TALAIA: a 3D visual dictionary for protein structures.

MOTIVATION: Graphical analysis of the molecular structure of proteins can be very complex. Full-atom representations retain most geometric information but are generally crowded, and key structural patterns can be challenging to identify. Non-full-atom representations could be more instructive on physicochemical aspects but be insufficiently detailed regarding shapes (e.g. entity beans-like models in coarse grain approaches) or simple properties of amino acids (e.g. representation of superficial electrostatic properties). In this work, we present TALAIA a visual dictionary that aims to provide another layer of structural representations.TALAIA offers a visual grammar that combines simple representations of amino acids while retaining their general geometry and physicochemical properties. It uses unique objects, with differentiated shapes and colors to represent amino acids. It makes easier to spot crucial molecular information, including patches of amino acids or key interactions between side chains. Most conventions used in TALAIA are standard in chemistry and biochemistry, so experimentalists and modelers can rapidly grasp the meaning of any TALAIA depiction. RESULTS: We propose TALAIA as a tool that renders protein structures and encodes structure and physicochemical aspects as a simple visual grammar. The approach is fast, highly informative, and intuitive, allowing the identification of possible interactions, hydrophobic patches, and other characteristic structural features at first glance. The first implementation of TALAIA can be found at https://github.com/insilichem/talaia.

Rationalization of a Streptavidin Based Enantioselective Artificial Suzukiase: An Integrative Computational Approach.

An Artificial Metalloenzyme (ArM) built employing the streptavidin-biotin technology has been used for the enantioselective synthesis of binaphthyls by means of asymmetric Suzuki-Miyaura cross-coupling reactions. Despite its success, it remains a challenge to understand how the length of the biotin cofactors or the introduction of mutations to streptavidin leads the preferential synthesis of one atropisomer over the other. In this study, we apply an integrated computational modeling approach, including DFT calculations, protein-ligand dockings and molecular dynamics to rationalize the impact of mutations and length of the biotion cofactor on the enantioselectivities of the biaryl product. The results unravel that the enantiomeric differences found experimentally can be rationalized by the disposition of the first intermediate, coming from the oxidative addition step, and the entrance of the second substrate. The work also showcases the difficulties facing to control the enantioselection when engineering ArM to catalyze enantioselective Suzuki-Miyaura couplings and how the combination of DFT calculations, molecular dockings and MD simulations can be used to rationalize artificial metalloenzymes.

Protein-Protein Stabilization in VIVO/8-Hydroxyquinoline-Lysozyme adduct: A Spectroscopic, Crystallographic, and Computational Study.

The binding of the potential drug [VIVO(8-HQ)2], where 8-HQ is 8-hydroxyquinolinato, with hen egg white lysozyme (HEWL) was evaluated through spectroscopic (electron paramagnetic resonance, EPR, and UV-visible), spectrometric (electrospray ionization-mass spectrometry, ESI-MS), crystallographic (X-ray diffraction, XRD), and computational (DFT and docking) studies. ESI-MS indicates the interaction of [VIVO(8-HQ)(H2O)]+ and [VIVO(8-HQ)2(H2O)] species with HEWL. Room temperature EPR spectra suggest both covalent and non-covalent binding of the two different V-containing fragments. XRD analyses confirm these findings, showing that [VIVO(8-HQ)(H2O)]+ interacts covalently with the solvent exposed Asp119, while cis-[VIVO(8-HQ)2(H2O)] non-covalently with Arg128 and Lys96 from a symmetry mate. The covalent binding of [VIVO(8-HQ)(H2O)]+ to Asp119 is favored by a π-π contact with Trp62 and a H-bond with Asn103 of a symmetry-related molecule. Additionally, the covalent binding of VVO2+ to Asp48 and non-covalent binding of other V-containing fragments to Arg5, Cys6, and Glu7 is revealed. Molecular docking indicates that, in the absence of the interactions occurring at the protein-protein interface close to Asp119, the binding to Glu35 or Asp52 should be preferred. Such a protein-protein stabilization could be more common than what believed up today, at least in the solid state, and should be considered in the characterization of metal-protein adducts.

Computational Study of Amyloidß42 Familial Mutations and Metal Interaction: Impact on Monomers and Aggregates Dynamical Behaviors.

