SAXS structure of homodimeric oxyHemoglobin III from bivalve Lucina pectinata.

Hemoglobin III (HbIII) is one of the two oxygen reactive hemoproteins present in the bivalve, Lucina pectinata. The clam inhabits a sulfur-rich environment and HbIII is the only hemoprotein present in the system which does not yet have a structure described elsewhere. It is known that HbIII exists as a heterodimer with hemoglobin II (HbII) to generate the stable Oxy(HbII-HbIII) complex but it remains unknown if HbIII can form a homodimeric species. Here, a new chromatographic methodology to separate OxyHbIII from the HbII-HbIII dimer has been developed, employing a fast performance liquid chromatography and ionic exchange chromatography column. The nature of OxyHbIII in solution at concentrations from 1.6 mg/mL to 20.4 mg/mL was studied using small angle X-ray scattering (SAXS). The results show that at all concentrations, the Oxy(HbIII-HbIII) dimer dominates in solution. However, as the concentration increases to nonphysiological values, 20.4 mg/mL, HbIII forms a 30% tetrameric fraction. Thus, there is a direct relationship between the Oxy(HbIII-HbIII) oligomeric form and hemoglobin concentration. We suggest it is likely that the OxyHbIII dimer contributes to active oxygen transport in tissues of L pectinata, where the Oxy(HbII-HbIII) complex is not present.

Characterization of Recombinant His-Tag Protein Immobilized onto Functionalized Gold Nanoparticles.

The recombinant polyhistidine-tagged hemoglobin I ((His)6-rHbI) from the bivalve Lucina pectinata is an ideal biocomponent for a hydrogen sulfide (H2S) biosensor due to its high affinity for H2S. In this work, we immobilized (His)6-rHbI over a surface modified with gold nanoparticles functionalized with 3-mercaptopropionic acid complexed with nickel ion. The attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) analysis of the modified-gold electrode displays amide I and amide II bands characteristic of a primarily α-helix structure verifying the presence of (His)6-rHbI on the electrode surface. Also, X-ray photoelectron spectroscopy (XPS) results show a new peak after protein interaction corresponding to nitrogen and a calculated overlayer thickness of 5.3 nm. The functionality of the immobilized hemoprotein was established by direct current potential amperometry, using H2S as the analyte, validating its activity after immobilization. The current response to H2S concentrations was monitored over time giving a linear relationship from 30 to 700 nM with a corresponding sensitivity of 3.22 × 10-3 nA/nM. These results confirm that the analyzed gold nanostructured platform provides an efficient and strong link for polyhistidine-tag protein immobilization over gold and glassy carbon surfaces for a future biosensors development.

Purple Fibrils: A New Type of Protein Chromophore.

A purple color is formed during the fibrillation of lysozyme, a well-studied protein lacking a prosthetic group. The application of Raman spectroscopy, electron paramagnetic resonance and UV-vis absorption spectroscopy indicates the formation of a sulfur∴π-bonded radical cation due to the methionine-phenylalanine interaction, which is consistent with a small molecule model reported in the literature. A purple chromophore with characteristic 550 nm absorption is formed due to a specific orientation of the sulfur-centered radical cation and a phenyl ring stabilized by the fibril framework. A specific fibril conformation and the resulting formation of the chromophore are controlled reversibly by varying the pH. This is the first known example of a side chain self-assembled chromophore formed due to protein aggregation.

Reactivity and dynamics of H2S, NO, and O2 interacting with hemoglobins from Lucina pectinata.

