Virgil Simplaceanu

Human Neuroglobin Functions as a Redox-regulated Nitrite Reductase

Neuroglobin is a highly conserved hemoprotein of uncertain physiological function that evolved from a common ancestor to hemoglobin and myoglobin. It possesses a six-coordinate heme geometry with proximal and distal histidines directly bound to the heme iron, although coordination of the sixth ligand is reversible. We show that deoxygenated human neuroglobin reacts with nitrite to form nitric oxide (NO). This reaction is regulated by redox-sensitive surface thiols, cysteine 55 and 46, which regulate the fraction of the five-coordinated heme, nitrite binding, and NO formation. Replacement of the distal histidine by leucine or glutamine leads to a stable five-coordinated geometry; these neuroglobin mutants reduce nitrite to NO ∼2000 times faster than the wild type, whereas mutation of either Cys-55 or Cys-46 to alanine stabilizes the six-coordinate structure and slows the reaction. Using lentivirus expression systems, we show that the nitrite reductase activity of neuroglobin inhibits cellular respiration via NO binding to cytochrome c oxidase and confirm that the six-to-five-coordinate status of neuroglobin regulates intracellular hypoxic NO-signaling pathways. These studies suggest that neuroglobin may function as a physiological oxidative stress sensor and a post-translationally redox-regulated nitrite reductase that generates NO under six-to-five-coordinate heme pocket control. We hypothesize that the six-coordinate heme globin superfamily may subserve a function as primordial hypoxic and redox-regulated NO-signaling proteins.

Quaternary structure of hemoglobin in solution

Many important proteins perform their physiological functions under allosteric control, whereby the binding of a ligand at a specific site influences the binding affinity at a different site. Allosteric regulation usually involves a switch in protein conformation upon ligand binding. The energies of the corresponding structures are comparable, and, therefore, the possibility that a structure determined by x-ray diffraction in the crystalline state is influenced by its intermolecular contacts, and thus differs from the solution structure, cannot be excluded. Here, we demonstrate that the quaternary structure of tetrameric human normal adult carbonmonoxy-hemoglobin can readily be determined in solution at near-physiological conditions of pH, ionic strength, and temperature by NMR measurement of 15N-1H residual dipolar couplings in weakly oriented samples. The structure is found to be a dynamic intermediate between two previously solved crystal structures, known as the R and R2 states. Exchange broadening at the subunit interface points to a rapid equilibrium between different structures that presumably include the crystallographically observed states.

New look at hemoglobin allostery.

