The enzymatic function of the honorary enzyme: S-nitrosylation of hemoglobin in physiology and medicine.

The allosteric transition within tetrameric hemoglobin (Hb) that allows both full binding to four oxygen molecules in the lung and full release of four oxygens in hypoxic tissues would earn Hb the moniker of 'honorary enzyme'. However, the allosteric model for oxygen binding in hemoglobin overlooked the essential role of blood flow in tissue oxygenation that is essential for life (aka autoregulation of blood flow). That is, blood flow, not oxygen content of blood, is the principal determinant of oxygen delivery under most conditions. With the discovery that hemoglobin carries a third biologic gas, nitric oxide (NO) in the form of S-nitrosothiol (SNO) at ß-globin Cys93 (ßCys93), and that formation and export of SNO to dilate blood vessels are linked to hemoglobin allostery through enzymatic activity, this title is honorary no more. This chapter reviews evidence that hemoglobin formation and release of SNO is a critical mediator of hypoxic autoregulation of blood flow in tissues leading to oxygen delivery, considers the physiological implications of a 3-gas respiratory cycle (O2/NO/CO2) and the pathophysiological consequences of its dysfunction. Opportunities for therapeutic intervention to optimize oxygen delivery at the level of tissue blood flow are highlighted.

The Quantum Biology of Reactive Oxygen Species Partitioning Impacts Cellular Bioenergetics.

Quantum biology is the study of quantum effects on biochemical mechanisms and biological function. We show that the biological production of reactive oxygen species (ROS) in live cells can be influenced by coherent electron spin dynamics, providing a new example of quantum biology in cellular regulation. ROS partitioning appears to be mediated during the activation of molecular oxygen (O2) by reduced flavoenzymes, forming spin-correlated radical pairs (RPs). We find that oscillating magnetic fields at Zeeman resonance alter relative yields of cellular superoxide (O2•-) and hydrogen peroxide (H2O2) ROS products, indicating coherent singlet-triplet mixing at the point of ROS formation. Furthermore, the orientation-dependence of magnetic stimulation, which leads to specific changes in ROS levels, increases either mitochondrial respiration and glycolysis rates. Our results reveal quantum effects in live cell cultures that bridge atomic and cellular levels by connecting ROS partitioning to cellular bioenergetics.

Spin biochemistry modulates reactive oxygen species (ROS) production by radio frequency magnetic fields.

The effects of weak magnetic fields on the biological production of reactive oxygen species (ROS) from intracellular superoxide (O2•-) and extracellular hydrogen peroxide (H2O2) were investigated in vitro with rat pulmonary arterial smooth muscle cells (rPASMC). A decrease in O2•- and an increase in H2O2 concentrations were observed in the presence of a 7 MHz radio frequency (RF) at 10 µTRMS and static 45 µT magnetic fields. We propose that O2•- and H2O2 production in some metabolic processes occur through singlet-triplet modulation of semiquinone flavin (FADH•) enzymes and O2•- spin-correlated radical pairs. Spin-radical pair products are modulated by the 7 MHz RF magnetic fields that presumably decouple flavin hyperfine interactions during spin coherence. RF flavin hyperfine decoupling results in an increase of H2O2 singlet state products, which creates cellular oxidative stress and acts as a secondary messenger that affects cellular proliferation. This study demonstrates the interplay between O2•- and H2O2 production when influenced by RF magnetic fields and underscores the subtle effects of low-frequency magnetic fields on oxidative metabolism, ROS signaling, and cellular growth.

Selective trapping of SNO-BSA and GSNO by benzenesulfinic acid sodium salt: mechanistic study of thiosulphonate formation and feasibility as a protein S-nitrosothiol detection strategy.

The conversion of S-nitrosothiols to thiosulphonates by reaction with the sodium salt of benzenesulfinic acid (PhSO2Na) has been examined in detail with the exemplary substrates S-nitrosoglutathione (GSNO) and S-nitrosylated bovine serum albumin (SNO-BSA). The reaction stoichiometry (2:1, PhSO2Na:RSNO) and the rate law (first order in both PhSO2Na and RSNO) have been determined under mild acidic conditions (pH 4.0). The products have been identified as the corresponding thiosulphonates (GSSO2Ph and BSA-SSO2Ph) along with PhSO2NHOH obtained in a 1:1 ratio. GSH, GSSG, and BSA were unreactive to PhSO2Na.

