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Phytochromes are red-light-sensitive biliprotein photoreceptors that control a variety of physiological processes in plants, fungi, and bacteria. Lately, greater attention has been paid to these photoreceptors due to their potential as fluorescent probes for deep-tissue microscopy. Such fluorescing phytochromes have been generated by multiple amino acid substitutions in weakly fluorescent wild-type (WT) proteins. Remarkably, the single substitution of conserved Tyr176 by His in cyanobacterial phytochrome Cph1 increases the fluorescence quantum yield from 2.4 to 14.5%. In this work, we studied this Y176H variant by crystallography, MAS NMR, resonance Raman spectroscopy, and ultrafast absorption spectroscopy complemented by theoretical methods. Two factors were identified to account for the strong fluorescence increase. First, the equilibrium between the photoactive and fluorescent substates of WT Cph1 was shown to shift entirely to the fluorescent substate in Y176H. Second, structural flexibility of the chromophore is drastically reduced and the photoisomerization barrier is raised, thereby increasing the excited-state lifetime. The most striking finding, however, is that Y176H includes the structural properties of both the dark-adapted Pr and the light-activated Pfr state. While the chromophore adopts the Pr-typical ZZZssa configuration, the tongue segment of the protein adopts a Pfr-typical α-helical structure. This implies that Tyr176 plays a key role in coupling chromophore photoisomerization to the sheet-to-helix transition of the tongue and the final Pfr structure. This conclusion extends to plant phytochromes, where the homologous substitution causes light-independent signaling activity akin to that of Pfr.

The mitochondrial outer membrane iron-sulphur ([Fe-S]) protein mitoNEET has been extensively studied as a target of the anti-inflammatory and type-2 diabetes drug pioglitazone and as a protein affecting mitochondrial respiratory rate. Despite these extensive past studies, its molecular function has yet to be discovered. Here, we applied an interdisciplinary approach and discovered an explicit nitric oxide (NO) access site to the mitoNEET [2Fe-2S] cluster. We found that O 2 and pioglitazone block NO access to the cluster, suggesting a molecular function for the mitoNEET [2Fe-2S] cluster in mitochondrial signal transduction. Our discovery hints at a new pathway via which mitochondria can sense hypoxia through O 2 protection of the mitoNEET [2Fe-2S] cluster, a new paradigm in understanding the importance of [Fe-S] clusters for gasotransmitter signal transduction in eukaryotes., (© 2025 The Author(s). FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Chitosan is a functional polymer with diverse applications in biomedicine, agriculture, water treatment, and beyond. Via derivatization of pristine chitosan, its functionality can be tailored to desired applications, e.g. immobilization of biomolecules. Here, we performed molecular dynamics simulations of three aminated chitosan polymers, where one, two, and three long-distanced side chains have been incorporated. These polymers have been previously synthesized and their properties were investigated experimentally, however, the observed dependencies could not be fully explained on the molecular level. Here, we develop a computational protocol for the simulation of functionalized chitosan polymers and perform advanced analysis of their conformational states, intramolecular interactions, and water binding. We demonstrate that intra- and intermolecular forces, especially hydrogen bonds induced by polymer side chain modifications, modulate dihedral angle conformational states of the polymer backbone and interactions with water. We explain the role of the chemical composition of the functionalized chitosans in their tendency to collapse and reveal the key role of the protonation of the amino group near the polymer backbone on the reduction of polymer collapse. We demonstrate that specific binding of water molecules, especially the intermediate water, is more pronounced in the polymer exhibiting such an amino group., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

A profound understanding of protein structure and mechanism requires dedicated experimental and theoretical tools to elucidate electrostatic and hydrogen bonding interactions in proteins. In this work, we employed an approach to disentangle noncovalent and hydrogen-bonding electric field changes during the reaction cascade of a multidomain protein, i.e., the phytochrome Agp2. The approach exploits the spectroscopic properties of nitrile probes commonly used as reporter groups of the vibrational Stark effect. These probes were introduced into the protein through site-specific incorporation of noncanonical amino acids resulting in four variants with different positions and orientations of the nitrile groups. All substitutions left structures and the reaction mechanism unchanged. Structural models of the dark states (Pfr) were used to evaluate the total electric field at the nitrile label and its transition dipole moment. These quantities served as an internal standard to calculate the respective properties of the photoinduced products (Lumi-F, Meta-F, and Pr) based on the relative intensities of the nitrile stretching bands. In most cases, the spectral analysis revealed two substates with a nitrile in a hydrogen-bonded or hydrophobic environment. Using frequencies and intensities, we managed to extract the noncovalent contribution of the electric field from the individual substates. This analysis resulted in profiles of the noncovalent and hydrogen-bond-related electric fields during the photoinduced reaction cascade of Agp2. These profiles, which vary significantly among the four variants due to the different positions and orientations of the nitrile probes, were discussed in the context of the molecular events along the Pfr → Pr reaction cascade.

Molecularly imprinted polymers (MIPs) are artificial receptors equipped with selective recognition sites for target molecules. One of the most promising strategies for protein MIPs relies on the exploitation of short surface-exposed protein fragments, termed epitopes, as templates to imprint binding sites in a polymer scaffold for a desired protein. However, the lack of high-resolution structural data of flexible surface-exposed regions challenges the selection of suitable epitopes. Here, we addressed this drawback by developing a polyscopoletin-based MIP that recognizes recombinant proteins via imprinting of the widely used Strep-tag II affinity peptide (Strep-MIP). Electrochemistry, surface-sensitive IR spectroscopy, and molecular dynamics simulations were employed to ensure an utmost control of the Strep-MIP electrosynthesis. The functionality of this novel platform was verified with two Strep-tagged enzymes: an O 2 -tolerant [NiFe]-hydrogenase, and an alkaline phosphatase. The enzymes preserved their biocatalytic activities after multiple utilization confirming the efficiency of Strep-MIP as a general biocompatible platform to confine recombinant proteins for exploitation in biotechnology., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

[NiFe]-hydrogenases catalyze the reversible activation of H 2 using a unique NiFe(CN) 2 CO metal site, which is assembled by a sophisticated multiprotein machinery. The [4Fe-4S] cluster-containing HypCD complex, which possesses an ATPase activity with a hitherto unknown function, serves as the hub for the assembly of the Fe(CN) 2 CO subfragment. HypCD is also thought to be responsible for the subsequent transfer of the iron fragment to the apo-form of the catalytic hydrogenase subunit, but the underlying mechanism has remained unexplored. Here, we performed a thorough spectroscopic characterization of different HypCD preparations using infrared, Mössbauer, and NRVS spectroscopy, revealing molecular details of the coordination of the Fe(CN) 2 CO fragment. Moreover, biochemical assays in combination with spectroscopy, AlphaFold structure predictions, protein-ligand docking calculations, and crosslinking MS deciphered unexpected mechanistic aspects of the ATP requirement of HypCD, which we found to actually trigger the transfer of the Fe(CN) 2 CO fragment to the apo-hydrogenase.

