SARS-CoV-2 Specific Nanobodies Neutralize Different Variants of Concern and Reduce Virus Load in the Brain of h-ACE2 Transgenic Mice.

Since the beginning of the COVID-19 pandemic, there has been a significant need to develop antivirals and vaccines to combat the disease. In this work, we developed llama-derived nanobodies (Nbs) directed against the receptor binding domain (RBD) and other domains of the Spike (S) protein of SARS-CoV-2. Most of the Nbs with neutralizing properties were directed to RBD and were able to block S-2P/ACE2 interaction. Three neutralizing Nbs recognized the N-terminal domain (NTD) of the S-2P protein. Intranasal administration of Nbs induced protection ranging from 40% to 80% after challenge with the WA1/2020 strain in k18-hACE2 transgenic mice. Interestingly, protection was associated with a significant reduction in virus replication in nasal turbinates and a reduction in virus load in the brain. Employing pseudovirus neutralization assays, we identified Nbs with neutralizing capacity against the Alpha, Beta, Delta, and Omicron variants, including a Nb capable of neutralizing all variants tested. Furthermore, cocktails of different Nbs performed better than individual Nbs at neutralizing two Omicron variants (B.1.529 and BA.2). Altogether, the data suggest the potential of SARS-CoV-2 specific Nbs for intranasal treatment of COVID-19 encephalitis.

Binding mechanism of disulfide species to ferric hemeproteins: The case of metmyoglobin.

The interactions of the heme iron of hemeproteins with sulfide and disulfide compounds are of potential interest as physiological signaling processes. While the interaction with hydrogen sulfide has been described computationally and experimentally, the reaction with disulfide, and specifically the molecular mechanism for ligand binding has not been studied in detail. In this work, we study the association process for disulfane and its conjugate base disulfanide at different pH conditions. Additionally, by means of advanced sampling techniques based on multiple steered molecular dynamics, we provide free energy profiles for ligand migration for both acid/base species, showing a similar behavior to the previously reported for the related H2S/HS¯ pair. Finally, we studied the ligand interchange reaction (H2O/H2S, HS¯ and H2O/HSSH, HSS¯) by means of hybrid quantum mechanics-molecular mechanics calculations. We show that the anionic species are able to displace more efficiently the H2O bound to the iron, and that the H-bond network in the distal cavity can help the neutral species to perform the reaction. Altogether, we provide a molecular explanation for the experimental information and show that the global association process depends on a fine balance between the migration towards the active site and the ligand interchange reaction.

Autocatalytic Mechanism in the Anaerobic Reduction of Metmyoglobin by Sulfide Species.

The mechanism of the metal centered reduction of metmyoglobin (MbFeIII) by sulfide species (H2S/HS-) under an argon atmosphere has been studied by a combination of spectroscopic, kinetic, and computational methods. Asymmetric S-shaped time-traces for the formation of MbFeII at varying ratios of excess sulfide were observed at pH 5.3 < pH < 8.0 and 25 °C, suggesting an autocatalytic reaction mechanism. An increased rate at more alkaline pHs points to HS- as relevant reactive species for the reduction. The formation of the sulfanyl radical (HS•) in the slow initial phase was assessed using the spin-trap phenyl N-tert-butyl nitrone. This radical initiates the formation of S-S reactive species as disulfanuidyl/ disulfanudi-idyl radical anions and disulfide (HSSH•-/HSS•2- and HSS-, respectively). The autocatalysis has been ascribed to HSS-, formed after HSSH•-/HSS•2- disproportionation, which behaves as a fast reductant toward the intermediate complex MbFeIII(HS-). We propose a reaction mechanism for the sulfide-mediated reduction of metmyoglobin where only ferric heme iron initiates the oxidation of sulfide species. Beside the chemical interest, this insight into the MbFeIII/sulfide reaction under an argon atmosphere is relevant for the interpretation of biochemical aspects of ectopic myoglobins found on hypoxic tissues toward reactive sulfur species.

Reduction of metmyoglobin by inorganic disulfide species.

