A cooperative oxygen binding hemoglobin from Mycobacterium tuberculosis. Stabilization of heme ligands by a distal tyrosine residue

The homodimeric hemoglobin (HbN) from Mycobacterium tuberculosis displays an extremely high oxygen binding affinity and cooperativity. Sequence alignment with other hemoglobins suggests that the proximal F8 ligand is histidine, the distal E7 residue is leucine, and the B10 position is occupied by tyrosine. To determine how these heme pocket residues regulate the ligand binding affinities and physiological functions of HbN, we have measured the resonance Raman spectra of the O(2), CO, and OH(-) derivatives of the wild type protein and the B10 Tyr --> Leu and Phe mutants. Taken together these data demonstrate a unique distal environment in which the heme bound ligands strongly interact with the B10 tyrosine residue. The implications of these data on the physiological functions of HbN and another heme-containing protein, cytochrome c oxidase, are considered.     

Insertion of an h-bonding residue into the distal pocket of the ferriheme protein nitrophorin 4: effect on nitriteiron coordination and nitrite disproportionation

Heme proteins are important entities for the metabolism of nitrite. Inspection of the structural features of the reported hemoprotein?nitrite crystal structures reveals that, except for nitrophorin 4 (NP4), H-bonding to the nitrite ligand is accomplished via histidine or arginine residues. These H-bonds probably play an important role for the nitrite coordination and/or reactivities. In nitrophorins, which catalyze the nitrite disproportionation reaction, such a residue is missing. Here, we report on the L130R mutant of the NP isoprotein NP4 that provides the Arg130 residue as part of the flexible G?H loop as a potential H-bonding residue in the distal heme pocket. Similar to the wild-type protein, nitrite remains N-bonded in the crystal structure of NP4(L130R). However, spectroscopic investigations show that, in solution, a second ligand-rotational orientation exists, which is in fast-exchange equilibrium with the normal, parallel ligand orientation. Moreover, the nitrite disproportionation is inhibited in NP4(L130R). Comparison with another, also less active mutant NP4(D30N) suggests that the displacement of H(2) O molecules from the heme cavity prevents the proton donation pathway through Asp30.     

Myoglobin structure and regulation of solvent accessibility of heme pocket

The effects of heme removal on the molecular structure of tuna and sperm whale myoglobin have been investigated by comparing the solvent accessibility to the heme pocket of the two proteins with that of the corresponding apoproteins. Although the heme microenvironment of tuna myoglobin is more polar than that of sperm whale myoglobin, the accessibility of solvent to heme is identical in the two proteins as revealed by thermal perturbation of Soret absorption. The removal of heme produces loss of helical folding and increase of solvent accessibility but the effects are rather different for the two proteins. More precisely, the loss of helical structure upon heme removal is 50% for tuna myoglobin and 15% for sperm whale myoglobin; moreover, the solvent accessibility of the heme pocket of tuna apomyoglobin is 2-3-fold greater than that of sperm whale apomyoglobin. These results have been explained in terms of the lack of helical folding in segment D, the structural organization of which may have a relevant effect in regulating the accessibility of ligands to the heme. The effects produced by charged quenchers reveal that the ligand path from the surface of the molecule to the ion atom of the heme involves a positively charged residue which may reasonably be identified as Arg-45 (sperm whale myoglobin) or Lys-41 (tuna myoglobin) on the basis of recent X-ray crystallographic information.     

Cofacial heme binding is linked to dimerization by a bacterial heme transport protein