Temperature dependence of electron magnetic resonance spectra of iron oxide nanoparticles mineralized in Listeria innocua protein cages.

Electron magnetic resonance (EMR) spectroscopy was used to determine the magnetic properties of maghemite (γ-Fe(2)O(3)) nanoparticles formed within size-constraining Listeria innocua (LDps)-(DNA-binding protein from starved cells) protein cages that have an inner diameter of 5 nm. Variable-temperature X-band EMR spectra exhibited broad asymmetric resonances with a superimposed narrow peak at a gyromagnetic factor of g ≈ 2. The resonance structure, which depends on both superparamagnetic fluctuations and inhomogeneous broadening, changes dramatically as a function of temperature, and the overall linewidth becomes narrower with increasing temperature. Here, we compare two different models to simulate temperature-dependent lineshape trends. The temperature dependence for both models is derived from a Langevin behavior of the linewidth resulting from "anisotropy melting." The first uses either a truncated log-normal distribution of particle sizes or a bi-modal distribution and then a Landau-Liftshitz lineshape to describe the nanoparticle resonances. The essential feature of this model is that small particles have narrow linewidths and account for the g ≈ 2 feature with a constant resonance field, whereas larger particles have broad linewidths and undergo a shift in resonance field. The second model assumes uniform particles with a diameter around 4 nm and a random distribution of uniaxial anisotropy axes. This model uses a more precise calculation of the linewidth due to superparamagnetic fluctuations and a random distribution of anisotropies. Sharp features in the spectrum near g ≈ 2 are qualitatively predicted at high temperatures. Both models can account for many features of the observed spectra, although each has deficiencies. The first model leads to a nonphysical increase in magnetic moment as the temperature is increased if a log normal distribution of particles sizes is used. Introducing a bi-modal distribution of particle sizes resolves the unphysical increase in moment with temperature. The second model predicts low-temperature spectra that differ significantly from the observed spectra. The anisotropy energy density K(1), determined by fitting the temperature-dependent linewidths, was ∼50 kJ/m(3), which is considerably larger than that of bulk maghemite. The work presented here indicates that the magnetic properties of these size-constrained nanoparticles and more generally metal oxide nanoparticles with diameters d < 5 nm are complex and that currently existing models are not sufficient for determining their magnetic resonance signatures.

End-group distributions of multiple generations of spin-labeled PAMAM dendrimers.

Dendrimers are attractive templates to display functional molecular components. Since the behavior of dendrimer systems can depend greatly on the accessibility of these molecular components to the external environment, and on the spatial arrangement of functional groups attached to the dendrimer terminal branches (end-groups), techniques to determine the locations of end-groups are highly desirable. In this report, we describe a method to analyze the EPR spectra of multiple generations of poly(amidoamine) (PAMAM) dendrimers which have spin-labels attached to end-groups in variable percentages of the total number of available sites. The spectra are treated as a convolution of a narrow spin-label spectrum and a variable line broadening function. Trends in the parameters that describe the best-fit line broadening function with spin-label loading reveal the spatial arrangements and homogeneity of spin environments of the labels. We observe a shift in the end-group distribution from generation 3 (G(3)) to G(4) dendrimers that indicates a change in morphology from an open, extended structure to a more dense, compact arrangement.

Monitoring structural transitions in icosahedral virus protein cages by site-directed spin labeling.

This work describes an approach for calculating and measuring dipolar interactions in multispin systems to monitor conformational changes in icosahedral protein cages using site-directed spin labeling. Cowpea chlorotic mottle virus (CCMV) is used as a template that undergoes a pH-dependent reversible capsid expansion wherein the protein cage swells by 10%. The sequence-position-dependent geometric presentation of attached spin-label groups provides a strategy for targeting amino acid residues most probative of structural change. The labeled protein cage residues and structural transition were found to affect the local mobility and dipolar interactions of the spin label, respectively. Line-shape changes provided a spectral signature that could be used to follow the conformational change in CCMV coat dynamics. The results provide evidence for a concerted swelling process in which the cages exist in only two structural forms, with essentially no intermediates. This methodology can be generalized for all symmetry types of icosahedral protein architectures to monitor protein cage dynamics.

