Heme Globin Research Articles

Heme pocket hydrogen bonding residue interactions within the Pectobacterium Diguanylate cyclase-containing globin coupled sensor: A resonance Raman study

Heme-based sensor proteins are used by organisms to control signaling and physiological effects in response to their gaseous environment. Globin-coupled sensors (GCS) are oxygen-sensing proteins that are widely distributed in bacteria. These proteins consist of a heme globin domain linked by a middle domain to various output domains, including diguanylate cyclase domains, which are responsible for synthesizing c-di-GMP, a bacterial second messenger crucial for regulating biofilm formation. To understand the roles of heme pocket residues in controlling activity of the diguanylate cyclase domain, variants of the Pectobacterium carotovorum GCS (PccGCS) were characterized by enzyme kinetics and resonance Raman (rR) spectroscopy. Results of these studies have identified roles for hydrogen bonding and heme edge residues in modulating heme pocket conformation and flexibility. Better understanding of the ligand-dependent GCS signaling mechanism and the residues involved may allow for future development of methods to control O2-dependent c-di-GMP production.

Nitric Oxide Trickle Drives Heme into Hemoglobin and Muscle Myoglobin.

Ever since the days of NO being proclaimed as the “molecule of the year”, the molecular effects of this miracle gas on the globins have remained elusive. While its vasodilatory role in the cardiopulmonary system and the vasculature is well recognized, the molecular underpinnings of the NO–globin axis are incompletely understood. We show, by transwell co-culture of nitric oxide (NO) generating, HEK eNOS/nNOS cells, and K562 erythroid or C2C12 muscle myoblasts, that low doses of NO can effectively insert heme into hemoglobin (Hb) and myoglobin (Mb), making NO not only a vasodilator, but also a globin heme trigger. We found this process to be dependent on the NO flux, occurring at low NO doses and fading at higher doses. This NO-triggered heme insertion occurred into Hb in just 30 min in K562 cells and into muscle Mb in C2C12 myoblasts between 30 min and 1 h, suggesting that the classical effect of NO on upregulation of globin (Hb or Mb) is just not transcriptional, but may involve sufficient translational events where NO can cause heme-downloading into the apo-globins (Hb/Mb). This effect of NO is unexpected and highlights its significance in maintaining globins in its heme-containing holo-form, where such heme insertions might be required in the circulating blood or in the muscle cells to perform spontaneous functions.

Hsp90 in Human Diseases: Molecular Mechanisms to Therapeutic Approaches.

The maturation of hemeprotein dictates that they incorporate heme and become active, but knowledge of this essential cellular process remains incomplete. Studies on chaperon Hsp90 has revealed that it drives functional heme maturation of inducible nitric oxide synthase (iNOS), soluble guanylate cyclase (sGC) hemoglobin (Hb) and myoglobin (Mb) along with other proteins including GAPDH, while globin heme maturations also need an active sGC. In all these cases, Hsp90 interacts with the heme-free or apo-protein and then drives the heme maturation by an ATP dependent process before dissociating from the heme-replete proteins, suggesting that it is a key player in such heme-insertion processes. As the studies on globin maturation also need an active sGC, it connects the globin maturation to the NO-sGC (Nitric oxide-sGC) signal pathway, thereby constituting a novel NO-sGC-Globin axis. Since many aggressive cancer cells make Hbβ/Mb to survive, the dependence of the globin maturation of cancer cells places the NO-sGC signal pathway in a new light for therapeutic intervention. Given the ATPase function of Hsp90 in heme-maturation of client hemeproteins, Hsp90 inhibitors often cause serious side effects and this can encourage the alternate use of sGC activators/stimulators in combination with specific Hsp90 inhibitors for better therapeutic intervention.

