Proximal ligand tunes active site structure and reactivity in bacterial L. monocytogenes coproheme ferrochelatase.

Ferrochelatases catalyze the insertion of ferrous iron into the porphyrin during the heme b biosynthesis pathway, which is fundamental for both prokaryotes and eukaryotes. Interestingly, in the active site of ferrochelatases, the proximal ligand coordinating the porphyrin iron of the product is not conserved, and its catalytic role is still unclear. Here we compare the L. monocytogenes bacterial coproporphyrin ferrochelatase native enzyme together with selected variants, where the proximal Tyr residue was replaced by a His (i.e. the most common ligand in heme proteins), a Met or a Phe (as in human and actinobacterial ferrochelatases, respectively), in their Fe(III), Fe(II) and Fe(II)-CO adduct forms. The study of the active site structure and the activity of the proteins in solution has been performed by UV-vis electronic absorption and resonance Raman spectroscopies, biochemical characterization, and classical MD simulations. All the mutations alter the H-bond interactions between the iron porphyrin propionate groups and the protein, and induce effects on the activity, depending on the polarity of the proximal ligand. The overall results confirm that the weak or non-existing coordination of the porphyrin iron by the proximal residue is essential for the binding of the substrate and the release of the final product.

The carbonyl-lock mechanism underlying non-aromatic fluorescence in biological matter.

Challenging the basis of our chemical intuition, recent experimental evidence reveals the presence of a new type of intrinsic fluorescence in biomolecules that exists even in the absence of aromatic or electronically conjugated chemical compounds. The origin of this phenomenon has remained elusive so far. In the present study, we identify a mechanism underlying this new type of fluorescence in different biological aggregates. By employing non-adiabatic ab initio molecular dynamics simulations combined with a data-driven approach, we characterize the typical ultrafast non-radiative relaxation pathways active in non-fluorescent peptides. We show that the key vibrational mode for the non-radiative decay towards the ground state is the carbonyl elongation. Non-aromatic fluorescence appears to emerge from blocking this mode with strong local interactions such as hydrogen bonds. While we cannot rule out the existence of alternative non-aromatic fluorescence mechanisms in other systems, we demonstrate that this carbonyl-lock mechanism for trapping the excited state leads to the fluorescence yield increase observed experimentally, and set the stage for design principles to realize novel non-invasive biocompatible probes with applications in bioimaging, sensing, and biophotonics.

Minimum Free Energy Pathways of Reactive Processes with Nudged Elastic Bands.

The determination of minimum free energy pathways (MFEP) is one of the most widely used strategies to study reactive processes. For chemical reactions in complex environments, the combination of quantum mechanics (QM) with a molecular mechanics (MM) representation is usually necessary in a hybrid QM/MM framework. However, even within the QM/MM approximation, the affordable sampling of the phase space is, in general, quite restricted. To reduce drastically the computational cost of the simulations, several methods such as umbrella sampling require performing a priori a selection of a reaction coordinate. The quality of the computed results, in an affordable computational time, is intimately related to the reaction coordinate election which is, in general, a nontrivial task. In this work, we provide an approach to model reactive processes in complex environments that does not require the a priori selection of a reaction coordinate. The proposed methodology combines QM/MM simulations with an extrapolation of the nudged elastic bands (NEB) method to the free energy surface (FENEB). We present and apply our own FENEB scheme to optimize MFEP in different reactive processes, using QM/MM frameworks at semiempirical and density functional theory levels. Our implementation is based on performing the FENEB optimization by uncoupling the optimization of the band in a perpendicular and tangential direction. In each step, a full optimization with the spring force is performed, which guarantees that the images remain evenly distributed. The robustness of the method and the influence of sampling on the quality of the optimized MFEP and its associated free energy barrier are studied. We show that the FENEB method provides a good estimation of the reaction barrier even with relatively short simulation times, supporting that its combination with QM/MM frameworks provides an adequate tool to study chemical processes in complex environments.