Electron transfer bifurcation enables biological systems to drive unfavourable (endergonic) electron transfer by coupling it to favourable (exergonic) transfer of a second electron. In electron transfer flavoproteins (ETFs), a domain-scale conformational change is believed to sever the favourable pathway after a single electron has used it, thereby preventing the energy dissipation that would accompany exergonic transfer of the second electron. To understand the conformation change that participates in turnover, we have deployed small-angle neutron scattering (SANS) and computational techniques to characterize the bifurcating ETF from Acidaminococcus fermentans ( Afe ETF). SANS data reveal an overall radius of gyration ( R g ) of 30.1 ± 0.2 Å and a maximum dimension ( D max ) of 100 Å for oxidized Afe ETF. These measurements are 4 Å and 30 Å larger, respectively, than those of any published bifurcating ETF structure. Thus, we find that none of the reported ETF structures can explain the observed scattering, nor can any individual conformation generated by either of our molecular dynamics protocols. To optimize ensembles best able to explain the SANS data, we adapted a genetic algorithm. Successful ensembles contained a compact conformation comparable to one of the crystallographically documented conformations, accompanied by a much more extended one, and these two conformations sufficed to account for the data. The extended conformations identified all have R g s at least 4 Å larger than those of any currently published ETF structures. However, they are strongly populated, constituting 20% of the population of reduced ETF and over 50% of the population of oxidized Afe ETF. Thus, the published (compact) structures provide a seriously incomplete picture of the conformation of Afe ETF in solution. Moreover, because the composition of the conformational ensemble changes upon reduction of Afe ETF's flavins, interconversion of the conformations may contribute to turnover. We propose that the extended conformations can provide energetically accessible paths for rapid interconversion of the open and closed compact conformations that are believed essential at alternating points in turnover., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Enniatins are mycotoxins with well-known antibacterial, antifungal, antihelmintic and antiviral activity, which have recently come to attention as potential mitochondriotoxic anticancer agents. The cytotoxicity of enniatins is traced back to ionophoric properties, in which the cyclodepsipeptidic structure results in enniatin:cation-complexes of various stoichiometries proposed as membrane-active species. In this work, we employed a combination of surface-enhanced infrared absorption (SEIRA) spectroscopy, tethered bilayer lipid membranes (tBLMs) and density functional theory (DFT)-based computational spectroscopy to monitor the cation-dependence (M z+ =Na + , K + , Cs + , Li + , Mg 2+ , Ca 2+ ) on the mechanism of enniatin B (EB) incorporation into membranes and identify the functionally relevant EB n  : M z+ complexes formed. We find that Na + promotes a cooperative incorporation, modelled via an autocatalytic mechanism and mediated by a distorted 2 : 1-EB 2  : Na + complex. K + (and Cs + ) leads to a direct but less efficient insertion into membranes due to the adoption of "ideal" EB 2  : K + sandwich complexes. In contrast, the presence of Li + , Mg 2+ , and Ca 2+ causes a (partial) extraction of EB from the membrane via the formation of "belted" 1 : 1-EB : M z+ complexes, which screen the cationic charge less efficiently. Our results point to a relevance of the cation dependence for the transport into the malignant cells where the mitochondriotoxic anticancer activity is exerted., (© 2024 The Authors. ChemPlusChem published by Wiley-VCH GmbH.)

Detection of pathogenic viruses for point-of-care applications has attracted great attention since the COVID-19 pandemic. Current virus diagnostic tools are laborious and expensive, while requiring medically trained staff. Although user-friendly and cost-effective biosensors are utilized for virus detection, many of them rely on recognition elements that suffer major drawbacks. Herein, computationally designed epitope-imprinted polymers (eIPs) are conjugated with a portable piezoelectric sensing platform to establish a sensitive and robust biosensor for the human pathogenic adenovirus (HAdV). The template epitope is selected from the knob part of the HAdV capsid, ensuring surface accessibility. Computational simulations are performed to evaluate the conformational stability of the selected epitope. Further, molecular dynamics simulations are executed to investigate the interactions between the epitope and the different functional monomers for the smart design of eIPs. The HAdV epitope is imprinted via the solid-phase synthesis method to produce eIPs using in silico-selected ingredients. The synthetic receptors show a remarkable detection sensitivity (LOD: 10 2 pfu mL -1 ) and affinity (dissociation constant ( K d ): 6.48 × 10 -12 M) for HAdV. Moreover, the computational eIPs lead to around twofold improved binding behavior than the eIPs synthesized with a well-established conventional recipe. The proposed computational strategy holds enormous potential for the intelligent design of ultrasensitive imprinted polymer binders.

New variants of SARS-CoV-2 that can escape immune response continue to emerge. Consequently, there is an urgent demand to design small molecule therapeutics inhibiting viral entry to host cells to reduce infectivity rate. Despite numerous in silico and in situ studies, the structural requirement of designing viral-entry inhibitors effective against multiple variants of SARS-CoV-2 has yet to be described. Here we systematically screened the binding of various natural products (NPs) to six different SARS-CoV-2 receptor-binding domain (RBD) structures. We demonstrate that Multi-structural Molecular Docking (MOD) combined with molecular dynamics calculations allowed us to predict a vulnerable site of RBD and the structural requirement of ligands binding to this vulnerable site. We expect that our findings lay the foundation for in silico screening and identification of lead molecules to guide drug discovery into designing new broad-spectrum lead molecules to counter the threat of future variants of SARS-CoV-2., (© 2023. Springer Nature Limited.)

Bifurcating electron transferring flavoproteins (Bf-ETFs) tune chemically identical flavins to two contrasting roles. To understand how, we used hybrid quantum mechanical molecular mechanical calculations to characterize noncovalent interactions applied to each flavin by the protein. Our computations replicated the differences between the reactivities of the flavins: the electron transferring flavin ( ET flavin) was calculated to stabilize anionic semiquinone (ASQ) as needed to execute its single-electron transfers, whereas the Bf flavin ( Bf flavin) was found to disfavor the ASQ state more than does free flavin and to be less susceptible to reduction. The stability of ET flavin ASQ was attributed in part to H-bond donation to the flavin O2 from a nearby His side chain, via comparison of models employing different tautomers of His. This H-bond between O2 and the ET site was uniquely strong in the ASQ state, whereas reduction of ET flavin to the anionic hydroquinone (AHQ) was associated with side chain reorientation, backbone displacement, and reorganization of its H-bond network including a Tyr from the other domain and subunit of the ETF. The Bf site was less responsive overall, but formation of the Bf flavin AHQ allowed a nearby Arg side chain to adopt an alternative rotamer that can H-bond to the Bf flavin O4. This would stabilize the anionic Bf flavin and rationalize effects of mutation at this position. Thus, our computations provide insights on states and conformations that have not been possible to characterize experimentally, offering explanations for observed residue conservation and raising possibilities that can now be tested., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Photoisomerization is a fundamental process in several classes of photoreceptors. Phytochromes sense red and far-red light in their Pr and Pfr states, respectively. Upon light absorption, these states react via individual photoreactions to the other state. Cph1 phytochrome shows a photoisomerization of its phycocyanobilin (PCB) chromophore in the Pfr state with a time constant of 0.7 ps. The dynamics of the PCB chromophore has been described, but whether or not the apoprotein exhibits an ultrafast response too, is not known. Here, we compare the photoreaction of 13 C/ 15 N labeled apoprotein with unlabeled apoprotein to unravel ultrafast apoprotein dynamics in Cph1. In the spectral range from 1750 to 1620 cm -1 we assigned several signals due to ultrafast apoprotein dynamics. A bleaching signal at 1724 cm -1 is tentatively assigned to deprotonation of a carboxylic acid, probably Asp207, and signals around 1670 cm -1 are assigned to amide I vibrations of the capping helix close to the chromophore. These signals remain after photoisomerization. The apoprotein dynamics appear upon photoexcitation or concomitant with chromophore isomerization. Thus, apoprotein dynamics occur prior to and after photoisomerization on an ultrafast time-scale. We discuss the origin of the ultrafast apoprotein response with the 'Coulomb hammer' mechanism, i.e. an impulsive change of electric field and Coulombic force around the chromophore upon excitation., (© 2023. The Author(s).)