GAPDH is involved in the heme‐maturation of myoglobin and hemoglobin

GAPDH, a heme chaperone, has been previously implicated in the incorporation of heme into iNOS and soluble guanylyl cyclase (sGC). Since sGC is critical for myoglobin (Mb) heme-maturation, we investigated the role of GAPDH in the maturation of this globin, as well as hemoglobins α, β, and γ. Utilizing cell culture systems, we found that overexpression of wild-type GAPDH increased, whereas GAPDH mutants H53A and K227A decreased, the heme content of Mb and Hbα and Hbβ. Overexpression of wild-type GAPDH fully recovered the heme-maturation inhibition observed with the GAPDH mutants. Partial rescue was observed by overexpression of sGCβ1 but not by overexpression of a sGCΔβ1 deletion mutant, which is unable to bind the sGCα1 subunit required to form the active sGCα1β1 complex. Wild type and mutant GAPDH was found to be associated in a complex with each of the globins and Hsp90. GAPDH at endogenous levels was found to be associated with Mb in differentiating C2C12 myoblasts, and with Hbγ or Hbα in differentiating HiDEP-1 erythroid progenitor cells. Knockdown of GAPDH in C2C12 cells suppressed Mb heme-maturation. GAPDH knockdown in K562 erythroleukemia cells suppressed Hbα and Hbγ heme-maturation as well as Hb dimerization. Globin heme incorporation was not only dependent on elevated sGCα1β1 heterodimer formation, but also influenced by iron provision and magnitude of expression of GAPDH, d-aminolevulinic acid, and FLVCR1b. Together, our data support an important role for GAPDH in the maturation of myoglobin and γ, β, and α hemoglobins.

The role of globins in cardiovascular physiology.

Globin proteins exist in every cell type of the vasculature, from erythrocytes to endothelial cells, vascular smooth muscle cells, and peripheral nerve cells. Many globin subtypes are also expressed in muscle tissues (including cardiac and skeletal muscle), in other organ-specific cell types, and in cells of the central nervous system (CNS). The ability of each of these globins to interact with molecular oxygen (O2) and nitric oxide (NO) is preserved across these contexts. Endothelial α-globin is an example of extraerythrocytic globin expression. Other globins, including myoglobin, cytoglobin, and neuroglobin, are observed in other vascular tissues. Myoglobin is observed primarily in skeletal muscle and smooth muscle cells surrounding the aorta or other large arteries. Cytoglobin is found in vascular smooth muscle but can also be expressed in nonvascular cell types, especially in oxidative stress conditions after ischemic insult. Neuroglobin was first observed in neuronal cells, and its expression appears to be restricted mainly to the CNS and the peripheral nervous system. Brain and CNS neurons expressing neuroglobin are positioned close to many arteries within the brain parenchyma and can control smooth muscle contraction and thus tissue perfusion and vascular reactivity. Overall, reactions between NO and globin heme iron contribute to vascular homeostasis by regulating vasodilatory NO signals and scavenging reactive species in cells of the mammalian vascular system. Here, we discuss how globin proteins affect vascular physiology, with a focus on NO biology, and offer perspectives for future study of these functions.

Erythrocytes and Vascular Function: Oxygen and Nitric Oxide.

Erythrocytes regulate vascular function through the modulation of oxygen delivery and the scavenging and generation of nitric oxide (NO). First, hemoglobin inside the red blood cell binds oxygen in the lungs and delivers it to tissues throughout the body in an allosterically regulated process, modulated by oxygen, carbon dioxide and proton concentrations. The vasculature responds to low oxygen tensions through vasodilation, further recruiting blood flow and oxygen carrying erythrocytes. Research has shown multiple mechanisms are at play in this classical hypoxic vasodilatory response, with a potential role of red cell derived vasodilatory molecules, such as nitrite derived nitric oxide and red blood cell ATP, considered in the last 20 years. According to these hypotheses, red blood cells release vasodilatory molecules under low oxygen pressures. Candidate molecules released by erythrocytes and responsible for hypoxic vasodilation are nitric oxide, adenosine triphosphate and S-nitrosothiols. Our research group has characterized the biochemistry and physiological effects of the electron and proton transfer reactions from hemoglobin and other ferrous heme globins with nitrite to form NO. In addition to NO generation from nitrite during deoxygenation, hemoglobin has a high affinity for NO. Scavenging of NO by hemoglobin can cause vasoconstriction, which is greatly enhanced by cell free hemoglobin outside of the red cell. Therefore, compartmentalization of hemoglobin inside red blood cells and localization of red blood cells in the blood stream are important for healthy vascular function. Conditions where erythrocyte lysis leads to cell free hemoglobin or where erythrocytes adhere to the endothelium can result in hypertension and vaso constriction. These studies support a model where hemoglobin serves as an oxido-reductase, inhibiting NO and promoting higher vessel tone when oxygenated and reducing nitrite to form NO and vasodilate when deoxygenated.