Hemoglobin HbI from the clam Lucina pectinata is involved in H2S transport, whereas homologous heme protein HbII/III is involved in O2 metabolism. Despite similar tertiary structures, HbI and HbII/III exhibit very different reactivity toward heme ligands H2S, O2, and NO. To investigate this reactivity at the heme level, we measured the dynamics of ligand interaction by time-resolved absorption spectroscopy in the picosecond to nanosecond time range. We demonstrated that H2S can be photodissociated from both ferric and ferrous HbI. H2S geminately rebinds to ferric and ferrous out-of-plane iron with time constants (τgem) of 12 and 165 ps, respectively, with very different proportions of photodissociated H2S exiting the protein (24% in ferric and 80% in ferrous HbI). The Gln(E7)His mutation considerably changes H2S dynamics in ferric HbI, indicating the role of Gln(E7) in controling H2S reactivity. In ferric HbI, the rate of diffusion of H2S from the solvent into the heme pocket (kentry) is 0.30 µM(-1) s(-1). For the HbII/III-O2 complex, we observed mainly a six-coordinate vibrationally excited heme-O2 complex with O2 still bound to the iron. This explains the low yield of O2 photodissociation and low koff from HbII/III, compared with those of HbI and Mb. Both isoforms behave very differently with regard to NO and O2 dynamics. Whereas the amplitude of geminate rebinding of O2 to HbI (38.5%) is similar to that of myoglobin (34.5%) in spite of different distal heme sites, it appears to be much larger for HbII/III (77%). The distal Tyr(B10) side chain present in HbII/III increases the energy barrier for ligand escape and participates in the stabilization of bound O2 and NO.

Tyrosine B10 triggers a heme propionate hydrogen bonding network loop with glutamine E7 moiety.

Propionates, as peripheral groups of the heme active center in hemeproteins have been described to contribute in the modulation of heme reactivity and ligand selection. These electronic characteristics prompted the question of whether the presence of hydrogen bonding networks between propionates and distal amino acids present in the heme ligand moiety can modulate physiological relevant events, like ligand binding association and dissociation activities. Here, the role of these networks was evaluated by NMR spectroscopy using the hemoglobin I PheB10Tyr mutant from Lucina pectinata as model for TyrB10 and GlnE7 hemeproteins. (1)H-NMR results for the rHbICN PheB10Tyr derivative showed chemical shifts of TyrB10 OHη at 31.00ppm, GlnE7N(ε1)H/N(ε2)H at 10.66ppm/-3.27ppm, and PheE11 C(δ)H at 11.75ppm, indicating the presence of a crowded, collapsed, and constrained distal pocket. Strong dipolar contacts and inter-residues crosspeaks between GlnE7/6-propionate group, GlnE7/TyrB10 and TyrB10/CN suggest that this hydrogen bonding network loop between GlnE7, TyrB10, 6-propionate group, and the heme ligand contribute significantly to the modulation of the heme iron electron density as well as the ligand stabilization mechanism. Therefore, the network loop presented here support the fact that the electron withdrawing character of the hydrogen bonding is controlled by the interaction of the propionates and the nearby electronic environments contributing to the modulation of the heme electron density state. Thus, we hypothesize that in hemeproteins with similar electrostatic environment the flexibility of the heme-6-propionate promotes a hydrogen bonding network loop between the 6-propionate, the heme ligand and nearby amino acids, tailoring in this way the electron density in the heme-ligand moiety.

Using a reduced dimensionality model to compute the thermodynamic properties of finite polypeptide aggregates.

By implementing a simple reduced dimensionality model to describe the interactions in finite systems composed of two seven-amino-acid peptides, the thermodynamic properties of ordered and disordered aggregates were computed. Within this model, the hydrophobicity of each amino acid was varied, and the stability of the systems computed. Accurate averages in the canonical ensemble were obtained using various replica exchange Monte Carlo algorithms. Low and high temperature regions were encountered where the ordered and disordered aggregates were stabilized. It was observed that as the degree of hydrophobicity increased, the stability of the aggregates increased, with a significant energetic stabilization obtained for the ordered aggregates. Upon decreasing the concentration of the solution, the stability of the amorphous aggregates increased when compared to the ordered systems.

Bioinformatic Characterization and Molecular Evolution of the Lucina pectinata Hemoglobins.