One of the main hallmarks of Alzheimer's Disease is the formation of ß-amyloid plaques, whose formation may be enhanced by metal binding or the appearance of familial mutations. In the present study, the simultaneous effect of familial mutations (E22Q, E22G, E22K, and D23N) and binding to metal ions (Cu(II) or Al(III)) is studied at the Aß42 monomeric and fibrillar levels. With the application of GaMD and MD simulations, it is observed that the effects of metal binding and mutations differ in the monomeric and fibrillar forms. In the monomeric structures, without metal binding, all mutations reduce the amount of α-helix and increase, in some cases, the ß-sheet content. In the presence of Cu(II) and Al(III) metal ions, the peptide becomes less flexible, and the ß-sheet content decreases in favor of forming α-helix motifs that stabilize the system through interhelical contacts. Regarding the fibrillar structures, mutations decrease the opening of the fiber in the vertical axis, thereby stabilizing the S-shaped structure of the fiber. This effect is, in general, enhanced upon metal binding. These results may explain the different Aß42 aggregation patterns observed in familial mutations.

Catalysis by [Ga4 L6 ]12- Metallocage on the Nazarov Cyclization: The Basicity of Complexed Alcohol is Key.

The Nazarov cyclization is investigated in solution and within K12 [Ga4 L6 ] supramolecular organometallic cage by means of computational methods. The reaction needs acidic condition in solution but works at neutral pH in the presence of the metallocage. The reaction steps for the process are analogous in both media: (a) protonation of the alcohol group, (b) water loss and (c) cyclization. The relative Gibbs energies of all the steps are affected by changing the environment from solvent to the metallocage. The first step in the mechanism, the alcohol protonation, turns out to be the most critical one for the acceleration of the reaction inside the metallocage. In order to calculate the relative stability of protonated alcohol inside the cavity, we propose a computational scheme for the calculation of basicity for species inside cavities and can be of general use. These results are in excellent agreement with the experiments, identifying key steps of catalysis and providing an in-depth understanding of the impact of the metallocage on all the reaction steps.

Successes and challenges in multiscale modelling of artificial metalloenzymes: the case study of POP-Rh2 cyclopropanase.

Molecular modelling applications in metalloenzyme design are still scarce due to a series of challenges. On top of that, the simulations of metal-mediated binding and the identification of catalytic competent geometries require both large conformational exploration and simulation of fine electronic properties. Here, we demonstrate how the incorporation of new tools in multiscale strategies, namely substrate diffusion exploration, allows taking a step further. As a showcase, the enantioselective profiles of the most outstanding variants of an artificial Rh2-based cyclopropanase (GSH, HFF and RFY) developed by Lewis and co-workers (Nat. Commun., 2015, 6, 7789 and Nat. Chem., 2018, 10, 318-324) have been rationalized. DFT calculations on the free-cofactor-mediated process identify the carbene insertion and the cyclopropanoid formation as crucial events, the latter being the enantiodetermining step, which displays up to 8 competitive orientations easily altered by the protein environment. The key intermediates of the reaction were docked into the protein scaffold showing that some mutated residues have direct interaction with the cofactor and/or the co-substrate. These interactions take the form of a direct coordination of Rh in GSH and HFF and a strong hydrophobic patch with the carbene moiety in RFY. Posterior molecular dynamics sustain that the cofactor induces global re-arrangements of the protein. Finally, massive exploration of substrate diffusion, based on the GPathFinder approach, defines this event as the origin of the enantioselectivity in GSH and RFY. For HFF, fine molecular dockings suggest that it is likely related to local interactions upon diffusion. This work shows how modelling of long-range mutations on the catalytic profiles of metalloenzymes may be unavoidable and software simulating substrate diffusion should be applied.

Getting Deeper into the Molecular Events of Heme Binding Mechanisms: A Comparative Multi-level Computational Study of HasAsm and HasAyp Hemophores.