The mechanism of the metal centered reduction of metmyoglobin (MbFeIII) by inorganic disulfide species has been studied by combined spectroscopic and kinetic analyses, under argon atmosphere. The process is kinetically characterized by biexponential time traces, for variable ratios of excess disulfide to protein, in the pH interval 6.6-8.0. Using UV-vis and resonance Raman spectroscopies, we observed that MbFeIII is converted into a low spin hexacoordinated ferric complex, tentatively assigned as MbFeIII(HSS-)/MbFeIII(SS2-), in an initial fast step. The complex is slowly converted into a pentacoordinated ferrous form, assigned as MbFeII according to the resonance Raman records. The reduction is a pH-dependent process, but independent of the initial disulfide concentration, suggesting the unimolecular decomposition of the intermediate complex following a reductive homolysis. We estimated the rate of the fast formation of the complex at pH 7.4 (kon = 3.7 × 103 M-1 s-1), and a pKa2 = 7.5 for the equilibrium MbFeIII(HSS-)/MbFeIII(SS2-). Also, we estimated the rate for the slow reduction at the same pH (kred = 10-2 s-1). A reaction mechanism compliant with the experimental results is proposed. This mechanistic study provides a differential kinetic signature for the reactions of disulfide compared to sulfide species on metmyoglobin, which may be considered in other hemeprotein systems.

Nanobodies against SARS-CoV-2 reduced virus load in the brain of challenged mice and neutralized Wuhan, Delta and Omicron Variants.

In this work, we developed llama-derived nanobodies (Nbs) directed to the receptor binding domain (RBD) and other domains of the Spike (S) protein of SARS-CoV-2. Nanobodies were selected after the biopanning of two VHH-libraries, one of which was generated after the immunization of a llama (lama glama) with the bovine coronavirus (BCoV) Mebus, and another with the full-length pre-fused locked S protein (S-2P) and the RBD from the SARS-CoV-2 Wuhan strain (WT). Most of the neutralizing Nbs selected with either RBD or S-2P from SARS-CoV-2 were directed to RBD and were able to block S-2P/ACE2 interaction. Three Nbs recognized the N-terminal domain (NTD) of the S-2P protein as measured by competition with biliverdin, while some non-neutralizing Nbs recognize epitopes in the S2 domain. One Nb from the BCoV immune library was directed to RBD but was non-neutralizing. Intranasal administration of Nbs induced protection ranging from 40% to 80% against COVID-19 death in k18-hACE2 mice challenged with the WT strain. Interestingly, protection was not only associated with a significant reduction of virus replication in nasal turbinates and lungs, but also with a reduction of virus load in the brain. Employing pseudovirus neutralization assays, we were able to identify Nbs with neutralizing capacity against the Alpha, Beta, Delta and Omicron variants. Furthermore, cocktails of different Nbs performed better than individual Nbs to neutralize two Omicron variants (B.1.529 and BA.2). Altogether, the data suggest these Nbs can potentially be used as a cocktail for intranasal treatment to prevent or treat COVID-19 encephalitis, or modified for prophylactic administration to fight this disease.

Reactivity of inorganic sulfide species towards a pentacoordinated heme model system.

The reactivity of inorganic sulfide towards ferric bis(N-acetyl)- microperoxidase 11 in sodium dodecyl sulfate has been explored by means of visible absorption and resonance Raman spectroscopies. The reaction has been previously studied in buffered solutions at neutral pH and in the presence of excess sulfide, revealing the formation of a moderately stable hexacoordinated low spin ferric sulfide complex that yields the ferrous form in the hour's timescale. In the surfactant solution, instead, the ferrous form is rapidly formed. The spectroscopic characterization of the heme structure in the surfactant milieu revealed the stabilization of a major ferric mono-histidyl high spin heme, which may be ascribed to out of plane distortions prompting the detachment of the axially ligated water molecule, thus leading to a differential reactivity. The ferric bis(N-acetyl)- microperoxidase 11 in sodium dodecyl sulfate provides a model for pentacoordinated heme platforms with an imidazole-based ligand.

Multiple origins of green coloration in frogs mediated by a novel biliverdin-binding serpin.

Many vertebrates have distinctive blue-green bones and other tissues due to unusually high biliverdin concentrations-a phenomenon called chlorosis. Despite its prevalence, the biochemical basis, biology, and evolution of chlorosis are poorly understood. In this study, we show that the occurrence of high biliverdin in anurans (frogs and toads) has evolved multiple times during their evolutionary history, and relies on the same mechanism-the presence of a class of serpin family proteins that bind biliverdin. Using a diverse combination of techniques, we purified these serpins from several species of nonmodel treefrogs and developed a pipeline that allowed us to assemble their complete amino acid and nucleotide sequences. The described proteins, hereafter named biliverdin-binding serpins (BBS), have absorption spectra that mimic those of phytochromes and bacteriophytochromes. Our models showed that physiological concentration of BBSs fine-tune the color of the animals, providing the physiological basis for crypsis in green foliage even under near-infrared light. Additionally, we found that these BBSs are most similar to human glycoprotein alpha-1-antitrypsin, but with a remarkable functional diversification. Our results present molecular and functional evidence of recurrent evolution of chlorosis, describe a biliverdin-binding protein in vertebrates, and introduce a function for a member of the serpin superfamily, the largest and most ubiquitous group of protease inhibitors.