Nitric oxide binding to ferrous nitrobindins: A computer simulation investigation.

Nitrobindins (Nbs) represent an evolutionary conserved all-ß-barrel heme-proteins displaying a highly solvent-exposed heme-Fe(III) atom, coordinated by a proximal His residue. Interestingly, even if the distal side is exposed to the solvent, the value of the second order rate constants for ligand binding to the ferrous derivative is almost one order of magnitude lower than those reported for myoglobins (Mbs). Noteworthy, nitric oxide binding to the sixth coordination position of the heme-Fe(II)-atom causes the cleavage or the severe weakening of the proximal His-Fe(II) bond. Here, we provide a computer simulation investigation to shed light on the molecular basis of ligand binding kinetics, by dissecting the ligand binding process into the ligand migration and the bond formation steps. Classical molecular dynamics simulations were performed employing a steered molecular dynamics approach and the Jarzinski equality to obtain ligand migration free energy profiles. The formation of the heme-Fe(II)-NO bond took into consideration the iron atom displacement from the heme plane. The ligand migration is almost unhindered, and the low rate constant for NO binding is due to the large displacement of the Fe(II) atom with respect to the heme plane responsible for the barrier for the Fe(II)-NO bond formation. In addition, we investigated the weakening and breaking of the proximal His-Fe(II) bond, observed experimentally upon NO binding, by means of a combination of classical molecular dynamics simulations and quantum-classical (QM-MM) optimizations. In both human and M. tuberculosis Nbs, a stable alternative conformation of the proximal His residue interacting with a network of water molecules was observed.

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.

Disulfides form persulfides at alkaline pH leading to potential overestimations in the cold cyanolysis method.

It is well established that proteins and peptides can release sulfur under alkaline treatment, mainly through the ß-elimination of disulfides with the concomitant formation of persulfides and dehydroalanine derivatives. In this study, we evaluated the formation of glutathione persulfide (GSSH/GSS-) by exposure of glutathione disulfide (GSSG) to alkaline conditions. The kinetics of the reaction between GSSG and HO- was investigated by UV-Vis absorbance, reaction with 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB), and cold cyanolysis, obtaining an apparent second-order rate constant of ∼10-3 M-1 s-1 at 25 °C. The formation of GSSH and the dehydroalanine derivative was confirmed by HPLC and/or mass spectrometry. However, the mixtures did not equilibrate in a timescale of hours, and additional species, including thiol and diverse sulfane sulfur compounds were also formed, probably through further reactions of the persulfide. Cold cyanolysis is frequently used to quantify persulfides, since it measures sulfane sulfur. This method involves a step in which the sample to be analyzed is incubated with cyanide at alkaline pH. When cold cyanolysis was applied to samples containing GSSG, sulfane sulfur products that were not present in the original sample were measured. Thus, our results reveal the risk of overestimating the amount of sulfane sulfur compounds in samples that contain disulfides due to their decay to persulfides and other sulfane sulfur compounds at alkaline pH. Overall, our study highlights that the ß-elimination of disulfides is a potential source of persulfides, although we do not recommend the preparation of GSSH from incubation of GSSG in alkali. Our study also highlights the importance of being cautious when doing and interpreting cold cyanolysis experiments.

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.

The role of the distal cavity in carbon monoxide stabilization in the coproheme decarboxylase enzyme from C. diphtheriae.

