Understanding how sulfur-containing ligands modulate the structure, electronic configuration, and spectroscopy of ferric heme proteins is essential for interpreting their reactivity and assigning experimentally observed intermediates. Here, we investigated the spectroscopic properties of a series of ferric myoglobin species, MbFe(III)-X, being X = HS-, S2-, H2S2, HS2-, S22-, and OH-. We combined QM-MM molecular dynamics (treating the active site and the coordinated ligands as the QM region, with the rest of the protein and solvent treated classically) with the nuclear ensemble approach, in which we sampled configurations from the MD simulations and obtained the electronic spectra of the species using linear-response TD-DFT at the B3LYP/def2-TZVP level of theory. The simulated spectra reproduce the major experimental trends, providing strong support for assigning the sulfide-bound intermediate to MbFe(III)-HS-, explaining the near indistinguishability of MbFe(III)-HS2- and MbFe(III)-S22- spectra, and rationalizing the similarities between sulfide- and disulfide-derivatives absorption profiles. Taken together, our results demonstrate the importance of explicitly sampling nuclear configurations to account for dynamic fluctuations in the active sites of complex metalloproteins when modeling their electronic spectra. By incorporating this configurational heterogeneity, the resulting spectral predictions achieved a high level of reliability and showed close agreement with the experiments.

Myoglobin, a heme protein abundant in striated muscle cells, plays a crucial role in oxygen delivery to mitochondria. In this study, we combined spectroscopy, rapid kinetics and computer simulations to investigate how the interaction between oxymyoglobin and an outer mitochondrial membrane model - composed of 1:1 POPC:POPE liposomes- impacts oxygen affinity. UV-visible and Resonance Raman spectroscopy studies of metmyoglobin, deoxymyoglobin and oxymyoglobin reveal subtle structural perturbations of the heme binding site, when oxymyoglobin interacts with the model membrane. Stopped-flow kinetic measurements show that these distortions lead to a 2-fold increase in the rate constant for oxygen release. Furthermore, classical and multiscale QM/MM simulations suggest that the increase in the O2 dissociation rate in the presence of the liposomes is mainly due to proximal effects and heme plane distortions produced by specific interactions between oxymyoglobin and the membrane. Altogether, these results point to a fine-tuning in the active site conformation that facilitates oxygen release from oxymyoglobin upon membrane association, potentially enhancing mitochondrial oxygen availability and energy production in cells.

The interest in chemical interactions between inorganic sulfur species and heme compounds has grown significantly in recent years due to their physiological relevance. The model system ferric N-acetyl microperoxidase 11 (NAcMP11FeIII) enables the exploration of the mechanistic aspects of the interaction between the ferric heme group and binding sulfur ligands, without the constraints imposed by a protein matrix and the stabilizing effects of distal amino acids. In this study, we investigated the coordination of disulfane (HSSH) and its conjugate base hydrodisulfide (HSS-) to NAcMP11FeIII. Kinetic estimations of the binding constant retrieved a pH-independent kon= (1.5 ± 0.7) x105 M-1s-1, for 6.4 ≤ pH ≤ 7.2, and a similar value for the intrinsic constant for HSS-, the predominant species. To obtain a molecular description of the binding process, we resorted to two complementary theoretical approaches. Firstly, using multiple steered molecular dynamics, we calculated the free energy profiles for the migration of the neutral species HSSH and the monoanionic HSS-, and also for the siblings hydrogen sulfide, H2S, and hydrosulfide, HS-. Our results reveal that both neutral and anionic species can achieve the distal cavity, as expected considering the highly solvent exposed heme group in NAcMP11FeIII. Secondly, we explored the ligand-exchange reaction using a combination of nudged elastic band (NEB) and hybrid quantum mechanics/molecular mechanics (QM/MM) calculations, which suggest that the monoanionic species can displace the water molecule coordinated to the heme iron more efficiently than the neutral ones. Altogether, our results provide a molecular description of the ligand binding process of these sulfur species to ferric heme proteins.

