Probing the local electronic and geometric properties of the heme iron center in a H-NOX domain

Heme-Nitric oxide and/or OXygen binding (H-NOX) proteins are a family of diatomic gas binding hemoproteins that have attracted intense research interest. Here we employ X-ray absorption near-edge structure (XANES) spectroscopy to study the nitric oxide (NO) binding site of H-NOX. This is the first time this technique has been utilized to examine the NO/H-NOX signaling pathway. XANES spectra of wildtype and a point mutant (proline 115 to alanine, P115A) of the H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX) were obtained and analyzed for ferrous and ferric complexes of the protein. This work provides specific structural characterization of the solution state of several Tt H-NOX ferrous complexes (− unligated, − NO, and − CO) that were previously unavailable. Our iron K-edges indicate effective charge on the iron center in the various complexes and report on the electronic environment of heme iron. We analyzed the ligand field indicator ratio (LFIR), which is extracted from XANES spectra, for each complex, providing an understanding of ligand field strength, spin state of the central iron, movement of the iron atom upon ligation, and ligand binding properties. In particular, our LFIRs indicate that the heme iron is dramatically displaced towards the distal pocket during ligand binding. Based on these results, we propose that iron displacement towards the distal heme pocket is an essential step in signal initiation in H-NOX proteins. This provides a mechanistic link between ligand binding and the changes in heme and protein conformation that have been observed for H-NOX family members during signaling.     

Structure and reactivity of hexacoordinate hemoglobins

The heme prosthetic group in hemoglobins is most often attached to the globin through coordination of either one or two histidine side chains. Those proteins with one histidine coordinating the heme iron are called "pentacoordinate" hemoglobins, a group represented by red blood cell hemoglobin and most other oxygen transporters. Those with two histidines are called "hexacoordinate hemoglobins", which have broad representation among eukaryotes. Coordination of the second histidine in hexacoordinate Hbs is reversible, allowing for binding of exogenous ligands like oxygen, carbon monoxide, and nitric oxide. Research over the past several years has produced a fairly detailed picture of the structure and biochemistry of hexacoordinate hemoglobins from several species including neuroglobin and cytoglobin in animals, and the nonsymbiotic hemoglobins in plants. However, a clear understanding of the physiological functions of these proteins remains an elusive goal.     

Structure-function relationships in the growing hexa-coordinate hemoglobin sub-family

Hemoglobin and related heme proteins, generally referred to as 'globins', reversibly bind gaseous diatomic ligands (O2, NO, and CO) to a penta-coordinate heme iron atom, the ligand filling the sixth coordination site. Over the last decade, several new globins have been reported to display a functionally-relevant hexa-coordinate heme iron atom, whose sixth coordination site is taken by an endogenous protein ligand. The reversible intramolecular hexa- to penta-coordination process at the heme-Fe atom modulates exogenous ligand binding properties of hexa-coordinate globins. Here, we review current knowledge on hexa-coordinate globins in terms of their structural and functional properties.     

A Spectroscopic Study of Structural Heterogeneity and Carbon Monoxide Binding in Neuroglobin

Neuroglobin (Ngb) is a small globular protein that binds diatomic ligands like oxygen, carbon monoxide (CO) and nitric oxide at a heme prosthetic group. We have performed FTIR spectroscopy in the infrared stretching bands of CO and flash photolysis with monitoring in the electronic heme absorption bands to investigate structural heterogeneity at the active site of Ngb and its effects on CO binding and migration at cryogenic temperatures. Four CO stretching bands were identified; they correspond to discrete conformations that differ in structural details and CO binding properties. Based on a comparison of bound-state and photoproduct IR spectra of the wild-type protein, Ngb distal pocket mutants and myoglobin, we have provided structural interpretations of the conformations associated with the different CO bands. We have also studied ligand migration to the primary docking site, B. Rebinding from this site is governed by very low enthalpy barriers (∼1 kJ/mol), indicating an extremely reactive heme iron. Moreover, we have observed ligand migration to a secondary docking site, C, from which CO rebinding involves higher enthalpy barriers.     