Hemoglobin (Hb) is a truly remarkable molecule. Human adult hemoglobin (Hb A) has a tetrameric structure consisting of two α-chains with 141 amino acids each and two β-chains with 146 amino acids each. Figure 1 illustrates features of the molecule that will be discussed. The tertiary structure is the three dimensional structure of the individual protein chains. The quaternary structure is the arrangement of the multiple protein chains into a multi-subunit complex stabilized through non-covalent interactions. Each of the four chains in Hb possesses a heme group, the binding site for ligands, such as oxygen (O2), carbon monoxide (CO), or nitric oxide (NO). It is an essential protein for all vertebrates, designed to facilitate the loading of oxygen molecules in the lungs (or gills) and unloading of oxygen molecules in the tissues efficiently. Hb is one of the first proteins whose structure was determined by X-ray crystallography in the 1960s and has also been used as a paradigm for understanding the structure-function relationship in allosteric proteins. An allosteric protein is one in which binding of a substrate, product, or other effector to a subunit of a multi-subunit protein at a site (allosteric site) other than the functional site alters its conformation and functional properties and can therefore contribute to regulating its physiological properties. For a review of the structure-function relationship of Hb, see ref.1 Most of the published results and conclusions regarding the molecular basis for Hb function, until recently, were based on the information derived from X-ray crystallographic data of Hb, e.g., the classical Monod-Wyman-Changeux (MWC) and Perutzs two-structure stereochemical model for hemoglobin allostery.2-3 Figure 1 Structures of Hb A: (a) Crystal structure of deoxy-Hb A (2DN2); (b) Crystal structure of HbCO A (2DN3); (c) The 10 lowest-energy solution structures of HbCO A obtained by NMR spectroscopy (2M6Z); (d) Superimposition of the R (2DN3, colored in red), R2 ... The classical MWC/Perutz model postulates that all four subunits in Hb have to assume simultaneously either the tense (T)- or relaxed (R)-structure.2-3 Both structures can bind ligands while the affinity towards the ligand changes in transiting from the T- to the R-structure. Noticing the marked differences in the crystal structures of oxy- and deoxy-Hb, Perutz3 put forward his stereochemical mechanism that correlated the T- and R- states of the MWC model to the deoxy- and oxy-structures of Hb. A key feature of the MWC model is that all four subunits must make the switch from T to R or R to T at the same time. In other words, the ligation of one subunit would not affect the ligand affinity of the neighboring subunits within the same quaternary structure. It is a concerted quaternary structural transition model. Perutzs model further postulates that inter- and intra-subunit salt bridges stabilize the Hb molecule in the T-structure. The deoxy- or T-structure has a lower ligand affinity compared to the oxy- or R-structure and the binding of oxygen is cooperative, i.e., binding of the first oxygen molecule increases the affinity of the Hb molecule for additional oxygen molecules. The induced-fit or sequential model [also known as the Koshland-Nemethy-Filmer (KNF) model] is another classical model for Hb allostery.4-5 It postulates that the binding of a ligand to one subunit can induce the conformational changes in the tertiary structure of its neighboring subunits without their having a bound ligand. Thus, the ligand binding in a multi-subunit protein is a sequential process; there are not just two final states, T and R, but a series of intermediate states. A conformational change in a neighboring subunit can take place in the absence of ligand binding. Both the MWC and the KNF models can account for the cooperative oxygen binding to Hb, thus the ligand-binding data alone cannot distinguish the KNF model from the MWC/Perutz model. Much work has been done in the last sixty years in order to determine whether the transition from the T to the R state is concerted or sequential and to gain an understanding of the atomic and molecular details of the cooperative oxygenation of Hb A and the mechanism of allostery. The stereochemical mechanism of Perutz was extended by Szabo and Karplus6 and later refined by Lee and Karplus.7 This statistical-mechanical model derives a partition function that describes the influence of homotropic (oxygen) and heterotropic [e.g., hydrogen ions and 2,3-bisphosphoglycerate (2,3-BPG)] effectors on the Hb structural changes. Two different tertiary structures for each of the two quaternary structures have been included in their formulation. Contrary to Perutzs model, the Szabo-Karplus model takes into account the differences in strength of the salt bridges that stabilize the T-structure and the contributions of the pH-independent steric constraints in reducing the ligand affinity of Hb in the deoxy state. Yonetani and co-workers proposed a global allostery model.8 This model stipulates that in the absence of heterotropic effectors, the allostery of Hb follows the MWC/Perutz model. Heterotropic effectors, when present, interact with both T and R states of Hb to induce tertiary rather than quaternary structural changes. The changes in oxygen affinity, Bohr effect, and cooperativity of Hb are primarily the consequence of heterotropic effector-induced tertiary structural changes. The tertiary two-state model of Eaton and co-workers9 can be considered as a variation of the MWC/Perutz model. Within each quaternary structure, the subunits exist in equilibrium of high (r) and low (t) affinity conformers. The R- and T-structures as defined in the MWC/Perutz model favor the r and t formation, respectively. As in the MWC/Perutz model, ligand binding without a quaternary conformation change is non-cooperative. However, the tertiary conformations of individual subunits play the primary role instead of the quaternary conformations. The molecular code for cooperativity of Ackers and coworkers10-11 points out that there are eight ligation intermediates between the completely unliganded and the fully liganded tetrameric Hb. The tetrameric Hb switches from T- to R-form when at least one subunit of each dimer is liganded. Hence, five ligation intermediates plus the fully liganded tetrameric Hb exist in the R-structure. Within each quaternary state, oxygen binding or releasing “sequentially” modulates the tertiary constraints, which ultimately leads to the quaternary structural switch. Cooperativity is the result of both “concerted” quaternary switching and “sequential” modulation of ligand binding within each quaternary form. There are many crystal structures determined over the years and several well-characterized T- and R-types of crystal structures of Hb A reported in the literature are summarized in Table 1. This multitude of structures and recent results obtained by other methods clearly show that the classical two-structure MWC/Perutz description for hemoglobin allostery as presented in biochemistry, biophysics, and molecular biology textbooks cannot account for Hb function in details and needs revision. In our last review12 ten years ago, we gave a summary of our experimental results on the molecular basis of the Bohr effect of Hb A and the solution conformation, dynamics, and subunit communication of Hb A as derived from our nuclear magnetic resonance (NMR) studies. Here, we present new results of NMR and wide-angle X-ray scattering (WAXS) studies that are relevant to the structure-function relationship in hemoglobin. Table 1 Crystallization conditions and resolutions of various crystal structures. During the past 10 years, hemoglobin has remained an active research area for biochemical, biophysical, and computational studies with over 6,500 papers published in the literature. It is interesting to note that a search of PubMed indicates that there were 489 papers with hemoglobin titles published in 2004, 632 papers in 2009, and 813 papers in 2013. This increase in the number of Hb publications indicates that there are new results as well as new thinking in the field of hemoglobin research. This article is not intended to cover all areas of Hb research, but focuses on new findings on the nature of Hb as a dynamic ensemble as related to its properties in solution. For additional readings on Hb and Hb allostery, one could consider a number of relevant articles.13-31