In Silico Insight into the Reductive Nitrosylation of Ferric Hemeproteins.

A combination of in silico methods was used to extend the experimental description of the reductive nitrosylation mechanism in ferric hemeproteins with the molecular details of the role of surrounding amino acids. The computational strategy consisted in the estimation of potential energy profiles for the transition process associated with the interactions of the coordinated N(NO) moiety with O(H2O) or O(OH-) as nucleophiles, and with distal amino acids as proton acceptors or affecting the stability of transition states. We inspected the reductive nitrosylation in three representative hemeproteins -sperm whale metmyoglobin, α subunit of human methemoglobin and nitrophorin 4 of Rhodnius prolixus. For each case, classical molecular dynamics simulations were performed in order to obtain relevant reactive conformations, and a potential energy profile for the reactive step was obtained using adiabatic mapping or nudged elastic band approaches at the QM/MM level. Specifically, we report the role of a charged Arg45 of myoglobin in destabilizing the transition state when H2O acts as nucleophile, differently to the neutral Pro43 of the hemoglobin subunit. The case of the nitrophorin is unique in that the access of the required water molecules is scarce, thus, preventing the reaction.

Hemeproteins as Targets for Sulfide Species.

Significance: Sulfides are endogenous and ubiquitous signaling species that share the hemeproteins as biochemical targets with O2, nitric oxide, and carbon monoxide. The description of the binding mechanisms is mandatory to anticipate the biochemical relevance of the interaction. Recent Advances: The binding of sulfide to ferric hemeproteins has been described in more than 40 systems, including native proteins, mutants, and model systems. Mechanisms of sulfide binding to ferric hemeproteins have been examined by a combination of kinetic and computational experiments. The distal control of the association process, dissected into the migration of the ligand to the active site and the binding event, reveals that neutral hydrogen sulfide (H2S) reaches the active site and is the predominant binding ligand, while the HS- is excluded by the protein matrix. Experiments with model compounds, devoid of a protein scaffold, reveal that both H2S and HS- can bind the ferric heme if accessing the site. A critical role of the proximal ligand in the prevention of the metal-centered reduction has been experimentally assessed. For metmyoglobin and methemoglobin, the coordination of sulfide leads to noncanonical functions: sulfide storage and its oxidative detoxification have been evidenced under physiological and excess sulfide concentrations, respectively. Critical Issues: The bound species is suggested to predominate in the monoprotonated form, although spectroscopic evidence is pending. Future Directions: A description of the role of hemeproteins as biochemical targets for inorganic sulfide requires understanding the reactivity of bound sulfide, for example: the metal-centered reduction, the reaction with excess sulfide, oxidants, or other gasotransmitters, among other biomolecules.

A fluorescence nanoscopy marker for corticotropin-releasing hormone type 1 receptor: computer design, synthesis, signaling effects, super-resolved fluorescence imaging, and in situ affinity constant in cells.

Class B G protein-coupled receptors (GPCRs) are involved in a variety of human pathophysiological states. These groups of membrane receptors are less studied than class A GPCRs due to the lack of structural information, delayed small molecule drug discovery, and scarce fluorescence detection tools available. The class B corticotropin-releasing hormone type 1 receptor (CRHR1) is a key player in the stress response whose dysregulation is critically involved in stress-related disorders: psychiatric conditions (i.e. depression, anxiety, and addictions), neuroendocrinological alterations, and neurodegenerative diseases. Here, we present a strategy to label GPCRs with a small fluorescent antagonist that permits the observation of the receptor in live cells through stochastic optical reconstruction microscopy (STORM) with 23 nm resolution. The marker, an aza-BODIPY derivative, was designed based on computational docking studies, then synthesized, and finally tested in biological cells. Experiments on hippocampal neurons demonstrate antagonist effects in similar concentrations as the well-established antagonist CP-376395. A quantitative analysis of two color STORM images enabled the determination of the binding affinity of the new marker in the cellular environment.