Comprising at least a bipartite architecture, the large subunit of [NiFe]-hydrogenase harbors the catalytic nickel-iron site while the small subunit houses an array of electron-transferring Fe-S clusters. Recently, some [NiFe]-hydrogenase large subunits have been isolated showing an intact and redox active catalytic cofactor. In this computational study we have investigated one of these metalloproteins, namely the large subunit HoxG of the membrane-bound hydrogenase from Cupriavidus necator ( Cn MBH), targeting its conformational and mechanical stability using molecular modelling and long all-atom Gaussian accelerated molecular dynamics (GaMD). Our simulations predict that isolated HoxG is stable in aqueous solution and preserves a large portion of its mechanical properties, but loses rigidity in regions around the active site, in contrast to the MBH heterodimer. Inspired by biochemical data showing dimerization of the HoxG protein and IR measurements revealing an increased stability of the [NiFe] cofactor in protein preparations with higher dimer content, corresponding simulations of homodimeric forms were also undertaken. While the monomeric subunit contains several flexible regions, our data predicts a regained rigidity in homodimer models. Furthermore, we computed the electrostatic properties of models obtained by enhanced sampling with GaMD, which displays a significant amount of positive charge at the protein surface, especially in solvent-exposed former dimer interfaces. These data offer novel insights on the way the [NiFe] core is protected from de-assembly and provide hints for enzyme anchoring to surfaces, which is essential information for further investigations on these minimal enzymes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Dragelj, Karafoulidi-Retsou, Katz, Lenz, Zebger, Caserta, Sacquin-Mora and Mroginski.)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The function of the recently isolated sulerythrin (SulE) has been investigated using a combination of structural and electronic analyses based on quantum mechanical calculations. In the SulE structure of Fushinobu et al. (2003), isolated from a strictly aerobic archaeon, Sulfolobus tokadaii , a dioxygen-containing species was tentatively included at the active site during crystallographic refinement although the substrate specificity of SulE remains unclear. Studies have suggested that a structurally related enzyme, rubrerythrin, functions as a hydrogen peroxide reductase. Since SulE is a truncated version of rubrerythrin, the enzymes are hypothesized to function similarly. Hence, using available X-ray crystallography data (1.7 Å), we constructed various models of SulE containing a ZnII-Fe active site, differing in the nature of the substrate specificity (O 2 , H 2 O 2 ), the oxidation level and the spin state of the iron ion, and the protonation states of the coordinating glutamate residues. Also, the substrate H 2 O 2 is modeled in two possible configurations, differing in the orientation of the hydrogen atoms. Overall, the optimized geometries with an O 2 substrate do not show good agreement with the experimentally resolved geometry. In contrast, excellent agreement between crystal structure arrangement and optimized geometries is achieved considering a H 2 O 2 substrate and FeII in both spin states, when Glu92 is protonated. These results suggest that the dioxo species detected at the [ZnFe] active site of sulerythrin is H 2 O 2 , rather than an O 2 molecule in agreement with experimental data indicating that only the diferrous oxidation state of the dimetal site in rubrerythrin reacts rapidly with H 2 O 2 . Based on our computations, we proposed a possible reaction pathway for substrate binding at the ZnFeII site of SulE with a H 2 O 2 substrate. In this reaction pathway, Fe or another electron donor, such as NAD(P)H, catalyzes the reduction of H 2 O 2 to water at the zinc-iron site., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Moubarak, Rippers, Elghobashi-Meinhardt and Mroginski.)

Biological carbon dioxide (CO 2 ) reduction is an important step by which organisms form valuable energy-richer molecules required for further metabolic processes. The Mo-dependent formate dehydrogenase (FDH) from Rhodobacter capsulatus catalyzes reversible formate oxidation to CO 2 at a bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor. To elucidate potential substrate binding sites relevant for the mechanism, we studied herein the interaction with the inhibitory molecules azide and cyanate, which are isoelectronic to CO 2 and charged as formate. We employed infrared (IR) spectroscopy in combination with density functional theory (DFT) and inhibition kinetics. One distinct inhibitory molecule was found to bind to either a non-competitive or a competitive binding site in the secondary coordination sphere of the active site. Site-directed mutagenesis of key amino acid residues in the vicinity of the bis-MGD cofactor revealed changes in both non-competitive and competitive binding, whereby the inhibitor is in case of the latter interaction presumably bound between the cofactor and the adjacent Arg587., (© 2022 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Protein halogenation is a common non-enzymatic post-translational modification contributing to aging, oxidative stress-related diseases and cancer. Here, we report a genetically encodable halogenation of tyrosine residues in a reconstituted prokaryotic filamentous cell-division protein (FtsZ) as a platform to elucidate the implications of halogenation that can be extrapolated to living systems of much higher complexity. We show how single halogenations can fine-tune protein structures and dynamics of FtsZ with subtle perturbations collectively amplified by the process of FtsZ self-organization. Based on experiments and theories, we have gained valuable insights into the mechanism of halogen influence. The bending of FtsZ structures occurs by affecting surface charges and internal domain distances and is reflected in the decline of GTPase activities by reducing GTP binding energy during polymerization. Our results point to a better understanding of the physiological and pathological effects of protein halogenation and may contribute to the development of potential diagnostic tools., (© 2022. The Author(s).)

The biological function of phytochromes is triggered by an ultrafast photoisomerization of the tetrapyrrole chromophore biliverdin between two rings denoted C and D. The mechanism by which this process induces extended structural changes of the protein is unclear. Here we report ultrafast proton-coupled photoisomerization upon excitation of the parent state (Pfr) of bacteriophytochrome Agp2. Transient deprotonation of the chromophore's pyrrole ring D or ring C into a hydrogen-bonded water cluster, revealed by a broad continuum infrared band, is triggered by electronic excitation, coherent oscillations and the sudden electric-field change in the excited state. Subsequently, a dominant fraction of the excited population relaxes back to the Pfr state, while ~35% follows the forward reaction to the photoproduct. A combination of quantum mechanics/molecular mechanics calculations and ultrafast visible and infrared spectroscopies demonstrates how proton-coupled dynamics in the excited state of Pfr leads to a restructured hydrogen-bond environment of early Lumi-F, which is interpreted as a trigger for downstream protein structural changes., (© 2022. The Author(s).)