Chain-Selective Isotopic Labeling for NMR Studies of Large Multimeric Proteins: Application to Hemoglobin

Multidimensional, multinuclear NMR has the potential to elucidate the mechanisms of allostery and cooperativity in multimeric proteins under near-physiological conditions. However, NMR studies of proteins made up of non-equivalent subunits face the problem of severe resonance overlap, which can prevent the unambiguous assignment of resonances, a necessary step in interpreting the spectra. We report the application of a chain-selective labeling technique, in which one type of subunit is labeled at a time, to carbonmonoxy-hemoglobin A (HbCO A). This labeling method can be used to extend previous resonance assignments of key amino acid residues, which are important to the physiological function of hemoglobin. Among these amino acid residues are the surface histidyls, which account for the majority of the Bohr effect. In the present work, we report the results of two-dimensional heteronuclear multiple quantum coherence (HMQC) experiments performed on recombinant (15)N-labeled HbCO A. In addition to the C2-proton (H epsilon(1)) chemical shifts, these spectra also reveal the corresponding C4-proton (H delta(2)) resonances, correlated with the N epsilon(2) and N delta(1) chemical shifts of all 13 surface histidines per alpha beta dimer. The HMQC spectrum also allows the assignment of the H delta(1), H epsilon(1), and N epsilon(1) resonances of all three tryptophan residues per alpha beta dimer in HbCO A. These results indicate that heteronuclear NMR, used with chain-selective isotopic labeling, can provide resonance assignments of key regions in large, multimeric proteins, suggesting an approach to elucidating the solution structure of hemoglobin, a protein with molecular weight 64.5 kDa.

A carbon-13 nuclear magnetic resonance investigation of the metabolic fluxes associated withy glucose metabolism in human erythrocytes

We have used [2-13C]D-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy to investigate metabolic fluxes through the major pathways of glucose metabolism in intact human erythrocytes and to determine the interactions among these pathways under conditions that perturb metabolism. Using the method described, we have been able to measure fluxes through the pentose phosphate pathway, phosphofructokinase, the 2,3-diphosphoglycerate bypass, and phosphoglycerate kinase, as well as glucose uptake, concurrently and in a single experiment. We have measured these fluxes in normal human erythrocytes under the following conditions: (1) fully oxygenated; (2) treated with methylene blue; and (3) deoxygenated. This method makes it possible to monitor various metabolic effects of stresses in normal and pathological states. Not only has 13C-NMR spectroscopy proved to be a useful method for measuring in vivo flux through the pentose phosphate pathway, but it has also provided additional information about the cycling of metabolites through the non-oxidative portion of the pentose phosphate pathway. Our evidence from experiments with [1-13C]-, [2-13C]-, and [3-13C]D-glucoses indicates that there is an observable reverse flux of fructose 6-phosphate through the reactions catalyzed by transketolase and transaldolase, even in the presence of a net flux through the pentose phosphate pathway.

NMR investigation of the dynamics of tryptophan side-chains in hemoglobins.