Campylobacter jejuni is a leading bacterial cause of food-borne illness in the developed world. Like most pathogens, C. jejuni requires iron that must be acquired from the host environment. Although the iron preference of the food-borne pathogen C. jejuni is not established, this organism possesses heme transport systems to acquire iron. ChaN is an iron-regulated lipoprotein from C. jejuni proposed to be associated with ChaR, an outer-membrane receptor. Mutation of PhuW, a ChaN orthologue in Pseudomonas aeruginosa, compromises growth on heme as a sole iron source. The crystal structure of ChaN, determined to 1.9 A resolution reveals that ChaN is comprised of a large parallel beta-sheet with flanking alpha-helices and a smaller domain consisting of alpha-helices. Unexpectedly, two cofacial heme groups ( approximately 3.5 A apart with an inter-iron distance of 4.4 A) bind in a pocket formed by a dimer of ChaN monomers. Each heme iron is coordinated by a single tyrosine from one monomer, and the propionate groups are hydrogen bonded by a histidine and a lysine from the other monomer. Sequence analyses reveal that these residues are conserved among ChaN homologues from diverse bacterial origins. Electronic absorption and electron paramagnetic resonance (EPR) spectroscopy are consistent with heme binding through tyrosine coordination by ChaN in solution yielding a high-spin heme iron structure in a pH-dependent equilibrium with a low-spin species. Analytical ultracentrifugation demonstrates that apo-ChaN is predominantly monomeric and that dimerization occurs with heme binding such that the stability constant for dimer formation increases by 60-fold.     

Incorporation of Tyrosine and Glutamine Residues into the Soluble Guanylate Cyclase Heme Distal Pocket Alters NO and O2 Binding

Nitric oxide (NO) is the physiologically relevant activator of the mammalian hemoprotein soluble guanylate cyclase (sGC). The heme cofactor of α1β1 sGC has a high affinity for NO but has never been observed to form a complex with oxygen. Introduction of a key tyrosine residue in the sGC heme binding domain β1(1–385) is sufficient to produce an oxygen-binding protein, but this mutation in the full-length enzyme did not alter oxygen affinity. To evaluate ligand binding specificity in full-length sGC we mutated several conserved distal heme pocket residues (β1 Val-5, Phe-74, Ile-145, and Ile-149) to introduce a hydrogen bond donor in proximity to the heme ligand. We found that the NO coordination state, NO dissociation, and enzyme activation were significantly affected by the presence of a tyrosine in the distal heme pocket; however, the stability of the reduced porphyrin and the proteins affinity for oxygen were unaltered. Recently, an atypical sGC from Drosophila, Gyc-88E, was shown to form a stable complex with oxygen. Sequence analysis of this protein identified two residues in the predicted heme pocket (tyrosine and glutamine) that may function to stabilize oxygen binding in the atypical cyclase. The introduction of these residues into the rat β1 distal heme pocket (Ile-145 → Tyr and Ile-149 → Gln) resulted in an sGC construct that oxidized via an intermediate with an absorbance maximum at 417 nm. This absorbance maximum is consistent with globin Fe^II-O₂ complexes and is likely the first observation of a Fe^II-O₂ complex in the full-length α1β1 protein. Additionally, these data suggest that atypical sGCs stabilize O₂ binding by a hydrogen bonding network involving tyrosine and glutamine.     

Identification of residues crucially involved in the binding of the heme moiety of soluble guanylate cyclase

Soluble guanylate cyclase (sGC), a heterodimeric hemeprotein, is the only receptor for the biological messenger nitric oxide (NO) identified to date and is intimately involved in various signal transduction pathways. By using the recently discovered NO- and heme-independent sGC activator BAY 58-2667 and a novel cGMP reporter cell, we could distinguish between heme-containing and heme-free sGC in an intact cellular system. Using these novel tools, we identified the invariant amino acids tyrosine 135 and arginine 139 of the beta(1)-subunit as crucially important for both the binding of the heme moiety and the activation of sGC by BAY 58-2667. The heme is displaced by BAY 58-2667 due to a competition between the carboxylic groups of this compound and the heme propionic acids for the identified residues tyrosine 135 and arginine 139. This displacement results in the release of the axial heme ligand histidine 105 and to the observed activation of sGC. Based on these findings we postulate a signal transmission triad composed of histidine 105, tyrosine 135, and arginine 139 responsible for the enzyme activation by this compound and probably also for transducing changes in heme status and porphyrin geometry upon NO binding into alterations of sGC catalytic activity.     