Phytochromes, found in plants, fungi, and bacteria, exploit light as a source of information to control physiological processes via photoswitching between two states of different physiological activity, i.e. a red-absorbing Pr and a far-red-absorbing Pfr state. Depending on the relative stability in the dark, bacterial phytochromes are divided into prototypical and bathy phytochromes, where the stable state is Pr and Pfr, respectively. In this work we studied representatives of these groups (prototypical Agp1 and bathy Agp2 from Agrobacterium fabrum ) together with the bathy-like phytochrome Xcc BphP from Xanthomonas campestris by resonance Raman and IR difference spectroscopy. In all three phytochromes, the photoinduced conversions display the same mechanistic pattern as reflected by the chromophore structures in the various intermediate states. We also observed in each case the secondary structure transition of the tongue, which is presumably crucial for the function of phytochrome. The three phytochromes differ in details of the chromophore conformation in the various intermediates and the energetic barrier of their respective decay reactions. The specific protein environment in the chromophore pocket, which is most likely the origin for these small differences, also controls the proton transfer processes concomitant to the photoconversions. These proton translocations, which are tightly coupled to the structural transition of the tongue, presumably proceed via the same mechanism along the Pr → Pfr conversion whereas the reverse Pfr → Pr photoconversion includes different proton transfer pathways. Finally, classification of phytochromes in prototypical and bathy (or bathy-like) phytochromes is discussed in terms of molecular structure and mechanistic properties.

Phytochromes constitute a widespread photoreceptor family that typically interconverts between two photostates called Pr (red light–absorbing) and Pfr (far-red light–absorbing). The lack of full-length structures solved at the (near-)atomic level in both pure Pr and Pfr states leaves gaps in the structural mechanisms involved in the signal transmission pathways during the photoconversion. Here, we present the crystallographic structures of three versions from the plant pathogen Xanthomonas campestris virulence regulator Xcc BphP bacteriophytochrome, including two full-length proteins, in the Pr and Pfr states. The structures show a reorganization of the interaction networks within and around the chromophore-binding pocket, an α-helix/β-sheet tongue transition, and specific domain reorientations, along with interchanging kinks and breaks at the helical spine as a result of the photoswitching, which subsequently affect the quaternary assembly. These structural findings, combined with multidisciplinary studies, allow us to describe the signaling mechanism of a full-length bacterial phytochrome at the atomic level.

Flavins are central to countless enzymes but display different reactivities depending on their environments. This is understood to reflect modulation of the flavin electronic structure. To understand changes in orbital natures, energies, and correlation over the ring system, we begin by comparing seven flavin variants differing at C8, exploiting their different electronic spectra to validate quantum chemical calculations. Ground state calculations replicate a Hammett trend and reveal the significance of the flavin π-system. Comparison of higher-level theories establishes CC2 and ACD(2) as methods of choice for characterization of electronic transitions. Charge transfer character and electron correlation prove responsive to the identity of the substituent at C8. Indeed, bond length alternation analysis demonstrates extensive conjugation and delocalization from the C8 position throughout the ring system. Moreover, we succeed in replicating a particularly challenging UV/Vis spectrum by implementing hybrid QM/MM in explicit solvents. Our calculations reveal that the presence of nonbonding lone pairs correlates with the change in the UV/Vis spectrum observed when the 8-methyl is replaced by NH 2 , OH, or SH. Thus, our computations offer routes to understanding the spectra of flavins with different modifications. This is a first step toward understanding how the same is accomplished by different binding environments.

Since the emergence of SARS-CoV-2, little attention has been paid to the interplay between the interaction of virus and commensal microbiota. Here, we used molecular docking and dynamics simulations to study the interaction of some of the known metabolites and natural products (NPs) produced by commensal microbiota with the receptor binding domain (RBD) of the spike glycoprotein of SARS-CoV-2. The results predict that NPs of commensal microbiota such as bile acids and non-ribosomal peptides (NRPs), of which some are siderophores, bind to the wild-type RBD and interfere with its binding to the ACE2 receptor. N501Y mutation, which is present in many of the emerging variants of the virus, abolishes the predicted binding pocket of bile acids and NRPs. Based on these findings, available experimental data showing that bile acids reduce the binding affinity of wild-type RBD to the ACE2 receptor, and the data suggesting that the respiratory tract microbiota affect viral infection we put forward the following proposal: mutations such as N501Y enable the RBD to bind to the ACE2 receptor more effectively in the presence of NPs produced by the respiratory tract bacteria thereby, increasing the infectivity rate of the virus. We hope our data stimulate future works to better understand the interactions of NPs produced by commensal microbiota with respiratory viruses like SARS-CoV-2., (© 2021 Wiley-VCH GmbH.)

Phytochromes switch between a physiologically inactive and active state via a light-induced reaction cascade, which is initiated by isomerization of the tetrapyrrole chromophore and leads to the functionally relevant secondary structure transition of a protein segment (tongue). Although details of the underlying cause-effect relationships are not known, electrostatic fields are likely to play a crucial role in coupling chromophores and protein structural changes. Here, we studied local electric field changes during the photoconversion of the dark state Pfr to the photoactivated state Pr of the bathy phytochrome Agp2. Substituting Tyr165 and Phe192 in the chromophore pocket by para -cyanophenylalanine (pCNF), we monitored the respective nitrile stretching modes in the various states of photoconversion (vibrational Stark effect). Resonance Raman and IR spectroscopic analyses revealed that both pCNF-substituted variants undergo the same photoinduced structural changes as wild-type Agp2. Based on a structural model for the Pfr state of F192pCNF, a molecular mechanical-quantum mechanical approach was employed to calculate the electric field at the nitrile group and the respective stretching frequency, in excellent agreement with the experiment. These calculations serve as a reference for determining the electric field changes in the photoinduced states of F192pCNF. Unlike F192pCNF, the nitrile group in Y165pCNF is strongly hydrogen bonded such that the theoretical approach is not applicable. However, in both variants, the largest changes of the nitrile stretching modes occur in the last step of the photoconversion, supporting the view that the proton-coupled restructuring of the tongue is accompanied by a change of the electric field.

Cytochrome c oxidase (C c O) pumps protons from the N-side to the P-side and consumes electrons from the P-side of the mitochondrial membrane driven by energy gained from reduction of dioxygen to water. ATP synthesis uses the resulting proton gradient and electrostatic potential difference. Since the distance a proton travels through C c O is too large for a one-step transfer process, proton-loading sites (PLS) that can carry protons transiently are necessary. One specific pump-active PLS couples to the redox reaction, thus energizing the proton to move across the membrane against electric potential and proton gradient. The PLS should also prevent proton backflow. Therefore, the propionates of the two redox-active hemes in C c O were suggested as PLS candidates although, according to C c O crystal structures, none of the four propionates can be protonated on account of strong H-bonds. Here, we show that modeling the local structure around heme a 3 propionates enhances significantly their capability of carrying a proton jointly. This was not possible for the propionates of heme a . The modeled structures are stable in molecular dynamics simulations (MDS) and are energetically similar to the crystal structure. Precise electrostatic energy computations of MDS data are used to estimate the p K A values of all titratable residues in C c O. For the modeled structures, the heme a 3 propionates have p K A values high enough to host a proton transiently but not too high to fix the proton permanently. The change in p K A throughout the redox reaction is sufficient to push the proton to the P-side of the membrane and to provide the protons with the necessary amount of energy for ATP synthesis.