Mechanism of Sulfide Binding by Ferric Hemeproteins.

The reaction of hydrogen sulfide (H2S) with hemeproteins is a key physiological reaction; still, its mechanism and implications are not completely understood. In this work, we propose a combination of experimental and theoretical tools to shed light on the reaction in model system microperoxidase 11 (MP11-FeIII) and myoglobin (Mb-FeIII), from the estimation of the intrinsic binding constants of the species H2S and hydrosulfide (HS-), and the computational description of the overall binding process. Our results show that H2S and HS- are the main reactive species in Mb-FeIII and MP11-FeIII, respectively, and that the magnitude of their intrinsic binding constants are similar to most of the binding constants reported so far for hemeproteins systems and model compounds. However, while the binding of HS- to Mb-FeIII was negligible, the binding of H2S to MP11-FeIII was significant, providing a frame for a discriminated analysis of both species and revealing differential mechanistic aspects. A joint inspection of the kinetic data and the free energy profiles of the binding processes suggests that a dissociative mechanism with the release of a coordinated water molecule as rate limiting step is operative in the binding of H2S to Mb-FeIII and that the binding of HS- is prevented in the access to the protein matrix. For the MP11-FeIII case, where no access restrictions for the ligands are present, an associative component in the mechanism seems to be operative. Overall, the results suggest that if accessing the active site then both H2S and HS- are capable of binding a ferric heme moiety.

Naturally occurring fluorescence in frogs.

Fluorescence, the absorption of short-wavelength electromagnetic radiation reemitted at longer wavelengths, has been suggested to play several biological roles in metazoans. This phenomenon is uncommon in tetrapods, being restricted mostly to parrots and marine turtles. We report fluorescence in amphibians, in the tree frog Hypsiboas punctatus, showing that fluorescence in living frogs is produced by a combination of lymph and glandular emission, with pigmentary cell filtering in the skin. The chemical origin of fluorescence was traced to a class of fluorescent compounds derived from dihydroisoquinolinone, here named hyloins. We show that fluorescence contributes 18-29% of the total emerging light under twilight and nocturnal scenarios, largely enhancing brightness of the individuals and matching the sensitivity of night vision in amphibians. These results introduce an unprecedented source of pigmentation in amphibians and highlight the potential relevance of fluorescence in visual perception in terrestrial environments.

Access and Binding of H2S to Hemeproteins: The Case of HbI of Lucina pectinata.

Hydrogen sulfide (H2S) was recently discovered as a gasotransmitter, capable of coordinating to the heme iron of hemeproteins. H2S is unique for its ability to render varying concentrations of the nucleophilic conjugate bases (HS(-) or S(2-)), either as free or bound species with expected outcomes on its further reactivity. There is no direct evidence about which species (H2S, HS(-), or S(2-)) coordinates to the iron. We performed computer simulations to address the migration and binding processes of H2S species to the hemoglobin I of Lucina pectinata, which exhibits the highest affinity for the substrate measured to date. We found that H2S is the most favorable species in the migration from the bulk to the active site, through an internal pathway of the protein. After the coordination of H2S, an array of clustered water molecules modifies the active site environment, and assists in the subsequent deprotonation of the ligand, forming Fe(III)-SH(-). The feasibility of the second deprotonation of the coordinated ligand is also discussed.

Reactivity of inorganic sulfide species toward a heme protein model.

The reactivity of inorganic sulfide species toward heme peptides was explored under biorelevant conditions in order to unravel the molecular details of the reactivity of the endogenous hydrogen sulfide toward heme proteins. Unlike ferric porphyrinates, which are reduced by inorganic sulfide, some heme proteins can form stable Fe(III)-sulfide adducts. To isolate the protein factors ruling the redox chemistry, we used as a system model, the undecapeptide microperoxidase (MP11), a heme peptide derived from cytochrome c proteolysis that retains the proximal histidine bound to the Fe(III) atom. Upon addition of gaseous hydrogen sulfide (H2S) at pH 6.8, the UV-vis spectra of MP11 closely resembled those of the low-spin ferric hydroxo complex (only attained at an alkaline pH) and cysteine or alkylthiol derivatives, suggesting that the Fe(III) reduction was prevented. The low-frequency region of the resonance Raman spectrum revealed the presence of an Fe(III)-S band at 366 cm(-1) and the general features of a low-spin hexacoordinated heme. Anhydrous sodium sulfide (Na2S) was the source of sulfide of choice for the kinetic evaluation of the process. Theoretical calculations showed no distal stabilization mechanisms for bound sulfide species in MP11, highlighting a key role of the proximal histidine for the stabilization of the Fe(III)-S adducts of heme compounds devoid of distal counterparts, which is significant with regard to the biochemical reactivity of endogenous hydrogen sulfide.