Modulation of oxygen binding to insect hemoglobins: the structure of hemoglobin from the botfly Gasterophilus intestinalis

Hemoglobins (Hbs) reversibly bind gaseous diatomic ligands (e.g., O2) as the sixth heme axial ligand of the penta-coordinate deoxygenated form. Selected members of the Hb superfamily, however, display a functionally relevant hexa-coordinate heme Fe atom in their deoxygenated state. Endogenous heme hexa-coordination is generally provided in these Hbs by the E7 residue (often His), which thus modulates accessibility to the heme distal pocket and reactivity of the heme toward exogenous ligands. Such a pivotal role of the E7 residue is prominently shown by analysis of the functional and structural properties of insect Hbs. Here, we report the 2.6 A crystal structure of oxygenated Gasterophilus intestinalis Hb1, a Hb known to display a penta-coordinate heme in the deoxygenated form. The structure is analyzed in comparison with those of Drosophila melanogaster Hb, exhibiting a hexa-coordinate heme in its deoxygenated derivative, and of Chironomus thummi thummi HbIII, which displays a penta-coordinate heme in the deoxygenated form. Despite evident structural differences in the heme distal pockets, the distinct molecular mechanisms regulating O2 binding to the three insect Hbs result in similar O(2 affinities (P50 values ranging between 0.12 torr and 0.46 torr).     

Structural studies of constitutive nitric oxide synthases with diatomic ligands bound

Crystal structures are reported for the endothelial nitric oxide synthase (eNOS)–arginine–CO ternary complex as well as the neuronal nitric oxide synthase (nNOS) heme domain complexed with l-arginine and diatomic ligands, CO or NO, in the presence of the native cofactor, tetrahydrobiopterin, or its oxidized analogs, dihydrobiopterin and 4-aminobiopterin. The nature of the biopterin has no influence on the diatomic ligand binding. The binding geometries of diatomic ligands to nitric oxide synthase (NOS) follow the {MXY} ⁿ formalism developed from the inorganic diatomic–metal complexes. The structures reveal some subtle structural differences between eNOS and nNOS when CO is bound to the heme which correlate well with the differences in CO stretching frequencies observed by resonance Raman techniques. The detailed hydrogen-bonding geometries depicted in the active site of nNOS structures indicate that it is the ordered active-site water molecule rather than the substrate itself that would most likely serve as a direct proton donor to the diatomic ligands (CO, NO, as well as O₂) bound to the heme. This has important implications for the oxygen activation mechanism critical to NOS catalysis.     

Interaction of nitric oxide with cytochrome P450 BM3

The interaction of nitric oxide with cytochrome P450 BM3 from Bacillus megaterium has been analyzed by spectroscopic techniques and enzyme assays. Nitric oxide ligates tightly to the ferric heme iron, inducing large changes in each of the main visible bands of the heme and inhibiting the fatty acid hydroxylase function of the protein. However, the ferrous adduct is unstable under aerobic conditions, and activity recovers rapidly after addition of NADPH to the flavocytochrome due to reduction of the heme via the reductase domain and displacement of the ligand. The visible spectral properties revert to that of the oxidized resting form. Aerobic reduction of the nitrosyl complex of the BM3 holoenzyme or heme domain by sodium dithionite also displaces the ligand. A single electron reduction destabilizes the ferric-nitrosyl complex such that nitric oxide is released directly, as shown by the trapping of released nitric oxide. Aerobically and in the absence of exogenous reductant, nitric oxide dissociates completely from the P450 over periods of several minutes. However, recovery of the nativelike visible spectrum is accompanied by alterations in the catalytic activity of the enzyme and changes in the resonance Raman spectrum. Specifically, resonance Raman spectroscopy identifies the presence of internally located nitrated tyrosine residue(s) following treatment with nitric oxide. Analysis of a Y51F mutant indicates that this is the major nitration target under these conditions. While wild-type P450 BM3 does not form an aerobically stable ferrous-nitrosyl complex, a site-directed mutant of P450 BM3 (F393H) does form an isolatable ferrous-nitrosyl complex, providing strong evidence for the role of this residue in controlling the electronic properties of the heme iron. We report here the spectroscopic characterization of the ferric- and ferrous-nitrosyl complexes of P450 BM3 and describe the use of resonance Raman spectroscopy to identify nitrated tyrosine residue(s) in the enzyme. Nitration of tyrosine in P450 BM3 may exemplify a typical mechanism by which the ubiquitous messenger molecule nitric oxide exerts a regulatory function over the cytochromes P450.     