Many biological systems obtain their activity by the inclusion of metalloporphyrins into one or several binding pockets. However, decoding the molecular mechanism under which these compounds bind to their receptors is something that has not been widely explored and is a field with open questions. In the present work, we apply computational techniques to unravel and compare the mechanisms of two heme-binding systems, concretely the HasA hemophores from Gram negative bacteria Serratiamarcescens (HasAsm) and Yersinia pestis (HasAyp). Despite the high sequence identity between both systems, the comparison between the X-ray structures of their apo and holo forms suggests different heme-binding mechanisms. HasAyp has extremely similar structures for heme-free and heme-bound forms, while HasAsm presents a very large displacement of a loop that ultimately leads to an additional coordination to the metal with respect to HasAyp. We combined Gaussian accelerated molecular dynamics simulations (GaMDs) in explicit solvent and protein-ligand docking optimized for metalloligands. GaMDs were first carried out on heme-free forms of both hemophores. Then, protein-ligand dockings of the heme were performed on cluster representatives of these simulations and the best poses were then subjected to a new series of GaMDs. A series of analyses reveal the following: (1) HasAyp has a conformational landscape extremely similar between heme-bound and unbound states with no to limited impact on the binding of the cofactor, (2) HasAsm presents as a slightly broader conformational landscape in its apo state but can only visit conformations similar to the X-ray of the holo form when the heme has been bound. Such behavior results from a complex cascade of changes in interactions that spread from the heme-binding pocket to the flexible loop previously mentioned. This study sheds light on the diversity of molecular mechanisms of heme-binding and discusses the weight between the pre-organization of the receptor as well as the induced motions resulting in association.

Influence of Association on Binding of Disaccharides to YKL-39 and hHyal-1 Enzymes.

Disaccharide complexes have been shown experimentally to be useful for drug delivery or as an antifouling surface biofilm, and are promising drug-encapsulation and delivery candidates. Although such complexes are intended for medical applications, to date no studies at the molecular level have been devoted to the influence of complexation on the enzymatic decomposition of polysaccharides. A theoretical approach to this problem has been hampered by the lack of a suitable computational tool for binding such non-covalent complexes to enzymes. Herein, we combine quantum-mechanical calculations of disaccharides complexes with a nonstandard docking GaudiMM engine that can perform such a task. Our results on four different complexes show that they are mostly stabilized by electrostatic interactions and hydrogen bonds. This strong non-covalent stabilization demonstrates the studied complexes are some excellent candidates for self-assembly smart materials, useful for drug encapsulation and delivery. Their advantage lies also in their biocompatible and biodegradable character.

Photocatalytic Hydrogen Production and Carbon Dioxide Reduction Catalyzed by an Artificial Cobalt Hemoprotein.

The covalent insertion of a cobalt heme into the cavity of an artificial protein named alpha Rep (αRep) leads to an artificial cobalt hemoprotein that is active as a catalyst not only for the photo-induced production of H2, but also for the reduction of CO2 in a neutral aqueous solution. This new artificial metalloenzyme has been purified and characterized by Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS), circular dichroism, and UltraViolet-Visible spectroscopy. Using theoretical experiments, the structure of this biohybrid and the positioning of the residues near the metal complex were examined, which made it possible to complete the coordination of the cobalt ion by an axial glutamine Gln283 ligand. While the Co(III)-porphyrin catalyst alone showed weak catalytic activity for both reactions, 10 times more H2 and four times more CO2 were produced when the Co(III)-porphyrin complex was buried in the hydrophobic cavity of the protein. This study thus provides a solid basis for further improvement of these biohybrids using well-designed modifications of the second and outer coordination sphere by site-directed mutagenesis of the host protein.

Stereoselective Self-Assembly of DNA Binding Helicates Directed by the Viral ß-Annulus Trimeric Peptide Motif.

Combining coordination chemistry and peptide engineering offers extraordinary opportunities for developing novel molecular (supra)structures. Here, we demonstrate that the ß-annulus motif is capable of directing the stereoselective assembly of designed peptides containing 2,2'-bipyridine ligands into parallel three-stranded chiral peptide helicates, and that these helicates selectively bind with high affinity to three-way DNA junctions.

Molecular Modeling for Artificial Metalloenzyme Design and Optimization.