EPR spectroscopy of nitrite complexes of methemoglobin.

The chemical interplay of nitrogen oxides (NO's) with hemoglobin (Hb) has attracted considerable recent attention because of its potential significance in the mechanism of NO-related vasoactivity regulated by Hb. An important theme of this interplay-redox coupling in adducts of heme iron and NO's-has sparked renewed interest in fundamental studies of FeNO(x) coordination complexes. In this Article, we report combined UV-vis and comprehensive electron paramagnetic resonance (EPR) spectroscopic studies that address intriguing questions raised in recent studies of the structure and affinity of the nitrite ligand in complexes with Fe(III) in methemoglobin (metHb). EPR spectra of metHb/NO(2)(-) are found to exhibit a characteristic doubling in their sharper spectral features. Comparative EPR measurements at X- and S-band frequencies, and in D(2)O versus H(2)O, argue against the assignment of this splitting as hyperfine structure. Correlated changes in the EPR spectra with pH enable complete assignment of the spectrum as deriving from the overlap of two low-spin species with g values of 3.018, 2.122, 1.45 and 2.870, 2.304, 1.45 (values for samples at 20 K and pH 7.4 in phosphate-buffered saline). These g values are typical of g values found for other heme proteins with N-coordinated ligands in the binding pocket and are thus suggestive of N-nitro versus O-nitrito coordination. The positions and shapes of the spectral lines vary only slightly with temperature until motional averaging ensues at approximately 150 K. The pattern of motional averaging in the variable-temperature EPR spectra and EPR studies of Fe(III)NO(2)(-)/Fe(II)NO hybrids suggest that one of two species is present in both of the alpha and beta subunits, while the other is exclusive to the beta subunit. Our results also reconfirm that the affinity of nitrite for metHb is of millimolar magnitude, thereby making a direct role for nitrite in physiological hypoxic vasodilation difficult to justify.

Interactions of NO with hemoglobin: from microbes to man.

Hemoglobins are found in organisms from every major phylum and subserve life-sustaining respiratory functions across a broad continuum. Sustainable aerobic respiration in mammals and birds relies on the regulated delivery of oxygen (O2) and nitric oxide (NO) bioactivity by hemoglobin, through reversible binding of NO and O2 to hemes as well as S-nitrosylation of cysteine thiols (SNO synthase activity). In contrast, bacterial and yeast flavohemoglobins function in vivo as denitrosylases (O2 nitroxylases), and some multimeric, invertebrate hemoglobins function as deoxygenases (Cys-dependent NO dioxygenases), which efficiently consume rather than deliver NO and O2, respectively. Analogous mechanisms may operate in plants. Bacteria and fungi deficient in flavohemoglobin show compromised virulence in animals that results from impaired resistance to NO, whereas animals and humans deficient in S-nitrosylated Hb exhibit altered vasoactivity. NO-related functions of hemoglobins center on reactions with ferric (FeIII) heme iron, which is exploited in enzymatic reactions that address organismal requirements for delivery or detoxification of NO and O2. Delivery versus detoxification of NO/O2 is largely achieved through structural changes and amino acid rearrangements within the heme pockets, thereby influencing the propensity for heme/cysteine thiol redox coupling. Additionally, the behavior exhibited by hemoglobin in vivo may be profoundly dependent both on the abundance of NO and O2 and on the allosteric effects of heterotropic ligands. Here we review well-documented examples of redox interactions between NO and hemoglobin, with an emphasis on biochemical mechanisms and physiological significance.

Assessment of nitric oxide signals by triiodide chemiluminescence.