NMR relaxation measurements of 15N spin-lattice relaxation rate (R(1)), spin-spin relaxation rate (R(2)), and heteronuclear nuclear Overhauser effect (NOE) have been carried out at 11.7T and 14.1T as a function of temperature for the side-chains of the tryptophan residues of 15N-labeled and/or (2H,15N)-labeled recombinant human normal adult hemoglobin (Hb A) and three recombinant mutant hemoglobins, rHb Kempsey (betaD99N), rHb (alphaY42D/betaD99N), and rHb (alphaV96W), in the carbonmonoxy and the deoxy forms as well as in the presence and in the absence of an allosteric effector, inositol hexaphosphate (IHP). There are three Trp residues (alpha14, beta15, and beta37) in Hb A for each alphabeta dimer. These Trp residues are located in important regions of the Hb molecule, i.e. alpha14Trp and beta15Trp are located in the alpha(1)beta(1) subunit interface and beta37Trp is located in the alpha(1)beta(2) subunit interface. The relaxation experiments show that amino acid substitutions in the alpha(1)beta(2) subunit interface can alter the dynamics of beta37Trp. The transverse relaxation rate (R(2)) for beta37Trp can serve as a marker for the dynamics of the alpha(1)beta(2) subunit interface. The relaxation parameters of deoxy-rHb Kemspey (betaD99N), which is a naturally occurring abnormal human hemoglobin with high oxygen affinity and very low cooperativity, are quite different from those of deoxy-Hb A, even in the presence of IHP. The relaxation parameters for rHb (alphaY42D/betaD99N), which is a compensatory mutant of rHb Kempsey, are more similar to those of Hb A. In addition, TROSY-CPMG experiments have been used to investigate conformational exchange in the Trp residues of Hb A and the three mutant rHbs. Experimental results indicate that the side-chain of beta37Trp is involved in a relatively slow conformational exchange on the micro- to millisecond time-scale under certain experimental conditions. The present results provide new dynamic insights into the structure-function relationship in hemoglobin.

Fluorine-19 nuclear magnetic resonance study of 5-fluorotryptophan-labeled histidine-binding protein J of Salmonella typhimurium

Fluorine-19 nuclear magnetic resonance has been used to investigate the histidine-binding protein J from Salmonella typhimurium. The protein has been labeled with fluorine-19 by growing the bacterial cells of a tryptophan auxotroph in the presence of 5-fluorotryptophan. Incorporation of up to 70% was achieved. The binding of L-histidine to the 19F-labeled protein is not affected by the isotopic labeling. The protein contains one tryptophan residue, giving rise to a single 19F resonance. Upon binding L-histidine to 19F-labeled histidine-binding protein J, the observed 19F resonance is shifted downfield by about 0.6 parts per million, indicating a conformational change of the protein molecule and a more hydrophobic environment for the 19F nucleus. Additional fluorescence experiments confirm that the tryptophan residue is located inside the hydrophobic core of the protein. 19F spin-lattice relaxation times of the 19F-labeled protein as a function of temperature show no difference between the free protein and the protein-histidine complex. However, the linewidth for the free protein is much larger than that of the protein-substrate complex. This can be explained by slow fluctuations between different conformations of the free protein molecule having slightly different 19F chemical shifts. Both with and without the substrate, the tryptophan residue is immobile inside the protein molecule as shown by the total disappearance of the 19F signal upon broadband irradiation at the 1H frequency. Also, the 19F spin-lattice relaxation times indicate that the protein is a rather rigid structure, in which rapid motions of the tryptophan residue on the time scale of 10(-8) second are not prominent.

Structural Consequences of Anesthetic and Nonimmobilizer Interaction with Gramicidin A Channels

Although interactions of general anesthetics with soluble proteins have been studied, the specific interactions with membrane bound-proteins that characterize general anesthesia are largely unknown. The structural modulations of anesthetic interactions with synaptic ion channels have not been elucidated. Using gramicidin A as a simplified model for transmembrane ion channels, we have recently demonstrated that a pair of structurally similar volatile anesthetic and nonimmobilizer, 1-chloro-1,2,2-trifluorocyclobutane (F3) and 1,2-dichlorohexafluorocyclobutane (F6), respectively, have distinctly different effects on the channel function. Using high-resolution NMR structural analysis, we show here that neither F3 nor F6 at pharmacologically relevant concentrations can significantly affect the secondary structure of the gramicidin A channel. Although both the anesthetic F3 and the nonimmobilizer F6 can perturb residues at the middle section of the channel deep inside the hydrophobic region in the sodium dodecyl sulfate micelles, only F3, but not F6, can significantly alter the chemical shifts of the tryptophan indole N-H protons near the channel entrances. The results are consistent with the notion that anesthetics cause functional change of the channel by interacting with the amphipathic domains at the peptide-lipid-water interface.