This work focuses on the carbon monoxide adducts of the wild-type and selected variants of the coproheme decarboxylase from actinobacterial Corynebacterium diphtheriae complexed with coproheme, monovinyl monopropionyl deuteroheme (MMD), and heme b. The UV - vis and resonance Raman spectroscopies together with the molecular dynamics simulations clearly show that the wild-type coproheme-CO adduct is characterized by two CO conformers, one hydrogen-bonded to the distal H118 residue and the other showing a weak polar interaction with the distal cavity. Instead, upon conversion to heme b, i.e. after decarboxylation of propionates 2 and 4 and rotation by 90o of the porphyrin ring inside the cavity, CO probes a less polar environment. In the absence of the H118 residue, both coproheme and heme b complexes form only the non-H-bonded CO species. The unrotated MMD-CO adduct as observed in the H118F variant, confirms that decarboxylation of propionate 2 only, does not affect the heme cavity. The rupture of both the H-bonds involving propionates 2 and 4 destabilizes the porphyrin inside the cavity with the subsequent formation of a CO adduct in an open conformation. In addition, in this work we present data on CO binding to reversed heme b, obtained by hemin reconstitution of the H118A variant, and to heme d, obtained by addition of an excess of hydrogen peroxide. The results will be discussed and compared with those reported for the representatives of the firmicute clade.

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.

Novel Lennard-Jones Parameters for Cysteine and Selenocysteine in the AMBER Force Field.

Cysteine is a common amino acid with a thiol group that plays a pivotal role in a variety of scenarios in redox biochemistry. In contrast, selenocysteine, the 21st amino acid, is only present in 25 human proteins. Classical force-field parameters for cysteine and selenocysteine are still scarce. In this context, we present a methodology to obtain Lennard-Jones parameters for cysteine and selenocysteine in different physiologically relevant oxidation and protonation states. The new force field parameters obtained in this work are available at https://github.com/MALBECC/AMBER-parameters-database. The parameters were adjusted to reproduce water radial distribution functions obtained by density functional theory ab initio molecular dynamics. We validated the results by evaluating the impact of the choice of parameters on the structure and dynamics in classical molecular dynamics simulations of representative proteins containing catalytic cysteine/selenocysteine residues. There are significant changes in protein structure and dynamics depending on the parameters choice, specifically affecting the residues close to the catalytic sites.

Possible molecular basis of the biochemical effects of cysteine-derived persulfides.

Persulfides (RSSH/RSS-) are species closely related to thiols (RSH/RS-) and hydrogen sulfide (H2S/HS-), and can be formed in biological systems in both low and high molecular weight cysteine-containing compounds. They are key intermediates in catabolic and biosynthetic processes, and have been proposed to participate in the transduction of hydrogen sulfide effects. Persulfides are acidic, more acidic than thiols, and the persulfide anions are expected to be the predominant species at neutral pH. The persulfide anion has high nucleophilicity, due in part to the alpha effect, i.e., the increased reactivity of a nucleophile when the neighboring atom has high electron density. In addition, persulfides have electrophilic character, a property that is absent in both thiols and hydrogen sulfide. In this article, the biochemistry of persulfides is described, and the possible ways in which the formation of a persulfide could impact on the properties of the biomolecule involved are discussed.

Insights into the product release mechanism of dengue virus NS3 helicase.

The non-structural protein 3 helicase (NS3h) is a multifunctional protein that is critical in RNA replication and other stages in the flavivirus life cycle. NS3h uses energy from ATP hydrolysis to translocate along single stranded nucleic acid and to unwind double stranded RNA. Here we present a detailed mechanistic analysis of the product release stage in the catalytic cycle of the dengue virus (DENV) NS3h. This study is based on a combined experimental and computational approach of product-inhibition studies and free energy calculations. Our results support a model in which the catalytic cycle of ATP hydrolysis proceeds through an ordered sequential mechanism that includes a ternary complex intermediate (NS3h-Pi-ADP), which evolves releasing the first product, phosphate (Pi), and subsequently ADP. Our results indicate that in the product release stage of the DENV NS3h a novel open-loop conformation plays an important role that may be conserved in NS3 proteins of other flaviviruses as well.

Mycobacterium tuberculosis DosS binds H2S through its Fe3+ heme iron to regulate the DosR dormancy regulon.