Nitric oxide (NO) bioactivity is mainly conveyed through reactions with iron and thiols, furnishing iron nitrosyls and S-nitrosothiols with wide-ranging stabilities and reactivities. Triiodide chemiluminescence methodology has been popularized as uniquely capable of quantifying these species together with NO byproducts, such as nitrite and nitrosamines. Studies with triiodide, however, have challenged basic ideas of NO biochemistry. The assay, which involves addition of multiple reagents whose chemistry is not fully understood, thus requires extensive validation: Few protein standards have in fact been characterized; NO mass balance in biological mixtures has not been verified; and recovery of species that span the range of NO-group reactivities has not been assessed. Here we report on the performance of the triiodide assay vs. photolysis chemiluminescence in side-by-side assays of multiple nitrosylated standards of varied reactivities and in assays of endogenous Fe- and S-nitrosylated hemoglobin. Although the photolysis method consistently gives quantitative recoveries, the yields by triiodide are variable and generally low (approaching zero with some standards and endogenous samples). Moreover, in triiodide, added chemical reagents, changes in sample pH, and altered ionic composition result in decreased recoveries and misidentification of NO species. We further show that triiodide, rather than directly and exclusively producing NO, also produces the highly potent nitrosating agent, nitrosyliodide. Overall, we find that the triiodide assay is strongly influenced by sample composition and reactivity and does not reliably identify, quantify, or differentiate NO species in complex biological mixtures.

An S-nitrosothiol (SNO) synthase function of hemoglobin that utilizes nitrite as a substrate.

Red blood cells (RBCs) act as O(2)-responsive transducers of vasodilator and vasoconstrictor activity in lungs and tissues by regulating the availability of nitric oxide (NO). Vasodilation by RBCs is impaired in diseases characterized by hypoxemia. We have proposed that the extent to which RBCs constrict vs. dilate vessels is, at least partly, controlled by a partitioning between NO bound to heme iron and to Cysbeta93 thiol of hemoglobin (Hb). Hemes sequester NO, whereas thiols deploy NO bioactivity. In recent work, we have suggested that specific micropopulations of NO-liganded Hb could support the chemistry of S-nitrosohemoglobin (SNO-Hb) formation. Here, by using nitrite as the source of NO, we demonstrate that a (T state) micropopulation of a heme-NO species, with spectral and chemical properties of Fe(III)NO, acts as a precursor to SNO-Hb formation, accompanying the allosteric transition of Hb to the R state. We also show that at physiological concentrations of nitrite and deoxyHb, a S-nitrosothiol precursor is formed within seconds and produces SNO-Hb in high yield upon its prompt exposure to O(2) or CO. Deoxygenation/reoxygenation cycling of oxyHb in the presence of physiological amounts of nitrite also efficiently produces SNO-Hb. In contrast, high amounts of nitrite or delays in reoxygenation inhibit the production of SNO-Hb. Collectively, our data provide evidence for a physiological S-nitrosothiol synthase activity of tetrameric Hb that depends on NO-Hb micropopulations and suggest that dysfunction of this activity may contribute to the pathophysiology of cardiopulmonary and blood disorders.

A nitric oxide processing defect of red blood cells created by hypoxia: deficiency of S-nitrosohemoglobin in pulmonary hypertension.

The mechanism by which hypoxia [low partial pressure of O(2) (pO(2))] elicits signaling to regulate pulmonary arterial pressure is incompletely understood. We considered the possibility that, in addition to its effects on smooth muscle, hypoxia may influence pulmonary vascular tone through an effect on RBCs. We report that exposure of native RBCs to sustained hypoxia is accompanied by a buildup of heme iron-nitrosyl (FeNO) species that are deficient in pO(2-)governed intramolecular transfer of NO to cysteine thiol, yielding a deficiency in the vasodilator S-nitrosohemoglobin (SNO-Hb). S-nitrosothiol (SNO)-deficient RBCs produce impaired vasodilator responses in vitro and exaggerated pulmonary vasoconstrictor responses in vivo and are defective in oxygenating the blood. RBCs from hypoxemic patients with elevated pulmonary arterial pressure (PAP) exhibit a similar FeNO/SNO imbalance and are thus deficient in pO(2)-coupled vasoregulation. Chemical restoration of SNO-Hb levels in both animals and patients restores the vasodilator activity of RBCs, and this activity is associated with improved oxygenation and lower PAPs.

Assessments of the chemistry and vasodilatory activity of nitrite with hemoglobin under physiologically relevant conditions.