(1) Introduction: Lucina pectinata is a clam found in sulfide-rich mud environments that has three hemoglobins believed to be responsible for the transport of hydrogen sulfide (HbILp) and oxygen (HbIILp and HbIIILp) to chemoautotrophic endosymbionts. The physiological roles and evolution of these globins in sulfide-rich environments are not well understood. (2) Methods: We performed bioinformatic and phylogenetic analyses with 32 homologous mollusk globin sequences. Phylogenetics suggests a first gene duplication resulting in sulfide binding and oxygen binding genes. A more recent gene duplication gave rise to the two oxygen-binding hemoglobins. Multidimensional scaling analysis of the sequence space shows evolutionary drift of HbIILp and HbIIILp, while HbILp was closer to the Calyptogena hemoglobins. Further corroboration is seen by conservation in the coding region of hemoglobins from L. pectinata compared to those from Calyptogena. (3) Conclusions: Presence of glutamine in position E7 in organisms living in sulfide-rich environments can be considered an adaptation to prevent loss of protein function. In HbILp a substitution of phenylalanine in position B10 is accountable for its unique reactivity towards H2S. It appears that HbILp has been changing over time, apparently not subject to functional constraints of binding oxygen, and acquired a unique function for a specialized environment.

Extreme differences between hemoglobins I and II of the clam Lucina pectinalis in their reactions with nitrite.

The clam Lucina pectinalis supports its symbiotic bacteria by H2S transport in the open and accessible heme pocket of Lucina Hb I and by O2 transport in the narrow and crowded heme pocket of Lucina Hb II. Remarkably, air-equilibrated samples of Lucina Hb I were found to be more rapidly oxidized by nitrite than any previously studied Hb, while those of Lucina Hb II showed an unprecedented resistance to oxidation induced by nitrite. Nitrite-induced oxidation of Lucina Hb II was enabled only when O2 was removed from its active site. Structural analysis revealed that O2 "clams up" the active site by hydrogen bond formation to B10Tyr and other distal-side residues. Quaternary effects further restrict nitrite entry into the active site and stabilize the hydrogen-bonding network in oxygenated Lucina Hb II dimers. The dramatic differences in nitrite reactivities of the Lucina Hbs are not related to their O2 affinities or anaerobic redox potentials, which were found to be similar, but are instead a result of differences in accessibility of nitrite to their active sites; i.e. these differences are due to a kinetic rather than thermodynamic effect. Comparative studies revealed heme accessibility to be a factor in human Hb oxidation by nitrite as well, as evidenced by variations of rates of nitrite-induced oxidation that do not correlate with R and T state differences and inhibition of oxidation rate in the presence of O2. These results provide a dramatic illustration of how evolution of active sites with varied heme accessibility can moderate the rates of inner-sphere oxidative reactions of Hb and other heme proteins.

Fluoride binding to characteristic heme-pocket centers: Insights into ligand stability.

The studies on the L. pectinata hemoglobins (HbI, HbII, and HbIII) are essential because of their biological roles in hydrogen sulfide transport and metabolism. Variation in the pH could also play a role in the transport of hydrogen sulfide by HbI and oxygen by HbII and HbIII, respectively. Here, fluoride binding was used to further understand the structural properties essential for the molecular mechanism of ligand stabilization as a function of pH. The data allowed us to gain insights into how the physiological roles of HbI, HbII, HbIII, adult hemoglobin (A-Hb), and horse heart myoglobin (Mb) have an impact on the heme-bound fluoride stabilization. In addition, analysis of the vibrational assignments of the met-cyano heme complexes shows varied strength interactions of the heme-bound ligand. The heme pocket composition properties differ between HbI (GlnE7 and PheB10) and HbII/HbIII (GlnE7 and TyrB10). Also, the structural GlnE7 stereo orientation changes between HbI and HbII/HbIII. In HbI, its carbonyl group orients towards the heme iron, while in HbII/HbIII, the amino group occupies this position. Therefore, in HbI, the interactions to the heme-bound fluoride ion, cyanide, and oxygen with GlnE7 via H-bonding are not probable. Still, the aromatic cage PheB10, PheCD1, and PheE11 may contribute to the observed stabilization. However, a robust H-bonding networking stabilizes HbII and HbIII, heme-bound fluoride, cyanide, and oxygen ligand with the OH and NH2 groups of TyrB10 and GlnE7, respectively. At the same time, A-Hb and Mb have moderate but similar ligand interactions controlled by their respective distal E7 histidine.