The comparative study on the solution structures of the oxidized bovine microsomal cytochrome b5 and mutant V45H

A comparative study on the solution structures of bovine microsomal cytochrome b5 (Tb5) and the mutant V45H has been achieved by 1D and 2D 1H-NMR spectroscopy to clarify the differences in the solution conformations between these two proteins. The results reveal that the global folding of the V45H mutant in solution is unchanged, but the subtle changes exist in the orientation of the axial ligand His39, and heme vinyl groups. The side chain of His45 in V45H mutant extends to the outer edge of the heme pocket leaving a cavity at the site originally occupied by the inner methyl group of Val45 residue. In addition, the imidazole ring of axial ligand His39 rotates counterclockwise by approximately 3 degrees around the His-Fe-His axis, and the 4-heme vinyl group turns to the space vacated by the removed side chain due to the mutation. Furthermore, the helix III of the heme pocket undergoes outward displacement, while the linkage between helix II and III is shifted leftward. These observations are not only consistent with the pattern of the pseudocontact shifts of the heme protons, but also well account for the lower stability of V45H mutant against heat and urea.     

The leghemoglobin proximal heme pocket directs oxygen dissociation and stabilizes bound heme

Sperm whale myoglobin (Mb) and soybean leghemoglobin (Lba) are two small, monomeric hemoglobins that share a common globin fold but differ widely in many other aspects. Lba has a much higher affinity for most ligands, and the two proteins use different distal and proximal heme pocket regulatory mechanisms to control ligand binding. Removal of the constraint provided by covalent attachment of the proximal histidine to the F-helices of these proteins decreases oxygen affinity in Lba and increases oxygen affinity in Mb, mainly because of changes in oxygen dissociation rate constants. Hence, Mb and Lba use covalent constraints in opposite ways to regulate ligand binding. Swapping the F-helices of the two proteins brings about similar effects, highlighting the importance of this helix in proximal heme pocket regulation of ligand binding. The F7 residue in Mb is capable of weaving a hydrogen-bonding network that holds the proximal histidine in a fixed orientation. On the contrary, the F7 residue in Lba lacks this property and allows the proximal histidine to assume a conformation favorable for higher ligand binding affinity. Geminate recombination studies indicate that heme iron reactivity on picosecond timescales is not the dominant cause for the effects observed in each mutation. Results also indicate that in Lba the proximal and distal pocket mutations probably influence ligand binding independently. These results are discussed in the context of current hypotheses for proximal heme pocket structure and function.     

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).     

Alteration of sperm whale myoglobin heme axial ligation by site-directed mutagenesis

Three mutant proteins of sperm whale myoglobin (Mb) that exhibit altered axial ligations were constructed by site-directed mutagenesis of a synthetic gene for sperm whale myoglobin. Substitution of distal pocket residues, histidine E7 and valine E11, with tyrosine and glutamic acid generated His(E7)Tyr Mb and Val(E11)Glu Mb. The normal axial ligand residue, histidine F8, was also replaced with tyrosine, resulting in His(F8)Tyr Mb. These proteins are analogous in their substitutions to the naturally occurring hemoglobin M mutants (HbM). Tyrosine coordination to the ferric heme iron of His(E7)Tyr Mb and His(F8)Tyr Mb is suggested by optical absorption and EPR spectra and is verified by similarities to resonance Raman spectral bands assigned for iron-tyrosine proteins. His(E7)Tyr Mb is high-spin, six-coordinate with the ferric heme iron coordinated to the distal tyrosine and the proximal histidine, resembling Hb M Saskatoon [His(beta E7)Tyr], while the ferrous iron of this Mb mutant is high-spin, five-coordinate with ligation provided by the proximal histidine. His(F8)Tyr Mb is high-spin, five-coordinate in both the oxidized and reduced states, with the ferric heme iron liganded to the proximal tyrosine, resembling Hb M Iwate [His(alpha F8)Tyr] and Hb M Hyde Park [His(beta F8)Tyr]. Val(E11)Glu Mb is high-spin, six-coordinate with the ferric heme iron liganded to the F8 histidine. Glutamate coordination to the ferric iron of this mutant is strongly suggested by the optical and EPR spectral features, which are consistent with those observed for Hb M Milwaukee [Val(beta E11)Glu]. The ferrous iron of Val(E11)Glu Mb exhibits a five-coordinate structure with the F8 histidine-iron bond intact.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel heme and peroxide-dependent tryptophan-tyrosine cross-link in a mutant of cytochrome c peroxidase