171 - Nitrite improves Zebrafish Cardiac Regeneration Potentially by Cytoglobin 1

Treatment with nitrite (NO2‒) has been shown to have a beneficial effect on ischemia/reperfusion injury via the production of nitric oxide (NO) and modulation of reactive oxygen/nitrogen species (ROS/RNS). Nitrite can be converted to NO through the action of heme globins via a nitrite reductase reaction. The role of NO2‒ and globins has not yet been investigated in the context of heart regeneration. Zebrafish can fully regenerate their hearts following ventricular amputation and were used a model to study the effects of hypoxia and NO2‒ treatment on heart regeneration. We hypothesize that treatment with hypoxia/ NO2‒ will improve cardiac regeneration through increased production of NO or signaling ROS/RNS. We have found that NO2‒ /hypoxia treatment improves the rate of cardiomyocyte proliferation and improves neovascularization compared to hypoxia alone five days post amputation (dpa). Preceding these events, we observe an increase in thrombocytes (1 dpa) and neutrophils (3 dpa) migration to the injured area and a decrease in the blood clot size, suggesting that NO2‒ treatment is able to modulate the immune response to improve healing following injury. Zebrafish Cytoglobin 1 (Cygb1) is a globin that may mediate the cardiac regenerative response to NO2‒. Our data show that Cygb1 is expressed in the epicardium (a tissue that is known to drive the regeneration process) and its expression is increased following cardiac injury. Cygb1 may be mediating this response via its fast nitrite reductase rate (28.6 ± 3.1 M (-1) s(-1)) in its deoxy state or oxy-Cygb1 may react with NO2- to produce RNS, modulating the immune response via ROS/RNS signaling. Given the extremely low Cygb1 oxygen P50 (0.277 torr), we hypothesize that it is the latter, even in the context of the hypoxic heart. Our current research aims to identify the mechanism by which nitrite treatment modulates the immune response in the healing heart and determine if Cygb1 expression is necessary and/or sufficient for cardiac regeneration.

Determination of Thermodynamic and Kinetic Parameters for Co Migration within Bacterial Flavohemoglobins of Ralstonia Eutropha and Staphylococcus Aureus

Flavohemoglobins (FlavoHbs) are a class of protein found in a large number of prokaryotic and eukaryotic microbes. These proteins are involved in NO signaling and detoxification through an NO dioxygenase mechanism. They are comprised of an N-terminal heme globin domain which allows for the binding of gaseous ligands and a C-terminal NADH/FADH binding domain which facilitates association of redox cofactors necessary for reduction of the bound ligand. This NO dioxygenase function is important in facilitating immune resistance by protecting the cell from nitrosative stress brought about by a host organism; as a result bacterial flavoHbs have recently been considered as targets for the development of new classes of antibiotics. In this study, photoacoustic calorimetry (PAC) and transient absorption spectroscopy (TA) have been used to characterize the energetics, structural dynamics, and kinetics of CO migration within flavoHbs from Ralstonia eutropha (FHP) and Staphylococcus aureus (HMPSa) with and without the addition of antibiotic azole compounds. We observe that CO escape from both proteins occurs within ∼50 ns of photodissociation. In Fhp, the ligand escape is associated with ΔH = 25.2±5.5 kcal mol-1 and ΔV = 20.6±1.2 mL mol-1 while in HMPSa ΔH = 73.0±5.9 kcal mol-1 and ΔV = 40.4±5.7 mL mol-1 was observed. These values indicated a distinct structural relaxation associated with ligand escape from both proteins. Multi-exponential kinetics observed for CO association to FHP and HMPSa suggest that multiple pathways for ligand entry to the heme binding pocket may exist in this protein. In addition, both proteins display somewhat temperature-dependent quantum yields for bimolecular rebinding of CO - approx. 0.65 for FHP and 0.32 for HMPSa at 20 °C.

Characterization of zebrafish neuroglobin and cytoglobins 1 and 2: Zebrafish cytoglobins provide insights into the transition from six-coordinate to five-coordinate globins