Reactivity of iron(II)-bound nitrosyl hydride (HNO, nitroxyl) in aqueous solution.

The reactivity of coordinated nitroxyl (HNO) has been explored with the [Fe(II)(CN)(5)HNO](3-) complex in aqueous medium, pH 6. We discuss essential biorelevant issues as the thermal and photochemical decompositions, the reactivity toward HNO dissociation, the electrochemical behavior, and the reactions with oxidizing and reducing agents. The spontaneous decomposition in the absence of light yielded a two-electron oxidized species, the nitroprusside anion, [Fe(II)(CN)(5)NO](2-), and a negligible quantity of N(2)O, with k(obs)≈5×10(-7)s(-1), at 25.0°C. The value of k(obs) represents an upper limit for HNO release, comparable to values reported for other structurally related L ligands in the [Fe(II)(CN)(5)L](n-) series. These results reveal that the FeN bond is strong, suggesting a significant σ-π interaction, as already postulated for other HNO-complexes. The [Fe(II)(CN)(5)HNO](3-) ion showed a quasi-reversible oxidation wave at 0.32 V (vs normal hydrogen electrode), corresponding to the [Fe(II)(CN)(5)HNO](3-)/[Fe(II)(CN)(5)NO](3-),H(+) redox couple. Hexacyanoferrate(III), methylviologen and the nitroprusside ion have been selected as potential oxidants. Only the first reactant achieved a complete oxidation process, initiated by a proton-coupled electron transfer reaction at the HNO ligand, with nitroprusside as a final oxidation product. Dithionite acted as a reductant of [Fe(II)(CN)(5)HNO](3-), in a 4-electron process, giving NH(3). The high stability of bound HNO may resemble the properties in related Fe(II) centers of redox active enzymes. The very minor release of N(2)O shows that the redox conversions may evolve without disruption of the FeN bonds, under competitive conditions with the dissociation of HNO.

A protective protein matrix improves the discrimination of nitroxyl from nitric oxide by MnIII protoporphyrinate IX in aerobic media.

The selectivity of MnIII/II porphyrinates toward nitroxyl or nitric oxide donors provides a convenient starting point for the development of new materials for the speciation of these nitrogen-containing redox relatives. In the present report, we describe the insertion of MnIII protoporphyrinate IX in apomyoglobin and its chemical behavior toward HNO or NO donors, either under anaerobic or aerobic conditions. For comparison and discussion, the MnIII porphyrinate, devoid of the protein matrix, was studied in parallel. The MnIII reconstituted globin successfully reacted with the nitroxyl donor trioxodinitrate, while it was unreactive toward NO or NO donors, in good agreement with previously reported data on water soluble MnIII porphyrinates. The estimated association rate constant for the reaction with the nitroxyl donor was of the same order of magnitude for the reconstituted globin and the free porphyrinate, suggesting that the protein environment is not involved in the reaction mechanism. In contrast, the reaction product exhibited enhanced stability in the presence of dioxygen only when the porphyrinate was included in the protein matrix; this feature is ascribed to the role of the distal residues on the metal centered reactivity. This behavior is required for spectroscopic detection under biologically relevant conditions.

Successful stabilization of the elusive species {FeNO}8 in a heme model.

Nitroxyl (HNO/NO(-)) heme-adducts have been postulated as intermediates in a variety of catalytic processes carried out by different metalloenzymes. Hence, there is growing interest in obtaining and characterizing heme model nitroxyl complexes. The one-electron chemical reduction of the {FeNO}(7) nitrosyl derivative of Fe(III)(TFPPBr(8))Cl, Fe(II)(TFPPBr(8))NO (1) (TFPPBr(8) = 2,3,7,8,12,13,17,18-octabromo-5,10,15,20-[Tetrakis-(pentafluorophenyl)]porphyrin) with cobaltocene yields the significantly stable {FeNO}(8) complex, [Co(C(5)H(5))(2)](+)[Fe(TFPPBr(8))NO](-) (2). Complex 2 was isolated and characterized by UV-vis, FTIR, (1)H and (15)N NMR spectroscopies. In addition, DFT calculations were performed to get more insight into the structure of 2. According to the spectroscopic and DFT results, we can state unequivocally that the surprisingly stable complex 2 is the elusive {FeNO}(8) species. Both experimental and computational data allow to assign the electronic structure of 2 as intermediate between Fe(II)NO(-) and Fe(I)NO, which is contrasted with the predominant Fe(II)NO(-) character of known nonheme {FeNO}(8) complexes. The enhanced stability achieved for a heme model {FeNO}(8) is expected to allow further studies related to the reactivity of this elusive species.