Structural determinants for the formation of sulfhemeprotein complexes.

Several hemoglobins were explored by UV-Vis and resonance Raman spectroscopy to define sulfheme complex formation. Evaluation of these proteins upon the reaction with H(2)O(2) or O(2) in the presence of H(2)S suggest: (a) the formation of the sulfheme derivate requires a HisE7 residue in the heme distal site with an adequate orientation to form an active ternary complex; (b) that the ternary complex intermediate involves the HisE7, the peroxo or ferryl species, and the H(2)S molecule. This moiety precedes and triggers the sulfheme formation.

Two-step counterdiffusion protocol for the crystallization of haemoglobin II from Lucina pectinata in the pH range 4-9.

Lucina pectinata haemoglobin II (HbII) transports oxygen in the presence of H(2)S to the symbiotic system in this bivalve mollusc. The composition of the haem pocket at the distal site includes TyrB10 and GlnE7, which are very common in other haem proteins. Obtaining crystals of oxyHbII at various pH values is required in order to elucidate the changes in the conformations of TyrB10 and GlnE7 and structural scenarios induced by changes in pH. Here, the growth of crystals of oxyHbII using the capillary counterdiffusion (CCD) technique at various pH values using a two-step protocol is reported. In the first step, a mini-screen was used to validate sodium formate as the best precipitating reagent for the growth of oxyHbII crystals. The second step, a pH screen typically used for optimization, was used to produce crystals in the pH range 4-9. Very well faceted prismatic ruby-red crystals were obtained at all pH values. X-ray data sets were acquired using synchrotron radiation of wavelength 0.886 A (for the crystals obtained at pH 5) and 0.908 A (for those obtained at pH 4, 8 and 9) to maximum resolutions of 3.30, 1.95, 1.85 and 2.00 A for the crystals obtained at pH 4, 5, 8 and 9, respectively. All of the crystals were isomorphous and belonged to space group P4(2)2(1)2.

A reaction pathway to compound 0 intermediates in oxy-myoglobin through interactions with hydrogen sulfide and His64.

Myoglobin (Mb) binds oxygen with high affinity as a low spin singlet complex and thus functions as an oxygen storage protein. Yet, hybrid Density Functional Theory/Molecular Mechanical (DFT/MM) calculations of oxy-Mb models predict that the O2 bond is much less resistant to breaking in the presence of hydrogen sulfide (H2S) compared with water. Specifically, a hydrogen atom from H2S can be transferred to the distal oxygen atom through homolytic cleavage of the S-H bond to form the intermediate Compound (Cpd) 0 structure and a thiyl radical. In the presence of a neutral His64 (Nε protonation, His64-ε) and H2S, only a metastable Cpd 0 would be formed as the active site is devoid of any additional proton donor to fully break the O2 bond. In contrast, the calculations predict that the triplet state is significantly favored over the open shell singlet diradical state throughout the entire reaction coordinate in the presence of H2S and a positively charged His64. Furthermore, a positively charged His64 can readily donate a proton to Cpd 0 to fully break the O2 bond resulting in a configuration analogous to reported reaction models of a hemoglobin mutant bound to H2O2 with H2S present. Typically, exotic techniques are required to generate Cpd 0 but under the conditions just described the intermediate is readily detected in UV-Vis spectra at room temperature. The effect is observed as a 2 nm red shift of the Soret band from 414 nm to 416 nm (pH 5.0, His64-εδ) and from 416 nm to 418 nm (pH 6.6, His64-ε).

Lucina pectinata oxyhemoglobin (II-III) heterodimer pH susceptibility.