Artificial metalloenzymes (ArMs) are obtained by inserting homogeneous catalysts into biological scaffolds and are among the most promising strategies in the quest for new-to-nature biocatalysts. The quality of their design strongly depends on how three partners interact: the biological host, the "artificial cofactor," and the substrate. However, structural characterization of functional artificial metalloenzymes by X-ray or NMR is often partial, elusive, or absent. How the cofactor binds to the protein, how the receptor reorganizes upon the binding of the cofactor and the substrate, and which are the binding mode(s) of the substrate for the reaction to proceed are key questions that are frequently unresolved yet crucial for ArM design. Such questions may eventually be solved by molecular modeling but require a step change beyond the current state-of-the-art methodologies.Here, we summarize our efforts in the study of ArMs, presenting both the development of computational strategies and their application. We first focus on our integrative computational framework that incorporates a variety of methods such as protein-ligand docking, classical molecular dynamics (MD), and pure quantum mechanical (QM) methods, which, when properly combined, are able to depict questions that range from host-cofactor binding predictions to simulations of entire catalytic mechanisms. We also pay particular attention to the protein-ligand docking strategies that we have developed to accurately predict the binding of transition metal-containing molecules to proteins. While this aspect is fundamental to many bioinorganic fields beyond ArMs, it has been disregarded from the molecular modeling landscape until very recently.Next we describe how to apply this computational framework to particular ArMs including systems previously characterized experimentally as well as others where computation served to guide the design. We start with the prediction of the interactions between homogeneous catalysts and biological hosts. Protein-ligand docking is pivotal at that stage, but it needs to be combined with QM/MM or MD approaches when the binding of the cofactor implies significant conformational changes of the protein or involve changes of the electronic state of the metal.Then, we summarize molecular modeling studies aimed at identifying cofactor-substrate arrangements inside the ArM active pocket that are consistent with its reactivity. These calculations stand on "Theozyme"-like dockings, MD-refined or not, which provide molecular rationale of the catalytic profiles of the artificial systems.In the third section, we present case studies to decode the entire catalytic mechanism of two ArMs: (1) an iridium based asymmetric transfer hydrogenase obtained by insertion of Noyori's catalyst into streptavidin and (2) a metallohydrolase achieved by including a receptor. Transition states, second coordination sphere effects, as well as motions of the cofactors are identified as drivers of the enantiomeric profiles.Finally, we report computer-aided designs of ArMs to guide experiments toward chemical and mutational changes that improve their activity and/or enantioselective profiles and expand toward future directions.

Origin of the Rate Acceleration in the C-C Reductive Elimination from Pt(IV)-complex in a [Ga4 L6 ]12- Supramolecular Metallocage.

The reductive elimination on [(Me3 P)2 Pt(MeOH)(CH3 )3 ]+ , 2P, complex performed in MeOH solution and inside a [Ga4 L6 ]12- metallocage are computationally analysed by mean of QM and MD simulations and compared with the mechanism of gold parent systems previously reported [Et3 PAu(MeOH)(CH3 )2 ]+ , 2Au. The comparative analysis between the encapsulated Au(III) and Pt(IV)-counterparts shows that there are no additional solvent MeOH molecules inside the cavity of the metallocage for both systems. The Gibbs energy barriers for the 2P reductive elimination calculated at DFT level are in good agreement with the experimental values for both environments. The effect of microsolvation and encapsulation on the rate acceleration are evaluated and shows that the latter is far more relevant, conversely to 2Au. Energy decomposition analysis indicates that the encapsulation is the main responsible for most of the energy barrier reduction. Microsolvation and encapsulation effects are not equally contributing for both metal systems and consequently, the reasons of the rate acceleration are not the same for both metallic systems despite the similarity between them.

Copper(II) N,N,O-Chelating Complexes as Potential Anticancer Agents.

Three novel dinuclear Cu(II) complexes based on a N,N,O-chelating salphen-like ligand scaffold and bearing varying aromatic substituents (-H, -Cl, and -Br) have been synthesized and characterized. The experimental and computational data obtained suggest that all three complexes exist in the dimeric form in the solid state and adopt the same conformation. The mass spectrometry and electron paramagnetic resonance results indicate that the dimeric structure coexists with the monomeric form in solution upon solvent (dimethyl sulfoxide and water) coordination. The three synthesized Cu(II) complexes exhibit high potentiality as ROS generators, with the Cu(II)/Cu(I) redox potential inside the biological redox window, and thus being able to biologically undergo Cu(II)/Cu(I) redox cycling. The formation of ROS is one of the most promising reported cell death mechanisms for metal complexes to offer an inherent selectivity to cancer cells. In vitro cytotoxic studies in two different cancer cell lines (HeLa and MCF7) and in a normal fibroblast cell line show promising selective cytotoxicity for cancer cells (IC50 about 25 µM in HeLa cells, which is in the range of cisplatin and improved with respect to carboplatin), hence placing this N,N,O-chelating salphen-like metallic core as a promising scaffold to be explored in the design of future tailor-made Cu(II) cytotoxic compounds.