Deactivation processes of photoexcited (λ ex  = 580 nm) phycocyanobilin (PCB) in methanol were investigated by means of UV/Vis and mid-IR femtosecond (fs) transient absorption (TA) as well as static fluorescence spectroscopy, supported by density-functional-theory calculations of three relevant ground state conformers, PCB A , PCB B and PCB C , their relative electronic state energies and normal mode vibrational analysis. UV/Vis fs-TA reveals time constants of 2.0, 18 and 67 ps, describing decay of PCB B *, of PCB A * and thermal re-equilibration of PCB A , PCB B and PCB C , respectively, in line with the model by Dietzek et al. (Chem Phys Lett 515:163, 2011) and predecessors. Significant substantiation and extension of this model is achieved first via mid-IR fs-TA, i.e. identification of molecular structures and their dynamics, with time constants of 2.6, 21 and 40 ps, respectively. Second, transient IR continuum absorption (CA) is observed in the region above 1755 cm -1 (CA1) and between 1550 and 1450 cm -1 (CA2), indicative for the IR absorption of highly polarizable protons in hydrogen bonding networks (X-H … Y). This allows to characterize chromophore protonation/deprotonation processes, associated with the electronic and structural dynamics, on a molecular level. The PCB photocycle is suggested to be closed via a long living (> 1 ns), PCB C -like (i.e. deprotonated), fluorescent species.

This perspective article highlights the challenges in the theoretical description of photoreceptor proteins using multiscale modeling, as discussed at the CECAM workshop in Tel Aviv, Israel. The participants have identified grand challenges and discussed the development of new tools to address them. Recent progress in understanding representative proteins such as green fluorescent protein, photoactive yellow protein, phytochrome, and rhodopsin is presented, along with methodological developments., (© 2021 American Society for Photobiology.)

Cytochrome c oxidase (CcO) in its as-isolated form is known to exist in a slow and fast form, which differ drastically in their ability to bind oxygen and other ligands. While preparation methods have been established that yield either the fast or the slow form of the protein, the underlying structural differences have not been identified yet. Here, we have performed surface enhanced resonance Raman (SERR) spectroscopy of CcO immobilized on electrodes in both forms. SERR spectra obtained in resonance with the heme a 3 metal-to-ligand charge transfer (MLCT) transition at 650 nm displayed a sharp vibrational band at 748 or 750 cm -1 when the protein was in its slow or fast form, respectively. DFT calculations identified the band as a mode of the His-419 ligand that is sensitive to the oxygen ligand and the protonation state of Tyr-288 within the binuclear complex. Potential-dependent SERR spectroscopy showed a redox-induced change of this band around 525 mV versus Ag/AgCl exclusively for the fast form, which coincides with the redox potential of the Tyr-O/Tyr-O - transition. Our data points to a peroxide ligand in the resting state of CcO for both forms. The observed frequencies and redox sensitivities of the Raman marker band suggest that a radical Tyr-288 is present in the fast form and a protonated Tyr-288 in the slow form.

Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C-S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins., (© 2021. The Author(s).)

Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C-S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins., Competing Interests: Competing interests The authors declare no competing interests.

The "open" (A open ) and "closed" (A closed ) A-clusters of the acteyl-CoA synthase (ACS) enzyme from Moorella thermoacetica have been studied using a combined quantum mechanical (QM)/molecular mechanical (MM) approach. Geometry optimizations of the oxidized, one- and two-electron reduced A open state have been carried out for the fully solvated ACS enzyme, and the CO ligand has been modeled in the reduced models. Using a combination of both α open and α closed protein scaffolds and the positions of metal atoms in these structures, we have been able to piece together critical parts of the catalytic cycle of ACS. We have replaced the unidentified exogenous ligand in the crystal structure with CO using both a square planar and tetrahedral proximal Ni atom. A one-electron reduced A-cluster that is characterized by a proximal Ni atom in a tetrahedral coordination pattern observed in both the A open (lower occupancy proximal Ni) and A closed (proximal Zn atom) geometries with three cysteine thiolates and a modeled CO ligand demonstrates excellent agreement with the crystal structure atomic positions, particularly with the displacement of the side chain ring of Phe512 which appears to serve as a structural gate for ligand binding. The QM/MM optimized geometry of the A-cluster of ACS with an uncoordinated, oxidized proximal nickel atom in a square planar geometry demonstrates poor agreement with the atomic coordinates taken from the crystal structure. Based on these calculations, we conclude that the square planar proximal nickel coordination that has been captured in the A open structure does not correspond to the ligand-free, oxidized [Fe 4 S 4 ] 2+  - Ni p 2+  - Ni d 2+ state. Overall, these computations shed further light on the mechanistic details of protein conformational changes and electronic transitions involved in the ACS catalytic cycle., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Phytochromes are photoreceptors that upon light absorption initiate a physiological reaction cascade. The starting point is the photoisomerization of the tetrapyrrole cofactor in the parent Pr state, followed by thermal relaxation steps culminating in activation of the physiological signal. Here we have employed resonance Raman (RR) spectroscopy to study the chromophore structure in the primary photoproduct Lumi-R, trapped between 130 and 200 K. The investigations covered phytochromes from plants (phyA) and prokaryotes (Cph1, Agp1, CphB, and Rp BphP2) including phytochromobilin (PΦB), phycocyanobilin (PCB), and biliverdin (BV). In PΦB- and PCB-binding phyA and Cph1, two Lumi-R states (Lumi-R1, Lumi-R2) were identified and discussed in terms of sequential and parallel reaction models. In Lumi-R1, the chromophore structural changes are restricted to the C-D methine bridge isomerization site but extended throughout the chromophore in Lumi-R2. Formation and decay kinetics as well as photochemical activity depend on the specific protein-chromophore interactions and thus account for the different distribution between Lumi-R1 and Lumi-R2 in the photostationary mixtures of the various PΦB(PCB)-binding phytochromes. For BV-binding bacteriophytochromes, only a single Lumi-R(BV) state was found. In this state, which is similar for Agp1, CphB, and Rp BphP2, the chromophore structural changes comprise major torsions of the C-D methine bridge but also perturbations at the A-B methine bridge remote from the isomerization site. The different structures of the photoproducts in PΦB(PCB)-binding phytochromes and BV-binding bacteriophytochromes are attributed to the different disposition of ring D upon isomerization, which leads to distinct protein-chromophore interactions in the Lumi-R states of these two classes of phytochromes.