Lucina pectinata live in high concentrations of hydrogen sulfide (H2S) and contains one hemoglobin, Hemoglobin I (HbI), transporting H2S and two hemoglobins, Hemoglobin II (HbII) and Hemoglobin (HbIII), transferring dioxygen to symbionts. HbII and HbIII contain B10 tyrosine (Tyr) and E7 glutamine (Gln) in the heme pocket generating an efficient hydrogen bonding network with the (HbII-HbIII)-O2 species, leading to very low ligand dissociation rates. The results indicate that the oxy-hemeprotein is susceptible to pH from 4 to 9, at acidic conditions, and as a function of the potassium ferricyanide concentration, 100% of the met-aquo derivative is produced. Without a strong oxidant, pH 5 generates a small concentration of the met-aquo complex. The process is accelerated by the presence of salts, as indicated by the crystallization structures and UV-Vis spectra. The results suggest that acidic pH generates conformational changes associated with B10 and E7 heme pocket amino acids, weakening the (HbII-HbIII)-O2 hydrogen bond network. The observation is supported by X-ray crystallography, since at pH 4 and 5, the heme-Fe tends to oxidize, while at pH 7, the oxy-heterodimer is present. Conformational changes also are observed at higher pH by the presence of a 605 nm transition associated with the iron heme-Tyr interaction. Therefore, pH is one crucial factor regulating the (HbII-HbIII)-O2 complex hydrogen-bonding network. Thus, it can be proposed that the hydrogen bonding adjustments between the heme bound O2 and the Tyr and Gln amino acids contribute to oxygen dissociation from the (HbII-HbIII)-O2 system.

Factors controlling the reactivity of hydrogen sulfide with hemeproteins.

Hemoglobin I (HbI) from the clam Lucina pectinata is an intriguing hemeprotein that binds and transports H(2)S to sulfide-oxidizing chemoautotrophic bacteria to maintain a symbiotic relationship and to protect the mollusk from H(2)S toxicity. Single point mutations at E7, B10, and E11 were introduced into the HbI heme pocket to define the reactivity of sulfide with hemeproteins. The functional and structural properties of mutant and wild-type recombinant proteins were first evaluated using the well-known ferrous CO and O(2) derivatives. The effects of these mutations on the ferric environment were then studied in the metaquo and hydrogen sulfide derivatives. The results obtained with the ferrous HbI mutants show that all the E7 substitutions and the PheB10Tyr mutation influence directly CO and O(2) binding and stability while the B10 and E11 substitutions induce distal structural rearrangements that affect ligand entry and escape indirectly. For the metaquo-GlnE7His, -PheB10Val, -PheB10Leu, and -E11 variants, two individual distal structures are suggested, one of which is associated with H-bonding interactions between the E7 residues and the bound water. Similar H-bonding interactions are invoked for these HbI-H(2)S mutant derivatives and the rHbI, altering in turn sulfide reactivity within these protein samples. This is evident in the resonance Raman spectra of these HbI-H(2)S complexes, which show reduction of heme iron as judged by the appearance of the nu(4) oxidation state marker at 1356 cm(-1), indicative of heme-Fe(II) species. This reduction process depends strongly on distal mutations showing faster reduction for those HbI mutants exhibiting the strongest H-bonding interactions. Overall, the results presented here show that (a) H(2)S association is regulated by external kinetic barriers, (b) H(2)S release is controlled by two competing reactions involving simple sulfide dissociation and heme reduction, (c) at high H(2)S concentrations, reduction of the ferric center dominates, and (d) reduction of the heme is also enhanced in those HbI mutants having polar distal environments.

Crystallization and diffraction patterns of the oxy and cyano forms of the Lucina pectinata haemoglobins complex.

The native oxygen-carrier haemoglobins complex (HbII-III) is composed of haemoglobin II (HbII) and haemoglobin III (HbIII), which are found in the ctenidia tissue of the bivalve mollusc Lucina pectinata. This protein complex was isolated and purified from its natural source and crystallized using the vapour-diffusion and capillary counter-diffusion methods. Oxy and cyano derivatives of the complex crystallized using several conditions, but the best crystals in terms of quality and size were obtained from sodium formate pH 5 using the counter-diffusion method in a single capillary. Crystals of the oxy and cyano complexes, which showed a ruby-red colour and nonsingular prismatic shapes, scattered X-rays to resolution limits of 2.15 and 2.20 A, respectively, using a 0.886 A synchrotron-radiation source. The crystals belonged to the tetragonal system, space group P4(2)2(1)2, with unit-cell parameters a = b = 74.07, c = 152.07 and a = b = 73.83, c = 152.49 A for the oxy and cyano complexes, respectively. The asymmetric unit of both crystals is composed of a single copy of the heterodimer, with Matthew coefficients (V(M)) of 3.08 and 3.06 A(3) Da(-1) for the oxy and cyano complexes, respectively, which correspond to a solvent content of approximately 60.0% by volume.