Mycobacterium tuberculosis (Mtb) senses and responds to host-derived gasotransmitters NO and CO via heme-containing sensor kinases DosS and DosT and the response regulator DosR. Hydrogen sulfide (H2S) is an important signaling molecule in mammals, but its role in Mtb physiology is unclear. We have previously shown that exogenous H2S can modulate expression of genes in the Dos dormancy regulon via an unknown mechanism(s). Here, we test the hypothesis that Mtb senses and responds to H2S via the DosS/T/R system. Using UV-Vis and EPR spectroscopy, we show that H2S binds directly to the ferric (Fe3+) heme of DosS (KDapp = 5.30 µM) but not the ferrous (Fe2+) form. No interaction with DosT(Fe2+-O2) was detected. We found that the binding of sulfide can slowly reduce the DosS heme iron to the ferrous form. Steered Molecular Dynamics simulations show that H2S, and not the charged HS- species, can enter the DosS heme pocket. We also show that H2S increases DosS autokinase activity and subsequent phosphorylation of DosR, and H2S-mediated increases in Dos regulon gene expression is lost in Mtb lacking DosS. Finally, we demonstrate that physiological levels of H2S in macrophages can induce DosR regulon genes via DosS. Overall, these data reveal a novel mechanism whereby Mtb senses and responds to a third host gasotransmitter, H2S, via DosS(Fe3+). These findings highlight the remarkable plasticity of DosS and establish a new paradigm for how bacteria can sense multiple gasotransmitters through a single heme sensor kinase.

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.

Distal lysine (de)coordination in the algal hemoglobin THB1: A combined computer simulation and experimental study.

THB1 is a monomeric truncated hemoglobin from the green alga Chlamydomonas reinhardtii. In the absence of exogenous ligands and at neutral pH, the heme group of THB1 is coordinated by two protein residues, Lys53 and His77. THB1 is thought to function as a nitric oxide dioxygenase, and the distal binding of O2 requires the cleavage of the Fe-Lys53 bond accompanied by protonation and expulsion of the lysine from the heme cavity into the solvent. Nuclear magnetic resonance spectroscopy and crystallographic data have provided dynamic and structural insights of the process, but the details of the mechanism have not been fully elucidated. We applied a combination of computer simulations and site-directed mutagenesis experiments to shed light on this issue. Molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics restrained optimizations were performed to explore the nature of the transition between the decoordinated and lysine-bound states of the ferrous heme in THB1. Lys49 and Arg52, which form ionic interactions with the heme propionates in the X-ray structure of lysine-bound THB1, were observed to assist in maintaining Lys53 inside the protein cavity and play a key role in the transition. Lys49Ala, Arg52Ala and Lys49Ala/Arg52Ala THB1 variants were prepared, and the consequences of the replacements on the Lys (de)coordination equilibrium were characterized experimentally for comparison with computational prediction. The results reinforced the dynamic role of protein-propionate interactions and strongly suggested that cleavage of the Fe-Lys53 bond and ensuing conformational rearrangement is facilitated by protonation of the amino group inside the distal cavity.

Control of distal lysine coordination in a monomeric hemoglobin: A role for heme peripheral interactions.

THB1 is a monomeric truncated hemoglobin (TrHb) found in the cytoplasm of the green alga Chlamydomonas reinhardtii. The canonical heme coordination scheme in hemoglobins is a proximal histidine ligand and an open distal site. In THB1, the latter site is occupied by Lys53, which is likely to facilitate Fe(II)/Fe(III) redox cycling but hinders dioxygen binding, two features inherent to the NO dioxygenase activity of the protein. TrHb surveys show that a lysine at a position aligning with Lys53 is an insufficient determinant of coordination, and in this study, we sought to identify factors controlling lysine affinity for the heme iron. We solved the "Lys-off" X-ray structure of THB1, represented by the cyanide adduct of the Fe(III) protein, and hypothesized that interactions that differ between the known "Lys-on" structure and the Lys-off structure participate in the control of Lys53 affinity for the heme iron. We applied an experimental approach (site-directed mutagenesis, heme modification, pH titrations in the Fe(III) and Fe(II) states) and a computational approach (MD simulations in the Fe(II) state) to assess the role of heme propionate-protein interactions, distal helix capping, and the composition of the distal pocket. All THB1 modifications resulted in a weakening of lysine affinity and affected the coupling between Lys53 proton binding and heme redox potential. The results supported the importance of specific heme peripheral interactions for the pH stability of iron coordination and the ability of the protein to undergo redox reactions.