Phytochromes are biological photoswitches that interconvert between two parent states (Pr and Pfr). The transformation is initiated by photoisomerization of the tetrapyrrole chromophore, followed by a sequence of chromophore and protein structural changes. In the last step, a phytochrome-specific peptide segment (tongue) undergoes a secondary structure change, which in prokaryotic phytochromes is associated with the (de)activation of the output module. The focus of this work is the Pfr-to-Pr photoconversion of the bathy bacteriophytochrome Agp2 in which Pfr is the thermodynamically stable state. Using spectroscopic techniques, we studied the structural and functional consequences of substituting Arg211, Tyr165, His278, and Phe192 close to the biliverdin (BV) chromophore. In Pfr, substitutions of these residues do not affect the BV structure. The characteristic Pfr properties of bathy phytochromes, including the protonated propionic side chain of ring C (propC) of BV, are preserved. However, replacing Arg211 or Tyr165 blocks the photoconversion in the Meta-F state, prior to the secondary structure transition of the tongue and without deprotonation of propC. The Meta-F state of these variants displays low photochemical activity, but electronic excitation causes ultrafast alterations of the hydrogen bond network surrounding the chromophore. In all variants studied here, thermal back conversion from the photoproducts to Pfr is decelerated but substitution of His278 or Phe192 is not critical for the Pfr-to-Pr photoconversion. These variants do not impair deprotonation of propC or the α-helix/β-sheet transformation of the tongue during the Meta-F-to-Pr decay. Thus, we conclude that propC deprotonation is essential for restructuring of the tongue.

Phytochromes are biological photoreceptors found in all kingdoms of life. Numerous physicochemical and spectroscopic studies of phytochromes have been carried out for many decades, both experimentally and computationally, with the main focus on the photoconversion mechanism involving a tetrapyrrole chromophore. In this computational work, we concentrate on the long-scale dynamic motion of the photosensory domain of Deinococcus radiodurans by means of classical all-atom molecular dynamics (MD) simulations. Conventional and accelerated MD methods in combination with two different force fields, CHARMM27 and AMBER ff14SB, are tested in long atomistic simulations to confront the dynamics of monomer and dimer forms. These calculations highlight dissimilar equilibrium conformations in aqueous solutions and, in turn, different large-scale dynamic behaviors of the monomer form vs the dimer form. While the phytochrome in a monomer form tends to close the cavity entailed between the GAF and PHY domains, the opposite trend is predicted for the phytochrome dimer, which opens up as a consequence of the formation of strong salt bridges between the PHY domains of two molecules in water.

Heme-copper oxygen reductases are terminal respiratory enzymes, catalyzing the reduction of dioxygen to water and the translocation of protons across the membrane. Oxygen consumption is inhibited by various substances. Here we tested the relatively unknown inhibition of cytochrome c oxidase (CcO) with isocyanate. In contrast to other more common inhibitors like cyanide, inhibition with cyanate was accompanied with the rise of a metal to ligand charge transfer (MLCT) band around 638 nm. Increasing the cyanate concentration furthermore caused selective reduction of heme a. The presence of the CT band allowed for the first time to directly monitor the nature of the ligand via surface-enhanced resonance Raman (SERR) spectroscopy. Analysis of isotope sensitive SERR spectra in comparison with Density Functional Theory (DFT) calculations identified not only the cyanate monomer as an inhibiting ligand but suggested also presence of an uretdion ligand formed upon dimerization of two cyanate ions. It is therefore proposed that under high cyanate concentrations the catalytic site of CcO promotes cyanate dimerization. The two excess electrons that are supplied from the uretdion ligand lead to the observed physiologically inverse electron transfer from heme a 3 to heme a.

Cyanobacteriochromes (CBCRs) are photoreceptor proteins that photoconvert between two parent states and thereby regulate various biological processes. An intriguing property is their variable ultraviolet-visible (UV-vis) absorption that covers the entire spectral range from the far-red to the near-UV region and thus makes CBCRs promising candidates for optogenetic applications. Here, we have studied Slr1393, a CBCR that photoswitches between red- and green-absorbing states (Pr and Pg, respectively). Using UV-vis absorption, fluorescence, and resonance Raman (RR) spectroscopy, a further orange-absorbing state O 600 that is in thermal equilibrium with Pr was identified. The different absorption properties of the three states were attributed to the different lengths of the conjugated π-electron system of the phycocyanobilin chromophore. In agreement with available crystal structures and supported by quantum mechanics/molecular mechanics (QM/MM) calculations, the most extended conjugation holds for Pr whereas it is substantially reduced in Pg. Here, the two outer pyrrole rings D and A are twisted out of the plane defined by inner pyrrole rings B and C . For the O 600 state, the comparison of the experimental RR spectra with QM/MM-calculated spectra indicates a partially distorted ZZZssa geometry in which ring A is twisted while ring D and the adjacent methine bridge display essentially the same geometry as Pr. The quantitative analysis of temperature-dependent spectra yields an enthalpy barrier of ∼30 kJ/mol for the transition from Pr to O 600 . This reaction is associated with the movement of a conserved tryptophan residue from the chromophore binding pocket to a solvent-exposed position.

The intriguing ability of phytochromes to photoconvert between red-absorbing (Pr) and far-red-absorbing (Pfr) states depends on key interactions between a bilin chromophore and protein matrix. However, both the identification of the chemical nature and quantification of chromophore-protein interactions have not been yet fully investigated by experiments or extensive computations. Here, we presented a powerful and straightforward approach based on the fragment molecular orbital method to identify the nature and quantify the strength of the noncovalent interactions at a fully quantum mechanical level between the biliverdin (BV) chromophore and protein matrix of the Deinococcus radiodurans phytochrome (DrBphP) in the Pr state. By using pair interaction energy decomposition analysis approach, the pyrrole water, Asp207, and Glu27 were detected as key residues for the stabilization of the pyrrole rings of the BV chromophore through the formation of six H-bonds. Furthermore, the conserved Arg254 and His260 were also identified as essential residues in the conformational stability of both propionic side chains B and C. Moreover, new interactions were identified in the chromophore-binding pocket, two nonclassical H-bonds (CH/O interactions) between Asp207 and Tyr263, and an OH/π interaction between Tyr263 and ring D of the BV chromophore, which might have photochemical relevance.

Bacteriophytochromes harboring a biliverdin IXα (BV) chromophore undergo photoinduced reaction cascades to switch between physiologically inactive and active states. Employing vibrational spectroscopic and computational methods, we analyzed the role of propionic substituents of BV in the transformations between parent states Pr and Pfr in prototypical (Agp1) and bathy (Agp2) phytochromes from Agrobacterium fabrum . Both proteins form adducts with BV monoesters (BVM), esterified at propionic side chain B (Ps B ) or C (Ps C ), but in each case, only one monoester adduct is reactive. In the reactive Agp2-BVM- B complex (esterified at ring B ), the Pfr dark state displays the structural properties characteristic of bathy phytochromes, including a protonated Ps C . As in native Agp2, Ps C is deprotonated in the final step of the Pfr phototransformation. However, the concomitant α-helix/β-sheet secondary structure change of the tongue is blocked at the stage of unfolding of the coiled loop region. This finding and the shift of the tautomeric equilibrium of BVM toward the enol form are attributed to the drastic changes in the electrostatic potential. The calculations further suggest that deprotonation of Ps C and the protonation state of His278 control the reactivity of the enol tautomer, thereby accounting for the extraordinarily slow thermal reversion. Although strong perturbations of the electrostatic potential are also found for Agp1-BVM, the consequences for the Pr-to-Pfr phototransformation are less severe. Specifically, the structural transition of the tongue is not impaired and thermal reversion is even accelerated. The different response of Agp1 and Agp2 to monoesterification of BV points to different photoconversion mechanisms.