Modeling Kinetics and Thermodynamics of Guest Encapsulation into the [M4L6]12- Supramolecular Organometallic Cage.

The encapsulation of molecular guests into supramolecular hosts is a complex molecular recognition process in which the guest displaces the solvent from the host cavity, while the host deforms to let the guest in. An atomistic description of the association would provide valuable insights on the physicochemical properties that guide it. This understanding may be used to design novel host assemblies with improved properties (i.e., affinities) toward a given class of guests. Molecular simulations may be conveniently used to model the association processes. It is thus of interest to establish efficient protocols to trace the encapsulation process and to predict the associated magnitudes ΔGbind and ΔGbind⧧. Here, we report the calculation of the Gibbs energy barrier and Gibbs binding energy by means of explicit solvent molecular simulations for the [Ga4L6]12- metallocage encapsulating a series of cationic molecules. The ΔGbind⧧ for encapsulation was estimated by means of umbrella sampling simulations. The steps involved were identified, including ion-pair formation and naphthalene rotation (from L ligands of the metallocage) during the guest's entrance. The ΔGbind values were computed using the attach-pull-release method. The results reveal the sensitivity of the estimates on the force field parameters, in particular on atomic charges, showing that higher accuracy is obtained when charges are derived from implicit solvent quantum chemical calculations. Correlation analysis identified some indicators for the binding affinity trends. All computed magnitudes are in very good agreement with experimental observations. This work provides, on one side, a benchmarked way to computationally model a highly charged metallocage encapsulation process. This includes a nonstandard parameterization and charge derivation procedure. On the other hand, it gives specific mechanistic information on the binding processes of [Ga4L6]12- at the molecular level where key motions are depicted. Taken together, the study provides an interesting option for the future design of metal-organic cages.

BioMetAll: Identifying Metal-Binding Sites in Proteins from Backbone Preorganization.

With a large amount of research dedicated to decoding how metallic species bind to proteins, in silico methods are interesting allies for experimental procedures. To date, computational predictors mostly work by identifying the best possible sequence or structural match of the target protein with metal-binding templates. These approaches are fundamentally focused on the first coordination sphere of the metal. Here, we present the BioMetAll predictor that is based on a different postulate: the formation of a potential metal-binding site is related to the geometric organization of the protein backbone. We first report the set of convenient geometric descriptors of the backbone needed for the algorithm and their parameterization from a statistical analysis. Then, the successful benchmark of BioMetAll on a set of more than 90 metal-binding X-ray structures is presented. Because BioMetAll allows structural predictions regardless of the exact geometry of the side chains, it appears extremely valuable for systems whose structures (either experimental or theoretical) are not optimal for metal-binding sites. We report here its application on three different challenging cases: (i) the modulation of metal-binding sites during conformational transition in human serum albumin, (ii) the identification of possible routes of metal migration in hemocyanins, and (iii) the prediction of mutations to generate convenient metal-binding sites for de novo biocatalysts. This study shows that BioMetAll offers a versatile platform for numerous fields of research at the interface between inorganic chemistry and biology and allows to highlight the role of the preorganization of the protein backbone as a marker for metal binding. BioMetAll is an open-source application available at https://github.com/insilichem/biometall.

Impact of Cu(II) and Al(III) on the conformational landscape of amyloidß1-42.

Metal ions have been found to play an important role in the formation of extracellular ß-amyloid plaques, a major hallmark of Alzheimer's disease. In the present study, the conformational landscape of Aß42 with Al(iii) and Cu(ii) has been explored using Gaussian accelerated molecular dynamics. Both metals reduce the flexibility of the peptide and entail a higher structural organization, although to different degrees. As a general trend, Cu(ii) binding leads to an increased α-helix content and to the formation of two α-helices that tend to organize in a U-shape. By contrast, most Al(iii) complexes induce a decrease in helical content, leading to more extended structures that favor the appearance of transitory ß-strands.

Hybrid Cyclobutane/Proline-Containing Peptidomimetics: The Conformational Constraint Influences Their Cell-Penetration Ability.