The crystal structure of a cytochrome c peroxidase mutant where the distal catalytic His52 is converted to Tyr reveals that the tyrosine side-chain forms a covalent bond with the indole ring nitrogen atom of Trp51. We hypothesize that this novel bond results from peroxide activation by the heme iron followed by oxidation of Trp51 and Tyr52. This hypothesis has been tested by incorporation of a redox-inactive Zn-protoporphyrin into the protein, and the resulting crystal structure shows the absence of a Trp51-Tyr52 cross-link. Instead, the Tyr52 side-chain orients away from the heme active-site pocket, which requires a substantial rearrangement of residues 72-80 and 134-144. Additional experiments where heme-containing crystals of the mutant were treated with peroxide support our hypothesis that this novel Trp-Tyr cross-link is a peroxide-dependent process mediated by the heme iron.     

The optical spectra of fluoride complexes can effectively probe H-bonding interactions in the distal cavity of heme proteins

Fluoride complexes of heme proteins are characterized by unique spectroscopic properties, that provide a simple and direct means to monitor the interactions of the distal heme pocket environment with the iron-bound ligand. In particular, a strong correlation has been demonstrated between the wavelength of the iron-porphyrin charge transfer band at 600-620 nm (CT1) and the strength of H-bonding donation from the distal amino acid side chains to the fluoride ion. In parallel, resonance Raman spectra with excitation within either the CT1 band or the charge transfer band at 450-460 nm (CT2) have revealed that the iron-fluoride stretching frequency is directly affected by H-bonding to the fluoride ion. On this basis, globins and peroxidases display distinct spectroscopic features, which are strongly dependent on the capability of their distal residues (i.e. histidine, arginine and tryptophan) to be involved in H-bonding with the ligand. In particular, in peroxidases strong H-bonding corresponds to a low iron-fluoride stretching frequency and to a red-shifted CT1 band. The reverse is observed in myoglobin. Interestingly, a truncated hemoglobin of microbial origin (Thermobifida fusca) investigated in the present work, displays the specific spectroscopic signature of a peroxidase, in agreement with the presence of strong H-bonding residues, i.e., tyrosine and tryptophan, within the distal pocket.     

Role of distal arginine in early sensing intermediates in the heme domain of the oxygen sensor FixL

FixL is a bacterial heme-based oxygen sensor, in which release of oxygen from the sensing PAS domain leads to activation of an associated kinase domain. Static structural studies have suggested an important role of the conserved residue arginine 220 in signal transmission at the level of the heme domain. To assess the role of this residue in the dynamics and properties of the initial intermediates in ligand release, we have investigated the effects of R220X (X = I, Q, E, H, or A) mutations in the FixLH heme domain on the dynamics and spectral properties of the heme upon photolysis of O(2), NO, and CO using femtosecond transient absorption spectroscopy. Comparison of transient spectra for CO and NO dissociation with steady-state spectra indicated less strain on the heme in the ligand dissociation species for all mutants compared to the wild type (WT). For CO and NO, the kinetics were similar to those of the wild type, with the exception of (1) a relatively low yield of picosecond NO rebinding to R220A, presumably related to the increase in the free volume of the heme pocket, and (2) substantial pH-dependent picosecond to nanosecond rebinding of CO to R220H, related to formation of a hydrogen bond between CO and histidine 220. Upon excitation of the complex bound with the physiological sensor ligand O(2), a 5-8 ps decay phase and a nondecaying (>4 ns) phase were observed for WT and all mutants. The strong distortion of the spectrum associated with the decay phase in WT is substantially diminished in all mutant proteins, indicating an R220-induced role of the heme in the primary intermediate in signal transmission. Furthermore, the yield of dissociated oxygen after this phase ( approximately 10% in WT) is increased in all mutants, up to almost unity in R220A, indicating a key role of R220 in caging the oxygen near the heme through hydrogen bonding. Molecular dynamics simulations corroborate these findings and suggest motions of O(2) and arginine 220 away from the heme pocket as a second step in the signal pathway on the 50 ps time scale.     