Neuroglobin (Ngb) and cytoglobin (Cygb) are two six-coordinate heme proteins of unknown physiological function. Although studies on the mammalian proteins have elucidated aspects of Ngb and Cygb biophysics and indicated potential functions, the properties of non-mammalian Ngbs and Cygbs are largely uncharacterized. We have expressed the recombinant zebrafish proteins Ngb, Cygb1, and Cygb2 in Escherichia coli and characterized their nitrite reduction rates, spectral properties, autoxidation rate constants, redox potentials and lipid binding properties. The three zebrafish proteins can catalyze the reduction of nitrite to nitric oxide with a broad range of reaction rate constants. (Ngb, 0.68 ± 0.04 M−1 s−1; Cygb1, 28.6 ± 3.1 M−1 s−1; Cygb2, 0.94 ± 0.18 M−1 s−1). We observe that zebrafish Ngb and Cygb2 have comparable spectral features to those of human Ngb and Cygb, consistent with a six-coordinate heme, whereas unexpectedly Cygb1 has a five-coordinate heme, a slower autoxidation and in general has properties more akin to oxygen transport proteins. In agreement with a possible oxygen carrier and nitrite reductase role, we detect mRNA transcript for Cygb1 but not Cygb2 or Ngb in zebrafish blood. Unlike human Cygb, neither of the zebrafish globins binds oleic acid with high affinity. This finding suggests that lipid binding may be a trait acquired later during evolution and not an ancestral property of cytoglobins. Altogether, our results uncover unexpected properties of zebrafish globins and reveal the pivotal role of cytoglobins in the transition of heme globins from six-coordinate to five-coordinate oxygen carriers and nitrite reductases.

Utility of heme analogues to intentionally modify heme–globin interactions in myoglobin

Myoglobin reconstitution with various synthetic heme analogues was reviewed to follow the consequences of modified heme–globin interactions. Utility of dimethyl sulfoxide as the solvent for water-insoluble hemes was emphasized.Proton NMR spectroscopy revealed that loose heme–globin contacts in the heme pocket eventually caused the dynamic heme rotation around the iron–histidine bond. The full rotational rate was estimated to be about 1400s−1 at room temperature for 1,4,5,8-tetramethylhemin. The X-ray analysis of the myoglobin containing iron porphine, the smallest heme without any side chains, showed that the original globin fold was well conserved despite the serious disruption of native heme–globin contacts. Comparison between the two myoglobins with static and rotatory prosthetic groups indicated that the oxygen and carbon monoxide binding profiles were almost unaffected by the heme motion. On the other hand, altered tetrapyrrole array of porphyrin dramatically changed the dissociation constant of oxygen from 0.0005mmHg of porphycene-myoglobin to ∞ in oxypyriporphyrin-myoglobin.Heme–globin interactions in myoglobin were also monitored with circular dichroism spectroscopy. The observation on several reconstituted protein revealed an unrecognized role of the propionate groups in protoheme. Shortening of heme 6,7-propionates to carboxylates resulted in almost complete disappearance of the positive circular dichroism band in the Soret region. The theoretical analysis suggested that the disappeared circular dichroism band reflected the cancellation effects between different conformers of the carboxyl groups directly attached to heme periphery. The above techniques were proposed to be applicable to other hemoproteins to create new biocatalysts. This article is part of a Special Issue entitled Biodesign for Bioenergetics – the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

P112 - Reduction of nitrite to NO by zebrafish (Danio rerio) six-coordinate heme globins

P112 - Reduction of nitrite to NO by zebrafish (Danio rerio) six-coordinate heme globins

Structure of the Hemoglobin-IsdH Complex Reveals the Molecular Basis of Iron Capture by Staphylococcus aureus

Staphylococcus aureus causes life-threatening disease in humans. The S. aureus surface protein iron-regulated surface determinant H (IsdH) binds to mammalian hemoglobin (Hb) and extracts heme as a source of iron, which is an essential nutrient for the bacteria. However, the process of heme transfer from Hb is poorly understood. We have determined the structure of IsdH bound to human Hb by x-ray crystallography at 4.2 Å resolution, revealing the structural basis for heme transfer. One IsdH molecule is bound to each α and β Hb subunit, suggesting that the receptor acquires iron from both chains by a similar mechanism. Remarkably, two near iron transporter (NEAT) domains in IsdH perform very different functions. An N-terminal NEAT domain binds α/β globin through a site distant from the globin heme pocket and, via an intervening structural domain, positions the C-terminal heme-binding NEAT domain perfectly for heme transfer. These data, together with a 2.3 Å resolution crystal structure of the isolated N-terminal domain bound to Hb and small-angle x-ray scattering of free IsdH, reveal how multiple domains of IsdH cooperate to strip heme from Hb. Many bacterial pathogens obtain iron from human hemoglobin using proteins that contain multiple NEAT domains and other domains whose functions are poorly understood. Our results suggest that, rather than acting as isolated units, NEAT domains may be integrated into higher order architectures that employ multiple interaction interfaces to efficiently extract heme from host proteins.