A new family of hybrid ß,γ-peptidomimetics consisting of a repetitive unit formed by a chiral cyclobutane-containing trans-ß-amino acid plus a Nα-functionalized trans-γ-amino-l-proline joined in alternation were synthesized and evaluated as cell penetrating peptides (CPP). They lack toxicity on the human tumoral cell line HeLa, with an almost negligible cell uptake. The dodecapeptide showed a substantial microbicidal activity on Leishmania parasites at 50 µM but with a modest intracellular accumulation. Their previously published γ,γ-homologues, with a cyclobutane γ-amino acid, showed a well-defined secondary structure with an average inter-guanidinium distance of 8-10 Å, a higher leishmanicidal activity as well as a significant intracellular accumulation. The presence of a very rigid cyclobutane ß-amino acid in the peptide backbone precludes the acquisition of a defined conformation suitable for their cell uptake ability. Our results unveiled the preorganized charge-display as a relevant parameter, additional to the separation among the charged groups as previously described. The data herein reinforce the relevance of these descriptors in the design of CPPs with improved properties.

Dynamic Stereoselection of Peptide Helicates and Their Selective Labeling of DNA Replication Foci in Cells*.

Although largely overlooked in peptide engineering, coordination chemistry offers a new set of interactions that opens unexplored design opportunities for developing complex molecular structures. In this context, we report new artificial peptide ligands that fold into chiral helicates in the presence of labile metal ions such as FeII and CoII . Heterochiral ß-turn-promoting sequences encode the stereoselective folding of the peptide ligands and define the physicochemical properties of their corresponding metal complexes. Circular dichroism and NMR spectroscopy in combination with computational methods allowed us to identify and determine the structure of two isochiral ΛΛ-helicates, folded as topological isomers. Finally, in addition to the in-vitro characterization of their selective binding to DNA three-way junctions, cell-microscopy experiments demonstrated that a rhodamine-labeled FeII helicate was internalized and selectively stains DNA replication factories in functional cells.

Reaction Rate Inside the Cavity of [Ga4 L6 ]12- Supramolecular Metallocage is Regulated by the Encapsulated Solvent.

In the present study the dependence of the reaction rate of carbon-carbon reductive elimination from R3 PAu(MeOH)(CH3 )2 (R=Me, Et) complexes inside [Ga4 L6 ]12- metallocage on the nature of the phosphine ligand is investigated by computational means. The reductive elimination mechanism is analyzed in methanol solution and inside the metallocage. Classical molecular dynamics simulations reveal that the smaller the gold complex (which depends on the phosphine ligand size) the larger the number of solvent molecules encapsulated. The size of the phosphine ligands defines the space that is left available inside the cavity that can be occupied by solvent molecules. The Gibbs energy barriers calculated at DFT level, in excellent agreement with experiment both in solution and in the metallocage, show that the presence/absence of explicit solvent molecules inside the cavity significantly modifies the reaction rate.

Unveiling VIV O2+ Binding Modes to Human Serum Albumins by an Integrated Spectroscopic-Computational Approach.

Human serum albumin (HSA) is involved in the transport of metal ions and potential metallodrugs. Depending on the metal, several sites are available, among which are N-terminal (NTS) and multi-metal binding sites (MBS). Despite the large number of X-ray determinations for albumins, only one structure with Zn2+ is available. In this work, the binding to HSA of the VIV O2+ ion was studied by an integrated approach based on spectroscopic and computational methods, which allowed the systems to be characterized even in the absence of X-ray analysis. The behavior depends on the type of albumin, defatted (HSAd ) or fatted (HSAf ). With HSAd 'primary' and 'secondary' sites were revealed, NTS with (His3, His9, Asp13, Asp255) and MBS with (His67, His247, Asp249, Asn99 or H2 O); with increasing the ratio VIV O2+ /HSAd , 'tertiary' sites, with one His-N and other donors (Asp/Glu-O or carbonyl-O) are populated. With HSAf , fatty acids (FAs) cause a rotation of the subdomains IA and IIA, which results in the formation of a dinuclear ferromagnetic adduct (VIV O)2 D (HSAf ) with a µ1,1 -Asp249 and the binding of His247, Glu100, Glu252, and His67 or Asn99. FAs hinder also the binding of VIV O2+ to the MBS.