Disproportionation of hydroxylamine by water-soluble iron(III) porphyrinate compounds.

The reactions of hydroxylamine (HA) with several water-soluble iron(III) porphyrinate compounds, namely iron(III) meso-tetrakis-(N-ethylpyridinium-2yl)-porphyrinate ([Fe(III)(TEPyP)](5+)), iron(III) meso-tetrakis-(4-sulphonatophenyl)-porphyrinate ([Fe(III)(TPPS)](3-)), and microperoxidase 11 ([Fe(III)(MP11)]) were studied for different [Fe(III)(Porph)]/[HA] ratios, under anaerobic conditions at neutral pH. Efficient catalytic processes leading to the disproportionation of HA by these iron(III) porphyrinates were evidenced for the first time. As a common feature, only N(2) and N(2)O were found as gaseous, nitrogen-containing oxidation products, while NH(3) was the unique reduced species detected. Different N(2)/N(2)O ratios obtained with these three porphyrinates strongly suggest distinctive mechanistic scenarios: while [Fe(III)(TEPyP)](5+) and [Fe(III)(MP11)] formed unknown steady-state porphyrinic intermediates in the presence of HA, [Fe(III)(TPPS)](3-) led to the well characterized soluble intermediate, [Fe(II)(TPPS)NO](4-). Free-radical formation was only evidenced for [Fe(III)(TEPyP)](5+), as a consequence of a metal centered reduction. We discuss the catalytic pathways of HA disproportionation on the basis of the distribution of gaseous products, free radicals formation, the nature of porphyrinic intermediates, the Fe(II)/Fe(III) redox potential, the coordinating capabilities of each complex, and the kinetic analysis. The absence of NO(2)(-) revealed either that no HAO-like activity was operative under our reaction conditions, or that NO(2)(-), if formed, was consumed in the reaction milieu.

Three redox states of nitrosyl: NO+, NO*, and NO-/HNO interconvert reversibly on the same pentacyanoferrate(II) platform.

Not so elusive: [Fe(II)(CN)(5)(HNO)](3-) has been characterized spectroscopically after the two-electron reduction of nitroprusside (see scheme). The complex is stable at pH 6, slowly decomposing to [Fe(CN)(6)](4-) and N(2)O. It is deprotonated at increasing pH value with oxidation of bound NO(-) to [Fe(II)(CN)(5)(NO)](3-). [Fe(II)(CN)(5)(HNO)](3-) is the first non-heme iron-nitroxyl complex prepared in aqueous solution that is reversibly redox-active under biologically relevant conditions.

Theoretical insight into the hydroxylamine oxidoreductase mechanism.

The multiheme enzyme hydroxylamine oxidoreductase from the autotrophic bacteria Nitrosomonas europaea catalyzes the conversion of hydroxylamine to nitrite, with a complicate arrangement of heme groups in three subunits. As a distinctive feature, the protein has a covalent linkage between a tyrosyl residue of one subunit and a meso carbon atom of the heme active site of another. We studied the influence of this bond in the catalysis from a theoretical perspective through electronic structure calculations at the density functional theory level, starting from the crystal structure of the protein. Geometry optimizations of proposed reaction intermediates were used to calculate the dissociation energy of different nitrogen containing ligands, considering the presence and absence of the meso tyrosyl residue. The results indicate that the tyrosine residue enhances the binding of hydroxylamine, and increases the stability of a Fe(III)NO intermediate, while behaving indifferently in the Fe(II)NO form. The calculations performed on model systems including neighboring aminoacids revealed the probable formation of a bidentate hydrogen bond between the Fe(III)H(2)O complex and Asp 257, in a high-spin aquo complex as the resting state. Characterization of non-planar heme distortions showed that the meso-substituent induces significant ruffling in the evaluated intermediates.