Flavin-based electron bifurcation allows enzymes to redistribute energy among electrons by coupling endergonic and exergonic electron transfer reactions. Diverse bifurcating enzymes employ a two-flavin electron transfer flavoprotein (ETF) that accepts hydride from NADH at a flavin (the so-called bifurcating FAD, Bf-FAD). The Bf-FAD passes one electron exergonically to a second flavin thereby assuming a reactive semiquinone state able to reduce ferredoxin or flavodoxin semiquinone. The flavin that accepts one electron and passes it on via exergonic electron transfer is known as the electron transfer FAD (ET-FAD) and is believed to correspond to the single FAD present in canonical ETFs, in domain II. The Bf-FAD is believed to be the one that is unique to bifurcating ETFs, bound between domains I and III. This very reasonable model has yet to be challenged experimentally. Herein we used site-directed mutagenesis to disrupt FAD binding to the presumed Bf site between domains I and III, in the Bf-ETF from Rhodopseudomonas palustris ( Rpa ETF). The resulting protein contained only 0.80 ± 0.05 FAD, plus 1.21 ± 0.04 bound AMP as in canonical ETFs. The flavin was not subject to reduction by NADH, confirming absence of Bf-FAD. The retained FAD displayed visible circular dichroism (CD) similar to that of the ET-FAD of Rpa ETF. Likewise, the mutant underwent two sequential one-electron reductions forming and then consuming anionic semiquinone, reproducing the reactivity of the ET-FAD. These data confirm that the retained FAD in domain II corresponds the ET-FAD. Quantum chemical calculations of the absorbance and CD spectra of each of WT Rpa ETF's two flavins reproduced the observed differences between their CD and absorbance signatures. The calculations for the flavin bound in domain II agreed better with the spectra of the ET-flavin, and those calculated based on the flavin between domains I and III agreed better with spectra of the Bf-flavin. Thus calculations independently confirm the locations of each flavin. We conclude that the site in domain II harbours the ET-FAD whereas the mutated site between domains I and III is the Bf-FAD site, confirming the accepted model by two different tests., (This journal is © The Royal Society of Chemistry 2019.)

Cathelicidins are a family of host defense antimicrobial peptides in mammalian species. Among them, LL-37 is the only peptide of this family found in humans. Although LL-37 has been intensively investigated in the past, the mode of exerting its bactericidal activity through the specific interactions with bacterial membranes remains elusive. In this work, we combined microbiological and computational approaches with a tool box of experimental biophysical techniques, including conventional and surface-enhanced infrared absorption spectroscopy as well as fluorescence spectroscopy to characterize the structural and dynamic properties of LL-37 and shorter variants adsorbed on POPC/POPG (9:1) lipid bilayers as mimics of bacterial membranes. First, microbiological assays demonstrate that, while LL-32 and, in a lesser degree, LL-37 show hemolysis and antimicrobial activity, LL-20 remains practically inactive. Second, by comparing experimental and computational data of LL-37 with LL-20, we explained the bactericidal activity of the active peptide core as a consequence of an increased flexibility of the peptide structure, leading to reactive dangling charged side chains. Third, permeabilization assays showed a concentration-dependent membrane disruption activity of LL-37 and LL-32: at high peptide concentrations, LL-32 shows higher activity than LL-37, while, at low peptide concentrations, both peptides show similar activities. Responsible for this behavior is the C-terminal VPRTES tail (C t -VPRTES tail), which, according to atomistic simulations, is able to promote the insertion of the peptide in the membrane and plays an essential role in controlling ordered peptide oligomerization on the surface of the membrane.

[NiFe] hydrogenases are enzymes that catalyze the splitting of molecular hydrogen according to the reaction H 2 → 2H + + 2e - . Most of these enzymes are inhibited even by low traces of O 2 . However, a special group of O 2 -tolerant hydrogenases exists. A member of this group is the membrane-bound [NiFe] hydrogenase from Ralstonia eutropha ( ReMBH). The ReMBH harbors an unusual iron sulfur cluster with composition 4Fe3S(6Cys) that is able to undergo structural changes triggering the flow of two electrons to the [NiFe] active site. These electrons promote oxygen reduction at the active site, preventing, in this way, aerobic inactivation of the enzyme. In the superoxidized state, the [4Fe3S] cluster binds to a hydroxyl group that originates from either molecular oxygen or water reaching the site. Both reactions, oxygen reduction to water at the [NiFe]- or [4Fe3S]-centers and oxygen evolution from water at the proximal cluster, require the delivery of protons regulated by a subtle communication mechanism between these metal centers. In this work, we sequentially apply multiscale modeling techniques as quantum mechanical/molecular mechanics methods and classical molecular dynamics simulations to investigate the role of two distinct proton transfer pathways connecting the [NiFe] active site and the [4Fe3S] proximal cluster of ReMBH in the protection mechanism against an oxygen attack. Although the "glutamate" pathway is preferred by protons migrating toward the active site to avoid inactivation by O 2 , the "histidine" pathway plays an essential role in delivering protons for O 2 reduction at the proximal cluster. The results obtained in this work not only provide new pieces to the puzzling catalytic mechanisms governing O 2 -tolerant hydrogenases but also highlight the relevance of dynamics in the proper description of biochemical reactions in general.

A pair of structurally precise analogues of molybdenum and rhenium complexes, [Et4N]/K2[MoO(prdt)2] and K[ReO(prdt)2] (prdt = pyrazine-2,3-dithiolene), were synthesized. These complexes serve as structural models for the active sites of bacterial molybdenum cofactor containing enzymes. They were comprehensively characterized and investigated by NMR, computationally supported IR and resonance Raman spectroscopy, cyclic voltammetry, mass spectrometry, elemental analysis and single-crystal X-ray diffraction. All compiled data are discussed in the context of comparing chemical and electronic structures and consequences thereof. This study constitutes the first investigation of a potential alternative Moco model system bearing rhenium as the central metal in an identical coordination environment to its molybdenum analogue. Structural evaluation revealed a slightly stronger M[double bond, length as m-dash]O bond in the rhenium complex in accordance with spectroscopic results, i.e. observed bond strengths. Thermodynamic parameters for the redox processes MoIV ↔ MoV and ReIV ↔ ReV were obtained by temperature dependent cyclic voltammetry. In contrast to molybdenum, rhenium loses entropy upon reduction and its redox potential is more temperature sensitive, indicating more significant differences than the respective diagonal relationship between the two metals in the periodic table might suggest and questioning rhenium's suitability as a functional artificial active site metal.