Heme binding properties of Staphylococcus aureus IsdE

Staphylococcus aureus is the source of a large number of hospital-acquired infections, of which many are serious and can lead to death. Iron is critically important to the survival and growth of the bacterium, and complex, multistep mechanisms are present to fulfill the necessary iron requirement. Isd proteins located on the wall and membrane of S. aureus have been proposed to function in heme acquisition. We report characterization of the S. aureus heme-binding protein IsdE, the lipoprotein component of a membrane-localized ABC transporter that is believed key to receiving heme from cell wall-anchored Isd proteins. Magnetic circular dichroism (MCD) data, which greatly extend the results from our initial study of IsdE in bacterial cell lysates (Mack, J., Vermeiren, C., Heinrichs, D. E., and Stillman, M. J. (2004) Biochem. Biophys. Res. Commun. 320, 781-788), probe the ligand and redox properties of the bound heme. The MCD data show that IsdE, when overexpressed in E. coli, binds either ferric or ferrous heme but that the largest fraction is low spin ferrous heme. Studies of mutants allowed identification and characterization of the ligands in the fifth and sixth position on the heme iron as histidine, proximally, and methionine, distally. This histidine-methionine heme-iron ligation is unique to heme transport proteins. The smaller fraction of ferric heme in the protein is not bound by methionine, allowing for access by strong field ligands, such as cyanide. Electrospray ionization mass spectral data are reported for the first time and show that only one heme ligand binds per IsdE protein molecule. These data also show there is little change in the conformation of the protein between the heme-bound and heme-free species, indicating that the heme-free IsdE adopts a structure essentially independent of the heme. The mass spectral data clearly show that IsdE reversibly unwinds under denaturing conditions to form at least two distinct, heme-free conformations.     

Molecular dynamics simulation of deoxy and carboxy murine neuroglobin in water

Globins are respiratory proteins that reversibly bind dioxygen and other small ligands at the iron of a heme prosthetic group. Hemoglobin and myoglobin are the most prominent members of this protein family. Unexpectedly a few years ago a new member was discovered and called neuroglobin (Ngb), being predominantly expressed in the brain. Ngb is a single polypeptide of 151 amino acids and despite the small sequence similarity with other globins, it displays the typical globin fold. Oxygen, nitric oxide, or carbon monoxide can displace the distal histidine which, in ferrous Ngb as well as in ferric Ngb, is bound to the iron, yielding a reversible adduct. Recent crystallographic data on carboxy Ngb show that binding of an exogenous ligand is associated to structural changes involving heme sliding and a topological reorganization of the internal cavities; in particular, the huge internal tunnel that connects the bulk with the active site, peculiar to Ngb, is heavily reorganized. We report the results of extended (90 ns) molecular dynamics simulations in water of ferrous deoxy and carboxy murine neuroglobin, which are both coordinated on the distal site, in the latter case by CO and in the former one by the distal His(64)(E7). The long timescale of the simulations allowed us to characterize the equilibrated protein dynamics and to compare protein structure and dynamical behavior coupled to the binding of an exogenous ligand. We have characterized the heme sliding motion, the topological reorganization of the internal cavities, the dynamics of the distal histidine, and particularly the conformational change of the CD loop, whose flexibility depends ligand binding.     