EPR characterization of the stereochemistry of the distal heme pocket of the engineered human myoglobin mutants.

Recombinant human myoglobin mutants with the distal histidine residue replaced by Leu, Val, or Gln residues have been prepared by site-directed mutagenesis and expression in Escherichia coli. The recombinant apomyoglobin proteins have been successfully reconstituted with cobaltous protoporphyrin IX to obtain cobalt myoglobin mutant proteins, and the role of the distal histidine residue on the interaction between the bound ligand and the myoglobin molecule has been studied by EPR spectroscopy. We found that the distal histidine residue is significant in the orientation of the bound oxygen molecule. Low temperature photolysis experiments on both oxy cobalt proteins and ferric nitric oxide complexes indicated that the nature of the photolyzed form depends on the steric crowding of the distal heme pocket. To our surprise, the distal Leu mutant has a less restricted, less sterically crowded distal heme pocket than that of the distal Val mutant myoglobin, despite the fact that Leu has a larger side chain volume than Val. Our results demonstrate that the distal heme pocket steric crowding is not necessarily related to the side chain volume of the E7 residue.     

Preparation and spectral characterization of the heme d1.apomyoglobin complex: an unusual protein environment for the substrate-binding heme of Pseudomonas cytochrome oxidase

The heme d1 prosthetic group isolated from Pseudomonas cytochrome oxidase combines with apomyoglobin to form a stable, optically well-defined complex. Addition of ferric heme d1 quenches apomyoglobin tryptophan fluorescence suggesting association in a 1:1 molar ratio. Optical absorption maxima for heme d1.apomyoglobin are at 629 and 429 nm before, and 632 and 458 nm after dithionite reduction; they are distinct from those of heme d1 in aqueous solution but more similar to those unobscured by heme c in Pseudomonas cytochrome oxidase. Cyanide, carbon monoxide and imidazole alter the spectrum of heme d1.apomyoglobin demonstrating axial coordination to heme d1 by exogeneous ligands. The cyanide-induced optical difference spectra exhibit isosbestic points, and a Scatchard-like analysis yields a linear plot with an apparent dissociation constant of 4.2 X 10(-5) M. However, carbon monoxide induces two absorption spectra with Soret maxima at 454 or 467 nm, and this duplicity, along with a shoulder that correlates with the latter before binding, suggests multiple carbon monoxide and possibly heme d1 orientations within the globin. The 50-fold reduction in cyanide affinity over myoglobin is more consistent with altered heme pocket interactions than the intrinsic electronic differences between the two hemes. However, stability of the heme d1.apomyoglobin complex is verified further by the inability to separate heme d1 from globin during dialysis and column chromatography in excess cyanide or imidazole. This stability, together with a comparison between spectra of ligand-free and -bound derivatives of heme d1-apomyoglobin and heme d1 in solution, implies that the prosthetic group is coordinated in the heme pocket through a protein-donated, strong-field ligand. Furthermore, the visible spectrum of heme d1.apomyoglobin varies minimally with ligand exchange, in contrast to the Soret, which suggests that much spectral information concerning heme d1 coordination in the oxidase is lost by interference from heme c absorption bands. A comparison of the absorption spectra of heme d1.apomyoglobin and Pseudomonas cytochrome oxidase, together with a critical examination of the previous axial ligand assignments from magnetic resonance techniques in the latter, implies that it is premature to accept the assignment of bishistidine heme d1 coordination in oxidized, ligand-free oxidase and other iron-isobacteriochlorin-containing enzymes.     