Characterization of the full length mRNA coding for Lucina pectinata HbIII revealed an alternative polyadenylation site.

Lucina pectinata is a bivalve mollusk that lives in the Southwestern coast of Puerto Rico and houses intracellular symbiotic bacteria. This peculiar organism contains three types of hemoglobin, each characterized by distinct physico-chemical properties. Hemoglobin I (HbI) is a sulfide-reactive protein that reacts with H(2)S to form ferric hemoglobin sulfide. In contrast, hemoglobin II and III are oxygen-reactive proteins that remain oxygenated in the presence of hydrogen sulfide. The partial coding region contained in the cDNA sequences we have cloned confirmed the L. pectinata HbIII amino sequence reported in the NCBI protein database) with a single amino acid difference (Asn72Asp; AsnE12Asp). The characterization of the full length mRNA coding for L. pectinata HbIII revealed an alternative polyadenylation site and an alternate transcription start site. The open reading frame (ORF) of the HbIII cDNA is composed of 459nts containing 153 codons. The initiation codon is preceded by 62 nts of untranslated region (5'UTR), whereas two 3'UTR regions of 640 nt and 455 nt long were identified, revealing the presence of alternative polyadenylation sites. Isoforms of the 3'UTR of HbIII only differed in the length of their sequences. It has been hypothesized that alternative polyadenylation acts through shortening of mRNA to regulate RNA localization, translation and stability. Interestingly, the HbIII mRNA is the only one of all the hemoglobin mRNAs from L. pectinata characterized so far with more than one 3'UTR. Primer extension products suggest two closely located start sites of HbIII mRNA transcription. We suggest that the L. pectinata hemoglobin genes may be under different cellular controls that direct them to exert their particular functions. These hypotheses need to be tested by functional studies and analysis of the regulatory elements of the cognate genes for L. pectinata hemoglobins.

Charge Transfer and π to π* Transitions in the Visible Spectra of Sulfheme Met Isomeric Structures.

Since the 1863 discovery of a new green hemoglobin derivative called "sulfhemoglobin", the nature of the characteristic 618 nm absorption band has been the subject of several hypotheses. The experimental spectra are a function of the observation time and interplay between two major sulfheme isomer concentrations (a three- and five-membered ring adduct), with the latter being the dominant isomer at longer times. Thus, time-dependent density functional theory (TDDFT) was used to calculate the sulfheme excited states and visualize the highest occupied molecular orbitals (HOMOs) and lowest unoccupied MOs (LUMOs) of both isomers in order to interpret the transitions between them. These two isomers have distinguishable a1u and a2u HOMO energies. Formation of the three-membered ring SA isomeric structure decreases the energy of the HOMO a1u and a2u orbitals compared to the unmodified heme due to the electron-withdrawing, sulfur-containing, three-membered ring. Conversely, formation of the SC isomeric structure decreases the energy of the HOMO a1u and a2u orbitals due to the electron-withdrawing, sulfur-containing, five-membered ring. The calculations reveal that the absorption spectrum within the 700 nm region arises from a mixture of MOs but can be characterized as π to π* transitions, while the 600 nm region is characterized by π to dπ (d yz, d xz) transitions having components of a deoxy-like derivative.

Characterization of Histone Genes from the Bivalve Lucina Pectinata.