Nitrite Regulates Hypoxic Vasodilation via Myoglobin-Dependent Nitric Oxide Generation

Hypoxic vasodilation is a physiological response to low oxygen tension that increases blood supply to match metabolic demands. Although this response has been characterized for >100 years, the underlying hypoxic sensing and effector signaling mechanisms remain uncertain. We have shown that deoxygenated myoglobin in the heart can reduce nitrite to nitric oxide (NO·) and thereby contribute to cardiomyocyte NO· signaling during ischemia. On the basis of recent observations that myoglobin is expressed in the vasculature of hypoxia-tolerant fish, we hypothesized that endogenous nitrite may contribute to physiological hypoxic vasodilation via reactions with vascular myoglobin to form NO·. We show in the present study that myoglobin is expressed in vascular smooth muscle and contributes significantly to nitrite-dependent hypoxic vasodilation in vivo and ex vivo. The generation of NO· from nitrite reduction by deoxygenated myoglobin activates canonical soluble guanylate cyclase/cGMP signaling pathways. In vivo and ex vivo vasodilation responses, the reduction of nitrite to NO·, and the subsequent signal transduction mechanisms were all significantly impaired in mice without myoglobin. Hypoxic vasodilation studies in myoglobin and endothelial and inducible NO synthase knockout models suggest that only myoglobin contributes to systemic hypoxic vasodilatory responses in mice. Endogenous nitrite is a physiological effector of hypoxic vasodilation. Its reduction to NO· via the heme globin myoglobin enhances blood flow and matches O(2) supply to increased metabolic demands under hypoxic conditions.

The expression of neuroglobin in astrocytoma

Neuroglobin (NGB) is a characterized heme globin that is widely expressed in the vertebrate central and peripheral nervous system as well as retina and endocrine tissues. However, to date, it is not determined whether functional NGB is expressed in cells of glia origin. In this study, we aimed to explore the detailed expression of NGB in a rat astrocytoma cell line (C6) and human astrocytoma cell line (U251) by reverse transcription-polymerase chain reaction, immunofluorescence, and Western blotting, and to detect the expression of NGB in human astrocytoma tissues by an immunohistochemical method. We found that NGB was present in a rat astrocytoma cell line (C6), human astrocytoma cell line (U251), and human astrocytoma tissues. The expression and potential roles of NGB in astrocytomas may provide insight into the mechanisms of tumor cells to adapt and survive in hypoxic microenvironments and also represent a novel therapeutic approach to astrocytomas.

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.

Molecular dynamics simulation of carboxy and deoxy human cytoglobin in solution

Cytoglobin (Cgb), the fourth member of the vertebrate heme globin family, is widely expressed in mammalian tissues, and reversibly binds to CO, O 2 and other small ligands. The diverse functions of Cgb may include ligand transport, redox reactions and enzymatic catalysis. Recent studies indicate that Cgb is a potential gene medicine for fibrosis and cancer therapy. In the present work, molecular dynamics (MD) simulations were performed to investigate the functionally related structural properties and dynamic characteristics in carboxy and deoxy human Cgb. The simulation results showed that the loop regions and internal cavities were significantly affected through the binding of an exogenous ligand. The AB, GH and EF loops were found to undergo significant rearrangement and this led to distinct cavity adjustments in Xe2, Xe4 and the distal pocket. In addition, solvent accessibility and torsion angle analyses revealed an interactive distal network comprised of His 81(E7), Leu 46(B10) and Arg 84(E10). The MD study of carboxy and deoxy human Cgb revealed that CO-ligated Cgb modulates the protein conformation primarily by loop and cavity rearrangements rather than the heme sliding mechanism found in neuroglobin (Ngb). The significant differences between Cgb and Ngb in the loop and cavity properties are presumably linked to their various biological functions.