A model for ligand binding to hexacoordinate hemoglobins

Hexacoordinate hemoglobins are heme proteins capable of reversible intramolecular coordination of the ligand binding site by an amino acid side chain from within the heme pocket. Examples of these proteins are found in many living organisms ranging from prokaryotes to humans. The nonsymbiotic hemoglobins (nsHbs) are a class of hexacoordinate heme proteins present in all plants. The nsHb from rice (rHb1) has been used as a model system to develop methods for determining rate constants characterizing binding and dissociation of the His residue responsible for hexacoordination. Measurement of these reactions exploits laser flash photolysis to initiate the reaction from the unligated, pentacoordinate form of the heme protein. A model for ligand binding is presented that incorporates the reaction following rapid mixing with the reaction starting from the pentacoordinate hemoglobin (Hb). This model is based on results indicating that ligand binding to hexacoordinate Hbs is not a simple combination of competing first order (hexacoordination) and second order (exogenous ligand binding) reactions. Ligand binding following rapid mixing is a multiphasic reaction displaying time courses ranging from milliseconds to minutes. The new model incorporates a "closed", slow reacting form of the protein that is not at rapid equilibrium with the reactive conformation. It is also demonstrated that formation of the closed protein species is not dependent on hexacoordination.     

Structure and kinetic properties of Paracoccus pantotrophus cytochrome cd1 nitrite reductase with the d1 heme active site ligand tyrosine 25 replaced by serine

The 1.4-A crystal structure of the oxidized state of a Y25S variant of cytochrome cd(1) nitrite reductase from Paracoccus pantotrophus is described. It shows that loss of Tyr(25), a ligand via its hydroxy group to the iron of the d(1) heme in the oxidized (as prepared) wild-type enzyme, does not result in a switch at the c heme of the unusual bishistidinyl coordination to the histidine/methionine coordination seen in other conformations of the enzyme. The Ser(25) side chain is seen in two positions in the d(1) heme pocket with relative occupancies of approximately 7:3, but in neither case is the hydroxy group bound to the iron atom; instead, a sulfate ion from the crystallization solution is bound between the Ser(25) side chain and the heme iron. Unlike the wild-type enzyme, the Y25S mutant is active as a reductase toward nitrite, oxygen, and hydroxylamine without a reductive activation step. It is concluded that Tyr(25) is not essential for catalysis of reduction of any substrate, but that the requirement for activation by reduction of the wild-type enzyme is related to a requirement to drive the dissociation of this residue from the active site. The Y25S protein retains the d(1) heme less well than the wild-type protein, suggesting that the tyrosine residue has a role in stabilizing the binding of this cofactor.     

X-Ray crystallographic structural characteristics of Arabidopsis hemoglobin I and their functional implications

Genome of the model dicot flowering plant, Arabidopsis thaliana, a popular tool for understanding molecular biology of plant physiology, encodes all three classes of plant hemoglobins that differ in their sequence, ligand binding and spectral properties. As such these globins are of considerable attention. Crystal structures of few members of plant class I nonsymbiotic hemoglobin have been described earlier. Here we report the crystal structure of Arabidopsis class I hemoglobin (AHb1) to 2.2 ? and compare its key features with the structures of similar nonsymbiotic hemoglobin from other species. Crystal structure of AHb1 is homologous to the related members with similar globin fold and heme pocket architecture. The structure is homodimeric in the asymmetric unit with both distal and proximal histidines coordinating to the heme iron atom. Residues lining the dimeric interface are also conserved in AHb1 with the exception of additional electrostatic interaction between H112 and E113 of each subunit and that involving Y119 through two water molecules. In addition, differences in heme pocket non-covalent interactions, a novel Ser residue at F7 position, Xe binding site variability, internal cavity topology differences, CD loop conformation and stability and other such properties might explain kinetic variability in AHb1. Detailed cavity analysis of AHb1 showed the presence of a novel long tunnel connecting the distal pockets of both the monomers. Presence of such tunnel, along with conformational heterogeneity observed in the two chains, might suggest cooperative ligand binding and support its role in NO scavenging. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.