Lucina pectinata is a clam that lives in sulfide-rich environments and houses intracellular sulfide-oxidizing endosymbionts. To identify new Lucina pectinata proteins, we produced libraries for genome and transcriptome sequencing and assembled them de novo. We searched for histone-like sequences using the Lucina pectinata histone H3 partial nucleotide sequence against our previously described genome assembly to obtain the complete coding region and identify H3 coding sequences from mollusk sequences in Genbank. Solen marginatus histone nucleotide sequences were used as query sequences using the genome and transcriptome assemblies to identify the Lucina pectinata H1, H2A, H2B and H4 genes and mRNAs and obtained the complete coding regions of the five histone genes by RT-PCR combined with automated Sanger DNA sequencing. The amino acid sequence conservation between the Lucina pectinata and Solen marginatus histones was: 77%, 93%, 83%, 96% and 97% for H1, H2A, H2B, H3 and H4, respectively. As expected, the H3 and H4 proteins were the most conserved and the H1 proteins were most similar to H1's from aquatic organisms like Crassostrea gigas, Aplysia californica, Mytilus trossulus and Biomphalaria glabrata. The Lucina pectinata draft genome and transcriptome assemblies, obtained by semiconductor sequencing, were adequate for identification of conserved proteins as evidenced by our results for the histone genes.

Hemoglobin I from Lucina pectinata: a model for distal heme-ligand control.

Lucina pectinata hemoglobin I (HbI), which is a ferric sulfide-reactive hemeprotein, contains a distal pocket characterized by the presence of GlnE7 and PheB10. To elucidate the structural-functional properties of HbI, oxygen binding kinetics and FTIR studies with recombinant HbI (rHbI) and a set of mutants were conducted using CO and CN- as sensors of the hemeprotein environment. Three nuCO modes were observed for rHbI at 1936 cm(-1) (A3, closed conformer) 1950 cm(-1) (A1,2, closed conformer) and 1960 cm(-1) (A0, open conformer). These nuCO were affected by substitution of GlnE7 and PheB10 in the CO complexes. The contribution of GlnE7 is demonstrated when this residue is replaced with Asn, Val or His. For instance, decreasing the positive electrostatic environment with GlnE7Val, causes an increase of 65% in the population of A0 and the disappearance and 55% reduction of the population of the A1,2 and A3 respectively. The contribution of PheB10 to the stabilization of ligands is also observed in the Leu and Tyr mutants. The PheB10Leu mutation produced an 8% decrease in the population of the A3 conformer while that of the A1,2 configuration increased by 30%. This suggests that GlnE7 and PheB10 contribute to the A3 conformer stabilizing the CO in a closed configuration. With CN- as probe no substantial differences in the nuCN was observed upon substitution of GlnE7 by Val while a slight down shift in the nuCN from 2120 cm(-1) to 2117 cm(-1) was observed in the PheB10Leu mutant. This implies that in HbICN GlnE7 moves away from the binding site while PheB10 remains in the vicinity of the bound CN-. Here, a mechanism in which the flexibility of the distal protein matrix coupled with hemeporphyrin movement toward a different configuration is suggested as an important process in the H2S transport and delivery in hemoglobin I.

Engineered (Lys)6-Tagged Recombinant Sulfide-Reactive Hemoglobin I for Covalent Immobilization at Multiwalled Carbon Nanotubes.

The recombinant HbI was fused with a poly-Lys tag ((Lys)6-tagged rHbI) for specific-site covalent immobilization on two carbon nanotube transducer surfaces, i.e., powder and vertically aligned carbon nanotubes. The immobilization was achieved by following two steps: (1) generation of amine-reactive ester from the carboxylic acid groups of the surfaces and (2) coupling these groups with the amine groups of the Lys-tag. We analyzed the immobilization process using different conditions and techniques to differentiate protein covalent attachment from physical adsorption. Fourier transform infrared microspectroscopy data showed a 14 cm-1 displacement of the protein's amide I and amide II peaks to lower the frequency after immobilization. This result indicates a covalent attachment of the protein to the surface. Differences in the morphology of the carbon substrate with and without (Lys)6-tagged rHbI confirmed protein immobilization, as observed by transmission electron microscopy. The electrochemical studies, which were performed to evaluate the redox center of the immobilized protein, show a confinement suitable for an efficient electron transfer system. More importantly, the electrochemical studies allowed determination of a redox potential for the new (Lys)6-tagged rHbI. The data show that the protein is electrochemically active and retains its biological activity toward H2S.