Nitrite reductase activity of myoglobin regulates respiration and cellular viability in myocardial ischemia-reperfusion injury

The nitrite anion is reduced to nitric oxide (NO*) as oxygen tension decreases. Whereas this pathway modulates hypoxic NO* signaling and mitochondrial respiration and limits myocardial infarction in mammalian species, the pathways to nitrite bioactivation remain uncertain. Studies suggest that hemoglobin and myoglobin may subserve a fundamental physiological function as hypoxia dependent nitrite reductases. Using myoglobin wild-type ((+/+)) and knockout ((-/-)) mice, we here test the central role of myoglobin as a functional nitrite reductase that regulates hypoxic NO* generation, controls cellular respiration, and therefore confirms a cytoprotective response to cardiac ischemia-reperfusion (I/R) injury. We find that myoglobin is responsible for nitrite-dependent NO* generation and cardiomyocyte protein iron-nitrosylation. Nitrite reduction to NO* by myoglobin dynamically inhibits cellular respiration and limits reactive oxygen species generation and mitochondrial enzyme oxidative inactivation after I/R injury. In isolated myoglobin(+/+) but not in myoglobin(-/-) hearts, nitrite treatment resulted in an improved recovery of postischemic left ventricular developed pressure of 29%. In vivo administration of nitrite reduced myocardial infarction by 61% in myoglobin(+/+) mice, whereas in myoglobin(-/-) mice nitrite had no protective effects. These data support an emerging paradigm that myoglobin and the heme globin family subserve a critical function as an intrinsic nitrite reductase that regulates responses to cellular hypoxia and reoxygenation [corrected]

Catalytic generation of N2O3 by the concerted nitrite reductase and anhydrase activity of hemoglobin

Nitrite reacts with deoxyhemoglobin to form nitric oxide (NO) and methemoglobin. Though this reaction is experimentally associated with NO generation and vasodilation, kinetic analysis suggests that NO should not be able to escape inactivation in the erythrocyte. We have discovered that products of the nitrite-hemoglobin reaction generate dinitrogen trioxide (N2O3) via a novel reaction of NO and nitrite-bound methemoglobin. The oxygen-bound form of nitrite-methemoglobin shows a degree of ferrous nitrogen dioxide (Fe(II)-NO2*) character, so it may rapidly react with NO to form N2O3. N2O3 partitions in lipid, homolyzes to NO and readily nitrosates thiols, all of which are common pathways for NO escape from the erythrocyte. These results reveal a fundamental heme globin- and nitrite-catalyzed chemical reaction pathway to N2O3, NO and S-nitrosothiol that could form the basis of in vivo nitrite-dependent signaling. Because the reaction redox-cycles (that is, regenerates ferrous heme) and the nitrite-methemoglobin intermediate is not observable by electron paramagnetic resonance spectroscopy, this reaction has been 'invisible' to experimentalists over the last 100 years.

S-Nitrosylation-induced Conformational Change in Blackfin Tuna Myoglobin

S-nitrosylation is a post-translational protein modification that can alter the function of a variety of proteins. Despite the growing wealth of information that this modification may have important functional consequences, little is known about the structure of the moiety or its effect on protein tertiary structure. Here we report high-resolution x-ray crystal structures of S-nitrosylated and unmodified blackfin tuna myoglobin, which demonstrate that in vitro S-nitrosylation of this protein at the surface-exposed Cys-10 directly causes a reversible conformational change by "wedging" apart a helix and loop. Furthermore, we have demonstrated in solution and in a single crystal that reduction of the S-nitrosylated myoglobin with dithionite results in NO cleavage from the sulfur of Cys-10 and rebinding to the reduced heme iron, showing the reversibility of both the modification and the conformational changes. Finally, we report the 0.95-A structure of ferrous nitrosyl myoglobin, which provides an accurate structural view of the NO coordination geometry in the context of a globin heme pocket.

Locally enhanced sampling molecular dynamics study of the dioxygen transport in human cytoglobin

Cytoglobin (Cyg)--a new member of the vertebrate heme globin family--is expressed in many tissues of the human body but its physiological role is still unclear. It may deliver oxygen under hypoxia, serve as a scavenger of reactive species or be involved in collagen synthesis. This protein is usually six-coordinated and binds oxygen by a displacement of the distal HisE7 imidazole. In this paper, the results of 60 ns molecular dynamics (MD) simulations of dioxygen diffusion inside Cyg matrix are discussed. In addition to a classical MD trajectory, an approximate Locally Enhanced Sampling (LES) method has been employed. Classical diffusion paths were carefully analyzed, five cavities in dynamical structures were determined and at least four distinct ligand exit paths were identified. The most probable exit/entry path is connected with a large tunnel present in Cyg. Several residues that are perhaps critical for kinetics of small gaseous diffusion were discovered. A comparison of gaseous ligand transport in Cyg and in the most studied heme protein myoglobin is presented. Implications of efficient oxygen transport found in Cyg to its possible physiological role are discussed.