Analysis of the contribution of the globin and reductase domains to the ligand-binding properties of bacterial haemoglobins

Bacterial Hbs (haemoglobins), like VHb (Vitreoscilla sp. Hb), and flavoHbs (flavohaemoglobins), such as FHP (Ralstonia eutropha flavoHb), have different autoxidation and ligand-binding rates. To determine the influence of each domain of flavoHbs on ligand binding, we have studied the kinetic ligand-binding properties of oxygen, carbon monoxide and nitric oxide to the chimaeric proteins, FHPg (truncated form of FHP comprising the globin domain alone) and VHb-Red (fusion protein between VHb and the C-terminal reductase domain of FHP) and compared them with those of their natural counterparts, FHP and VHb. Moreover, we also analysed polarity and solvent accessibility to the haem pocket of these proteins. The rate constants for the engineered proteins, VHb-Red and FHPg, do not differ significantly from those of their natural counterparts, VHb and FHP respectively. Our results suggest that the globin domain structure controls the reactivity towards oxygen, carbon monoxide and nitric oxide. The presence or absence of a reductase domain does not affect the affinity to these ligands.     

Structural dynamics in the active site of murine neuroglobin and its effects on ligand binding

We have examined the effects of active site residues on ligand binding to the heme iron of mouse neuroglobin using steady-state and time-resolved visible spectroscopy. Absorption spectra of the native protein, mutants H64L and K67L and double mutant H64L/K67L were recorded for the ferric and ferrous states over a wide pH range (pH 4-11), which allowed us to identify a number of different species with different ligands at the sixth coordination, to characterize their spectroscopic properties, and to determine the pK values of active site residues. In flash photolysis experiments on CO-ligated samples, reaction intermediates and the competition of ligands for the sixth coordination were studied. These data provide insights into structural changes in the active site and the role of the key residues His64 and Lys67. His64 interferes with exogenous ligand access to the heme iron. Lys67 sequesters the distal pocket from the solvent. The heme iron is very reactive, as inferred from the fast ligand binding kinetics and the ability to bind water or hydroxyl ligands to the ferrous heme. Fast bond formation favors geminate rebinding; yet the large fraction of bimolecular rebinding observed in the kinetics implies that ligand escape from the distal pocket is highly efficient. Even slight pH variations cause pronounced changes in the association rate of exogenous ligands near physiological pH, which may be important in functional processes.     

Molecular basis for nitric oxide dynamics and affinity with Alcaligenes xylosoxidans cytochrome c

The bacterial heme protein cytochrome ? from Alcaligenes xylosoxidans (AXCP) reacts with nitric oxide (NO) to form a 5-coordinate ferrous nitrosyl heme complex. The crystal structure of ferrous nitrosyl AXCP has previously revealed that NO is bound in an unprecedented manner on the proximal side of the heme. To understand how the protein structure of AXCP controls NO dynamics, we performed absorption and Raman time-resolved studies at the heme level as well as a molecular computational dynamics study at the entire protein structure level. We found that after NO dissociation from the heme iron, the structure of the proximal heme pocket of AXCP confines NO close to the iron so that an ultrafast (7 ps) and complete (99 +/- 1%) geminate rebinding occurs, whereas the proximal histidine does not rebind to the heme iron on the timescale of NO geminate rebinding. The distal side controls the initial NO binding, whereas the proximal heme pocket controls its release. These dynamic properties allow the trapping of NO within the protein core and represent an extreme behavior observed among heme proteins.     

Structural bases for heme binding and diatomic ligand recognition in truncated hemoglobins

Truncated hemoglobins (trHbs) are low-molecular-weight oxygen-binding heme-proteins distributed in eubacteria, cyanobacteria, unicellular eukaryotes, and in higher plants, constituting a distinct group within the hemoglobin (Hb) superfamily. TrHbs display amino acid sequences 20-40 residues shorter than classical (non)vertebrate Hbs and myoglobins, to which they are scarcely related by sequence similarity. The trHb tertiary structure is based on a 2-on-2 alpha-helical sandwich, which represents a striking editing of the highly conserved 3-on-3 alpha-helical globin fold, achieved through deletion/truncation of alpha-helices and specific residue substitutions. Despite their 'minimal' polypeptide chain span, trHbs display an inner tunnel/cavity system held to support ligand diffusion to/from the heme distal pocket, accumulation of heme ligands within the protein matrix, and/or multiligand reactions. Moreover, trHbs bind and effectively stabilize the heme and recognize diatomic ligands (i.e., O2, CO, NO, and cyanide), albeit with varying thermodynamic and kinetic parameters. Here, structural bases for heme binding and diatomic ligand recognition by trHbs are reviewed.     