The kinetics of ligand binding to ferric heme proteins is relevant in a variety of biochemical processes. With a few exceptions, ferric heme proteins at physiological pH typically show the sixth (distal) coordination position of the heme iron occupied by a water molecule. This contrasts with ferrous heme proteins, where this position is usually vacant in the absence of external ligands. In this review, we shed light on mechanistic aspects of this process, by discussing our recent results of binding of hydrogen sulfide and hydrosulfide (H2S/HS–) and disulfane and hydrodisulfide (HSSH/HSS–) to ferric microperoxidase 11 (MP11FeIII) and metmyoglobin (MbFeIII), as well as binding of peroxynitrous acid/peroxynitrite (ONOOH/ONOO–) to ferric M. tuberculosis nitrobindin (NbFeIII). Stopped flow experimental results of ligand binding rates as a function of pH can be analyzed with a mechanistic proposal consisting of ligand migration and ligand binding steps. Ligand migration to the active site was studied by using steered classical molecular dynamics simulations. The process of ligand binding substitution of the coordinated water molecule has been studied using hybrid quantum–classical (QM-MM) tools. Our results suggest that water molecule release is the critical event of the process in most of the cases, consistently with previous proposals. However, the scenario is complex, since water release depends subtly on the heme environment and may be also assisted by the acid–base behavior of the incoming ligands. Ligand migration may also play a key role in cases in which the active site entrance is hindered.

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.

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.

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.

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 x 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.

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.

Non covalent biliproteins are found in a growing number of living organisms and even in viruses, such as SARS-CoV-2. Unlike the well described covalent biliproteins, such as the phytochromes, they present a vast structural and functional diversity, and often with limited experimental information. A very important tool (and sometimes the only one available) to study these systems is the UV-Vis spectrum, which is modulated both by conformational changes of the biliverdin chromophore and specific interactions with the apoprotein. In this work we present a theoretical study of the microscopic determinants of the UV-Vis spectrum of these compounds through the use of hybrid QM(TD-DFT)/MM techniques and molecular dynamics simulations. Comparing our results with existing experimental data, we prove that it is possible to predict spectroscopic properties, such as relative position and intensity ratio of main bands, with affordable methods, and to provide a microscopic explanation of them. This systematic information can be very useful for the study of described biliproteins or for those yet unknown.

The role of inorganic sulfur species in biological systems has gained considerable interest since the recognition of sulfanes, particularly dihydrogen sulfide or sulfane, H 2 S, disulfane, HSSH, trisulfane, HSSSH, and their conjugate bases, as endogenous species and mediators of signaling functions in different tissues. The one-electron oxidation of H 2 S/HS − has been assigned as the onset of signaling processes or oxidative detoxification mechanisms. These varied sulfur containing inorganic species are, together with organic counterparts, reunited as reactive sulfur species (RSS). In order to shed light on this rich and still not completely explored chemistry, we have performed electronic structure calculations at different levels of theory, to provide estimations and the molecular basis of the pK a values of the polysulfides HSSH and HSSSH and of the radical HS • . In addition, we also reported the characterization of selected inorganic RSS including both radical and non-radical species with different protonation states with the intention of assisting the interpretation of chemical/biochemical experiments involving these species.

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.

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.

Significance Green coloration of vertebrates is normally attributed to pigments and structural components inside skin chromatophores cells. However, these components do not account for the vivid blue-green colors of hundreds of species of frogs with sparse chromatophores. Our study shows that green coloration originates in proteins of the serpin superfamily that bind the pigment biliverdin, modulating its absorbance properties. Using a South American treefrog, we demonstrated that these serpins have a clear ecological role in modulating the reflectance properties and rendering animals cryptic in the foliage even in the near-infrared portion of the spectrum. These findings open up exciting research perspectives both in biochemistry and evolution of serpins, as well as in the study of extracellular protein-mediated coloration in vertebrates.

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. Antioxid. Redox Signal. 00, 000-000.

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.

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.

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 andmatching 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.

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.