by mammalian proteins indicates a conserved pathway through vertebrate evolution. Here we report the reduction of zebrafish cytoglobins 1 and 2 by the zebrafish cytochrome b(5) reductase and the two zebrafish cytochrome b(5) isoforms. In addition, the reducing system also supports reduction of Globin X, a conserved globin in fish and amphibians. Indeed, the zebrafish reducing system can maintain a fully reduced pool for both cytoglobins, and both cytochrome b(5) isoforms can support this process. We determined the P(50) for oxygen being 0.5 torr for cytoglobin-1 and 4.4 torr for cytoglobin-2 at 25 °C. Thus, even at low oxygen tension, the reduced cytoglobins may exist in a predominant oxygen-bound form. In these conditions, the cytochrome b(5)/cytochrome b(5) reductase system can support a conserved role for cytoglobins through evolution, providing electrons for redox signaling reactions such as nitric oxide dioxygenation, nitrite reduction or phospholipid oxidation.

Cytoglobin is a heme protein evolutionarily related to hemoglobin and myoglobin. Cytoglobin is expressed ubiquitously in mammalian tissues; however, its physiological functions are yet unclear. Phylogenetic analyses indicate that the cytoglobin gene is highly conserved in vertebrate clades, from fish to reptiles, amphibians, birds, and mammals. Most proposed roles for cytoglobin require the maintenance of a pool of reduced cytoglobin (Fe-II). We have shown previously that the human cytochrome b(5)/cytochrome b(5) reductase system, considered a quintessential hemoglobin/myoglobin reductant, can reduce human and zebrafish cytoglobins

Since its discovery ≈15 years ago, cytoglobin has been studied extensively. Because it is found outside the red cell, cytoglobin is categorized as a nonerythroid globin, along with (in humans) proteins, such as myoglobin, neuroglobin, androglobin, and hemoglobin α. The putative functions of these nonerythroid globins are linked to tissue protection from conditions such as hypoxia, ischemia, and oxidative stress.1 Cytoglobin not only fulfills these functions but also has been related to other roles, including tumor suppression and the regulation of fibrosis in cell and animal models.2–7 Like other heme globins, cytoglobin can reversibly bind oxygen and other small molecules. The ability of cytoglobin to store and sense oxygen, as well as its involvement in nitrite and nitric oxide (NO) metabolism, being able to both scavenge NO and produce NO from nitrite, is probably key to its function(s).8,9 However, in spite of significant progress in understanding the structure, localization, and functional characteristics of cytoglobin, the central physiological roles of this protein have yet to be fully elucidated.10–12 See accompanying article on page 1944 In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Jourd’heuil et al13 examine the role of cytoglobin in controlling apoptosis and vascular remodeling after injury. Cytoglobin seems to be the predominant globin in vessel walls of humans, rats, and …

Cytoglobin (Cygb) is a ubiquitously expressed heme protein that has been identified in the genome of most higher vertebrate clades including fish, birds and mammals. Since its discovery, extensive study has revealed many structural and functional characteristics of the human Cygb protein, although its physiologic functions remain unknown. Fish have two Cygb genes (Cygb1 and Cygb2); these proteins show small but significant differences, with Cygb2 being more similar to mammalian Cygbs. To gain insights about Cygb function, we compared the properties of zebrafish cytoglobins to the mammalian proteins. Human Cygb exhibits high-affinity, cooperative oxygen binding (P50 of roughly 1 torr, Hill coefficient up to 1.6), as well as nitric oxide (NO) scavenging through NO dioxygenation and NO production via nitrite reduction. These reactions may modulate NO signaling in vivo. Reduced cytochrome b5 (CYB5) has been shown to rapidly reduce Cygb. We have reported that human CYB5 efficiently reduces both human and zebrafish Cygbs, suggesting a conserved role for the CYB5 reduction pathway. In this work, we characterize the two zebrafish Cygbs, measuring key parameters and comparing these results to human Cygb. Specifically, we examine oxygen binding for affinity and cooperativity, NO dioxygenation activity, and the ability of zebrafish CYB5 and CYB5R to reduce zebrafish Cygbs during NO dioxygenation. Oxygen affinity measurements suggest high oxygen affinity, with P50 values of

A variety of assays have been proposed to detect small quantities of nucleic acids at the point-of-care. One approach relies on target-induced aggregation of gold nanoparticles functionalized with oligonucleotide sequences complementary to adjacent regions on the targeted sequence. In the presence of the target sequence, the gold nanoparticles aggregate, producing an easily detectable shift in the optical scattering properties of the solution. The major limitations of this assay are that it requires heating, and that long incubation times are required to produce a result. This study aims to optimize the assay conditions and optical readout, with the goals of eliminating the need for heating and reducing the time to result without sacrificing sensitivity or dynamic range. By optimizing assay conditions and measuring the spectrum of scattered light at the endpoint of incubation, we find that the assay is capable of producing quantifiable results at room temperature in 30 minutes with a linear dynamic range spanning from 150 amoles to 15 fmoles of target. If changes in light scattering are measured dynamically during the incubation process, the linear range can be expanded 2-fold, spanning 50 amoles to 500 fmoles, while decreasing the time to result down to 10 minutes.