Powered by gas--a ligand for a fruit fly nuclear receptor

The difficulty in identifying ligands for nuclear hormone receptors remains an obstacle to understanding their function. For example, in the fruit fly Drosophila melanogaster, only one of its nuclear receptors has a known ligand. In this issue of Cell, report that the fruit fly E75 nuclear receptor contains heme in its ligand binding pocket and that the oxidation state of this molecule controls E75 activity. They also show that E75-heme responds to the small diatomic gases, nitric oxide and carbon monoxide. This study sheds light on how heme, gas signaling, and nuclear receptors interact to control metabolic and developmental pathways.     

Biological selectivity and functional aspects of protein tyrosine nitration

The formation of nitric oxide in biological systems has led to the discovery of a number of post-translational protein modifications that could regulate protein function or potentially be utilized as transducers of nitric oxide signaling. Principal among the nitric oxide-mediated protein modifications are: the nitric oxide-iron heme binding, the S-nitrosylation of reduced cysteine residues, and the C-nitration of tyrosine and tryptophan residues. With the exception of the nitric oxide binding to heme iron proteins, the other two modifications appear to require secondary reactions of nitric oxide and the formation of nitrogen oxides. The rapid development of analytical and immunological methodologies has allowed for the quantification of S-nitrosylated and C-nitrated proteins in vivo revealing an apparent selectivity and specificity of the proteins modified. This review is primarily focused upon the nitration of tyrosine residues discussing parameters that may govern the in vivo selectivity of protein nitration, and the potential biological significance and clinical relevance of this nitric oxide-mediated protein modification.     

The heme pocket geometry of Lucina pectinata hemoglobin II restricts nitric oxide and peroxide entry: model of ligand control for the design of a stable oxygen carrier

Blood pressure elevation has been attributed in large part to the consumption of nitric oxide (NO) by extracellular hemoglobin (Hb) therapeutics following infusion in humans. We studied NO and hydrogen peroxide (H2O2) oxidative reaction kinetics of monomeric Hbs isolated from the clam Lucina pectinata to probe the effects of their distinctive heme pocket chemistries on ligand controls and heme oxidative stability. HbI (Phe43(CD1), Gln64(E7), Phe29(B10), and Phe68(E11)) reacted with high avidity with NO (k'(ox,NO) = 91 microM-1 s-1), whereas HbII (Phe44(CD1), Gln65(E7), Tyr30(B10), and Phe69(E11)) reacted at a much slower rate (k'(ox,NO)= 2.8 microM-1 s-1). However, replacing B10 (Phe) by Tyr in recombinant HbI (HbI PheB10Tyr) produced only a 2-fold reduction in the NO-induced oxidation rate (k'(ox,NO)= 49.9 microM-1 s-1). Among the clam Hbs, HbII exhibited the fastest NO dissociation and the slowest NO association with ferrous iron. Autoxidation, H2O2-mediated ferryl iron (FeIV) formation, and the subsequent heme degradation kinetics were much slower in HbII and HbI PheB10Tyr when compared to those of HbI. The Tyr(B10) residue appears to afford a greater heme oxidative stability advantage toward H2O2, whereas the close proximity of this residue together with Gln(E7) to the heme iron contributes largely to the distal control of NO binding. Engineering of second-generation Hb-based oxygen therapeutics that are resistant to NO/H2O2-driven oxidation may ultimately require further optimization of the heme pocket architecture to limit heme exposure to solvent.     

Plant hemoglobins may be maintained in functional form by reduced flavins in the nuclei,and confer differential tolerance to nitro‐oxidative stress