Acidity and nucleophilic reactivity of glutathione persulfide.

Persulfides (RSSH/RSS-) participate in sulfur trafficking and metabolic processes, and are proposed to mediate the signaling effects of hydrogen sulfide (H2S). Despite their growing relevance, their chemical properties are poorly understood. Herein, we studied experimentally and computationally the formation, acidity, and nucleophilicity of glutathione persulfide (GSSH/GSS-), the derivative of the abundant cellular thiol glutathione (GSH). We characterized the kinetics and equilibrium of GSSH formation from glutathione disulfide and H2S. A pKa of 5.45 for GSSH was determined, which is 3.49 units below that of GSH. The reactions of GSSH with the physiologically relevant electrophiles peroxynitrite and hydrogen peroxide, and with the probe monobromobimane, were studied and compared with those of thiols. These reactions occurred through SN2 mechanisms. At neutral pH, GSSH reacted faster than GSH because of increased availability of the anion and, depending on the electrophile, increased reactivity. In addition, GSS- presented higher nucleophilicity with respect to a thiolate with similar basicity. This can be interpreted in terms of the so-called α effect, i.e. the increased reactivity of a nucleophile when the atom adjacent to the nucleophilic atom has high electron density. The magnitude of the α effect correlated with the Brønsted nucleophilic factor, ßnuc, for the reactions with thiolates and with the ability of the leaving group. Our study constitutes the first determination of the pKa of a biological persulfide and the first examination of the α effect in sulfur nucleophiles, and sheds light on the chemical basis of the biological properties of persulfides.

Nucleotide-dependent dynamics of the Dengue NS3 helicase.

Dengue represents a substantial public health burden, particularly in low-resource countries. Non-structural protein 3 (NS3) is a multifunctional protein critical in the virus life cycle and has been identified as a promising anti-viral drug target. Despite recent crystallographic studies of the NS3 helicase domain, only subtle structural nucleotide-dependent differences have been identified, such that its coupled ATPase and helicase activities remain mechanistically unclear. Here we use molecular dynamics simulations to explore the nucleotide-dependent conformational landscape of the Dengue virus NS3 helicase and identify substantial changes in the protein flexibility during the ATP hydrolysis cycle. We relate these changes to the RNA-protein interactions and proposed translocation models for other monomeric helicases. Furthermore, we report a novel open-loop conformation with a likely escape route for Pi after hydrolysis, providing new insight into the conformational changes that underlie the ATPase activity of NS3.

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.

Dioxygen Binding and Sensing Proteins.

Oxygen binding proteins (O2BIP) have been actively investigated for the past five decades due to their rich redox chemistry and function as O2 carriers in blood cells, as well as their function as gasotransmitters and sensors that modulate cellular signaling. A series of meetings on the periodic advances in the knowledge gained in the field of globin structure and function are conducted typically on a biannual basis. In the fall of 2018, the XXth International Conference was conducted, and very important articles with breakthrough discoveries were presented and very enthusiastically discussed. This was yet another highly successful meeting in the series. Select articles from this meeting were recently reviewed, updated, and published over several issues of Antioxidants and Redox Signaling, as Forum articles communicating the latest advances in this important area of redox biology. This Forum editorial introduces these articles and highlights their scientific significance in advancing the field. Each of these articles grew out of lectures presented in the meeting, and appears either as an original contribution or a comprehensive review in the journal. Overall, the articles published in the Forum provide in-depth details on the recent developments in the field as well as point the way to future directions. These Forum articles thus serve as an important summary of progress and the ongoing direction of this field, and serve to highlight recent advances in our understanding of O2BIP.