Effector-Induced Structural Fluctuation Regulates the Ligand Affinity of an Allosteric Protein: Binding of Inositol Hexaphosphate Has Distinct Dynamic Consequences for the T and R States of Hemoglobin†

The present study reports distinct dynamic consequences for the T- and R-states of human normal adult hemoglobin (Hb A) due to the binding of a heterotropic allosteric effector, inositol hexaphosphate (IHP). A nuclear magnetic resonance (NMR) technique based on modified transverse relaxation optimized spectroscopy (TROSY) has been used to investigate the effect of conformational exchange of Hb A in both deoxy and CO forms, in the absence and presence of IHP, at 14.1 and 21.1 T, and at 37 degrees C. Our results show that the majority of the polypeptide backbone amino acid residues of deoxy- and carbonmonoxy-forms of Hb A in the absence of IHP is not mobile on the micros-ms time scale, with the exception of several amino acid residues, that is, beta109Val and beta132Lys in deoxy-Hb A, and alpha40Lys in HbCO A. The mobility of alpha40Lys in HbCO A can be explained by the crystallographic data showing that the H-bond between alpha40Lys and beta146His in deoxy-Hb A is absent in HbCO A. However, the conformational exchange of beta109Val, which is located in the intradimer (alpha 1beta 1 or alpha 2beta 2) interface, is not consistent with the crystallographic observations that show rigid packing at this site. IHP binding appears to rigidify alpha40Lys in HbCO A, but does not significantly affect the flexibility of beta109Val in deoxy-Hb A. In the presence of IHP, several amino acid residues, especially those at the interdimer (alpha 1beta 2 or alpha 2beta 1) interface of HbCO A, exhibit significant conformational exchange. The affected residues include the proximal beta92His in the beta-heme pocket, as well as some other residues located in the flexible joint (betaC helix-alphaFG corner) and switch (alphaC helix-betaFG corner) regions that play an important role in the dimer-dimer rotation of Hb during the oxygenation process. These findings suggest that, upon IHP binding, HbCO A undergoes a conformational fluctuation near the R-state but biased toward the T-state, apparently along the trajectory of its allosteric transition, accompanied by structural fluctuations in the heme pocket of the beta-chain. In contrast, no significant perturbation of the dynamic features on the ms-micros time scale has been observed upon IHP binding to deoxy-Hb A. We propose that the allosteric effector-induced quaternary structural fluctuation may contribute to the reduced ligand affinity of ligated hemoglobin. Conformational exchange mapping of the beta-chain of HbCO A observed at 21.1 T shows significantly increased scatter in the chemical exchange contribution to the transverse relaxation rate ( R ex) values, relative to those at lower fields, due to the enhanced effect of the local chemical shift anisotropy (CSA) fluctuation. A spring-on-scissors model is proposed to interpret the dynamic phenomena induced by the heterotropic effector, IHP.

Rotating-frame relaxation studies of slow motions in fluorinated phospholipid model membranes.

Rotating-frame relaxation experiments have been carried out on 19F-labeled dimyristoylphosphatidylcholine model membranes. The lipids are labeled with a single CF2 group in the 4-, 8-, or 12-position of the 2-acyl chain. Both oriented lipid bilayers and multilamellar liposomes have been investigated. The relaxation rate has been measured as a function of the locking-field strength, the sample orientation, the label position, and the temperature. Our results have confirmed that extensive slow motions exist in the bilayer and dominate the low-frequency relaxation. The relaxation rate is quite sensitive to the label position. However, many other features of the relaxation are very similar for all three lipid isomers. The temperature dependence of the relaxation rate for the multilamellar liposomes differs from the oriented bilayers, which may imply that the motions are also different. To fit our data, a working model consisting of a superposition of an anisotropic reorientation term and a director fluctuation term has been proposed. We have also verified that almost all of the relaxation process is caused by modulations of the intramolecular interactions. Based on this, a view of the slow motions at a molecular level is discussed in this paper.