The heme of bacteria, plant and animal hemoglobins (Hbs) must be in the ferrous state to bind O₂ and other physiological ligands. Here we have characterized the full set of non‐symbiotic (class 1 and 2) and ‘truncated’ (class 3) Hbs of Lotus japonicus. Class 1 Hbs are hexacoordinate, but class 2 and 3 Hbs are pentacoordinate. Three of the globins, Glb1‐1, Glb2 and Glb3‐1, are nodule‐enhanced proteins. The O₂ affinity of Glb1‐1 (50 pm ) was the highest known for any Hb, and the protein may function as an O₂ scavenger. The five globins were reduced by free flavins, which transfer electrons from NAD(P)H to the heme iron under aerobic and anaerobic conditions. Class 1 Hbs were reduced at very fast rates by FAD, class 2 Hbs at slower rates by both FMN and FAD, and class 3 Hbs at intermediate rates by FMN. The members of the three globin classes were immunolocalized predominantly in the nuclei. Flavins were quantified in legume nodules and nuclei, and their concentrations were sufficient to maintain Hbs in their functional state. All Hbs, except Glb1‐1, were expressed in a flavohemoglobin‐deficient yeast mutant and found to confer tolerance to oxidative stress induced by methyl viologen, copper or low temperature, indicating an anti‐oxidative role for the hemes. However, only Glb1‐2 and Glb2 afforded protection against nitrosative stress induced by S‐nitrosoglutathione. Because this compound is specifically involved in transnitrosylation reactions with thiol groups, our results suggest a contribution of the single cysteine residues of both proteins in the stress response.     

Spectroscopic characterization of the soluble guanylate cyclase-like heme domains from Vibrio cholerae and Thermoanaerobacter tengcongensis

Soluble guanylate cyclase (sGC) is a nitric oxide- (NO-) sensing hemoprotein that has been found in eukaryotes from Drosophila to humans. Prokaryotic proteins with significant homology to the heme domain of sGC have recently been identified through genomic analysis. Characterization of two of these proteins is reported here. The first is a 181 amino acid protein cloned from Vibrio cholerae (VCA0720) that is encoded in a histidine kinase-containing operon. The ferrous unligated form of VCA0720 is 5-coordinate, high-spin. The CO complex is low-spin, 6-coordinate, and the NO complex is high-spin and 5-coordinate. These ligand-binding properties are very similar to those of sGC. The second protein is the N-terminal 188 amino acids of Tar4 (TtTar4H), a predicted methyl-accepting chemotaxis protein (MCP) from the strict anaerobe Thermoanaerobacter tengcongensis. TtTar4H forms a low-spin, 6-coordinate ferrous-oxy complex, the first of this sGC-related family that binds O2. TtTar4H has ligand-binding properties similar to those of the heme-containing O2 sensors such as AxPDEA1. sGC does not bind O2 despite having a porphyrin with a histidyl ligand like the globins. The results reported here, with sequence-related proteins from prokaryotes but in the same family as the sGC heme domain, show that these proteins have evolved to discriminate between ligands such as NO and O2; hence, we term this family H-NOX domains (heme-nitric oxide/oxygen).     

Nitric oxide binding to the cardiolipin complex of ferric cytochrome C

Cardiolipin, a phospholipid specific to the mitochondrion, interacts with the small electron transfer heme protein cytochrome c through both electrostatic and hydrophobic interactions. Once in a complex with cardiolipin, cytochrome c has been shown to undergo a conformational change that leads to the rupture of the bond between the heme iron and the intrinsic sulfur ligand of a methionine residue and to enhance the peroxidatic properties of the protein considered important to its apoptotic activity. Here we report that the ferric cytochrome c/cardiolipin complex binds nitric oxide tightly through a multistep process in which the first step is the relatively slow displacement (5 s(-1)) from heme coordination of an intrinsic ligand that replaces methionine in the complex. Nanosecond photolysis of the nitrosyl adduct demonstrated that a fraction of the nitric oxide escapes from the heme pocket and subsequently recombines to the heme in second-order processes (k = 1.8 × 10(6) and 5.5 × 10(5) M(-1) s(-1)) that, under these conditions, were much faster than recombination of the intrinsic ligand with which they compete. Ultrafast (femtosecond) laser photolysis showed that the geminate recombination of nitric oxide to the heme occurred with time constants (τ = 22 and 72 ps) and that ～23% of the photolyzed nitric oxide escaped into the bulk phase. This high value for the escape fraction relative to other heme proteins indicates the open nature of the heme pocket in this complex. These results are summarized in a scheme and are discussed in terms of the possible modulation of the apoptotic activity of cytochrome c by nitric oxide.