Hypoxic vasodilation involves detection of the oxygen content of blood by a sensor, which rapidly transduces this signal into vasodilatory bioactivity. Current perspectives on the molecular mechanism of this function hold that hemoglobin (Hb) operates as both oxygen sensor and a condition-responsive NO reactor that regulates the dispensing of bioactivity through release of the NO group from the beta-cys93 S-nitroso derivative of Hb, SNO-Hb. A common path to the formation of SNO-Hb involves oxidative transfer of the NO-group from heme to thiol. We have previously reported that the reaction of nitrite with deoxy-Hb, which furnishes heme-Fe(II)NO, represents one attractive route for the formation of SNO-Hb. Recent literature, however, posits that the nitrite-reductase reaction of Hb might produce physiological vasodilatory effects through NO that evades trapping on heme-Fe(II) and may be stored before release as Fe(III)NO. In this article, we briefly review current perspectives in NO biology on the nitrite-reductase reaction of Hb. We report in vitro spectroscopic (UV/Vis, EPR) studies that are difficult to reconcile with suggestions that this reaction either generates a heme-Fe(III)NO reservoir or significantly liberates NO. We further show in bioassay experiments that combinations of nitrite and deoxy-Hb--under conditions that suppress SNO-Hb formation--exhibit no direct vasodilatory activity. These results help underscore the differences between physiological, RBC-regulated, hypoxic vasodilation versus pharmacological effects of exogenous nitrite.

Chemical physiology of blood flow regulation by red blood cells: the role of nitric oxide and S-nitrosohemoglobin.

Blood flow in the microcirculation is regulated by physiological oxygen (O2) gradients that are coupled to vasoconstriction or vasodilation, the domain of nitric oxide (NO) bioactivity. The mechanism by which the O2 content of blood elicits NO signaling to regulate blood flow, however, is a major unanswered question in vascular biology. While the hemoglobin in red blood cells (RBCs) would appear to be an ideal sensor, conventional wisdom about its chemistry with NO poses a problem for understanding how it could elicit vasodilation. Experiments from several laboratories have, nevertheless, very recently established that RBCs provide a novel NO vasodilator activity in which hemoglobin acts as an O2 sensor and O2-responsive NO signal transducer, thereby regulating both peripheral and pulmonary vascular tone. This article reviews these studies, together with biochemical studies, that illuminate the complexity and adaptive responsiveness of NO reactions with hemoglobin. Evidence for the pivotal role of S-nitroso (SNO) hemoglobin in mediating this response is discussed. Collectively, the reviewed work sets the stage for a new understanding of RBC-derived relaxing activity in auto-regulation of blood flow and O2 delivery and of RBC dysfunction in disorders characterized by tissue O2 deficits, such as sickle cell disease, sepsis, diabetes, and heart failure.

Characterization of heterogeneously functionalized dendrimers by mass spectrometry and EPR spectroscopy.

Starburst dendrimers are receiving considerable attention as templates for the assembly of structured arrays of molecular components. This research motivates the development of improved methods for dendrimer characterization-specifically, for determining the numbers, distributions of numbers, and spatial distribution of molecular species synthetically attached to macromolecular templates. Such information provides the basis for advancing strategies aimed at controlling dendrimer functionalization, and thus represents enabling technology for tailoring the composition and structure of molecular arrays fashioned on dendrimer templates. Moreover, this information is vital to the proper interpretation of ongoing experiments in which dendrimers sparsely functionalized with reporter groups are used as probes. In this article, we report MALDI-TOF mass spectrometry and EPR spectroscopy of heterogeneously functionalized G(4)-PAMAM dendrimers bearing nitroxide spin-labels.

EPR and affinity studies of mannose-TEMPO functionalized PAMAM dendrimers.

Mannose-TEMPO functionalized G4-PAMAM dendrimers with increasing mannose loadings have been synthesized and characterized by MALDI-TOF MS and EPR spectroscopy. Analysis of linebroadening effects in the EPR spectra of these dendrimers allowed us to determine the relative presentation of mannose and TEMPO on the dendrimer surface. Hemagglutination assays and affinity chromatography/EPR experiments to assess the activity of the mannose-TEMPO dendrimers with Concanavalin A are presented.