The Ru III -based prodrug AziRu efficiently binds to proteins, but the mechanism of its release is still disputed. Herein, in order to test the hypothesis of a reduction-mediated Ru release from proteins, a Raman-assisted crystallographic study on AziRu binding to a model protein (hen egg white lysozyme), in two different oxidation states, Ru II and Ru III , was carried out. Our results indicate Ru reduction, but the Ru release upon reduction is dependent on the reducing agent. To better understand this process, a pH-dependent, spectroelectrochemical surface-enhanced Raman scattering (SERS) study was performed also on AziRu-functionalized Au electrodes as a surrogate and simplest model system of Ru II - and Ru III -based drugs. This SERS study provided a p K a of 6.0 ± 0.4 for aquated AziRu in the Ru III state, which falls in the watershed range of pH values separating most cancer environments from their physiological counterparts. These experiments also indicate a dramatic shift of the redox potential E 0 by >600 mV of aquated AziRu toward more positive potentials upon acidification, suggesting a selective AziRu reduction in cancer lumen but not in healthy ones. It is expected that the nature of the ligands (e.g., pyridine vs imidazole, present in well-known Ru III complex NAMI-A) will modulate the p K a and E 0 , without affecting the underlying reaction mechanism.

Phytochromes are modular photoreceptors of plants, bacteria and fungi that use light as a source of information to regulate fundamental physiological processes. Interconversion between the active and inactive states is accomplished by a photoinduced reaction sequence which couples the sensor with the output module. However, the underlying molecular mechanism is yet not fully understood due to the lack of structural data of functionally relevant intermediate states. Here we report the crystal structure of a Meta-F intermediate state of an Agp2 variant from Agrobacterium fabrum. This intermediate, the identity of which was verified by resonance Raman spectroscopy, was formed by irradiation of the parent Pfr state and displays significant reorientations of almost all amino acids surrounding the chromophore. Structural comparisons allow identifying structural motifs that might serve as conformational switch for initiating the functional secondary structure change that is linked to the (de-)activation of these photoreceptors.

Fungal pathogens kill more people per year globally than malaria or tuberculosis and threaten international food security through crop destruction. New sophisticated strategies to inhibit fungal growth are thus urgently needed. Among the potential candidate molecules that strongly inhibit fungal spore germination are small cationic, cysteine-stabilized proteins of the AFP family secreted by a group of filamentous Ascomycetes. Its founding member, AFP from Aspergillus giganteus , is of particular interest since it selectively inhibits the growth of filamentous fungi without affecting the viability of mammalian, plant, or bacterial cells. AFPs are also characterized by their high efficacy and stability. Thus, AFP can serve as a lead compound for the development of novel antifungals. Notably, all members of the AFP family comprise a γ-core motif which is conserved in all antimicrobial proteins from pro- and eukaryotes and known to interfere with the integrity of cytoplasmic plasma membranes. In this study, we used classical molecular dynamics simulations combined with wet laboratory experiments and nuclear magnetic resonance (NMR) spectroscopy to characterize the structure and dynamical behavior of AFP isomers in solution and their interaction with fungal model membranes. We demonstrate that the γ-core motif of structurally conserved AFP is the key for its membrane interaction, thus verifying for the first time that the conserved γ-core motif of antimicrobial proteins is directly involved in protein-membrane interactions. Furthermore, molecular dynamic simulations suggested that AFP does not destroy the fungal membrane by pore formation but covers its surface in a well-defined manner, using a multistep mechanism to destroy the membranes integrity. IMPORTANCE Fungal pathogens pose a serious danger to human welfare since they kill more people per year than malaria or tuberculosis and are responsible for crop losses worldwide. The treatment of fungal infections is becoming more complicated as fungi develop resistances against commonly used fungicides. Therefore, discovery and development of novel antifungal agents are of utmost importance., (Copyright © 2018 Utesch et al.)

Surface-enhanced infrared absorption spectroscopy is used in situ to determine the electrochemical stability of organic interfaces deposited onto the surface of nanostructured, thin-film gold electrodes via the electrochemical reduction of diazonium salts. These interfaces are shown to exhibit a wide electrochemical stability window in both acetonitrile and phosphate buffer, far surpassing the stability window of thiol-derived self-assembled monolayers. Using the same in situ technique, the application of radical scavengers during the electrochemical reduction of diazonium salts is shown to moderate interface formation. Consequently, the heterogeneous charge-transfer resistance can be reduced sufficiently to enhance the direct electron transfer between an immobilized redox-active enzyme and the electrode. This was demonstrated for the oxygen-tolerant [NiFe] hydrogenase from the "Knallgas" bacterium Ralstonia eutropha by relating its electrochemical activity for hydrogen oxidation to the interface properties.

Respiratory complex I (CpI) is a key player in the way organisms obtain energy, being an energy transducer, which couples nicotinamide adenine dinucleotide (NADH)/quinone oxidoreduction with proton translocation by a mechanism that remains elusive so far. In this work, we monitored the function of CpI in a biomimetic, supported lipid membrane system assembled on a 4-aminothiophenol (4-ATP) self-assembled monolayer by surface-enhanced infrared absorption spectroscopy. 4-ATP serves not only as a linker molecule to a nanostructured gold surface but also as pH sensor, as indicated by concomitant density functional theory calculations. In this way, we were able to monitor NADH/quinone oxidoreduction-induced transmembrane proton translocation via the protonation state of 4-ATP, depending on the net orientation of CpI molecules induced by two complementary approaches. An associated change of the amide I/amide II band intensity ratio indicates conformational modifications upon catalysis which may involve movements of transmembrane helices or other secondary structural elements, as suggested in the literature [ Di Luca , Proc. Natl. Acad. Sci. U.S.A. , 2017 , 114 , E6314 - E6321 ].

We present structural information for oat phyA3 in the far-red-light-absorbing (Pfr) signaling state, to our knowledge the first three-dimensional (3D) information for a plant phytochrome as Pfr. Solid-state magic-angle spinning (MAS) NMR was used to detect interatomic contacts in the complete photosensory module [residues 1-595, including the NTE (N-terminal extension), PAS ( P er/ A rnt/ S im), GAF (c G MP phosphodiesterase/ a denylyl cyclase/ F hlA) and PHY ( phy tochrome-specific) domains but with the C-terminal PAS repeat and transmitter-like module deleted] auto-assembled in vitro with 13 C- and 15 N-labeled phycocyanobilin (PCB) chromophore. Thereafter, quantum mechanics/molecular mechanics (QM/MM) enabled us to refine 3D structural models constrained by the NMR data. We provide definitive atomic assignments for all carbon and nitrogen atoms of the chromophore, showing the Pfr chromophore geometry to be periplanar ZZEssa with the D -ring in a β -facial disposition incompatible with many earlier notions regarding photoconversion yet supporting circular dichroism (CD) data. The Y268 side chain is shifted radically relative to published Pfr crystal structures in order to accommodate the β -facial ring D . Our findings support a photoconversion sequence beginning with Pr photoactivation via an anticlockwise D -ring Za → Ea photoflip followed by significant shifts at the coupling of ring A to the protein, a B -ring propionate partner swap from R317 to R287, changes in the C -ring propionate hydrogen-bonding network, breakage of the D272-R552 salt bridge accompanied by sheet-to-helix refolding of the tongue region stabilized by Y326-D272-S554 hydrogen bonding, and binding of the NTE to the hydrophobic side of ring A . We discuss phyA photoconversion, including the possible roles of mesoscopic phase transitions and protonation dynamics in the chromophore pocket. We also discuss possible associations between structural changes and translocation and signaling processes within the cell.

The solubility-permeability relationship of active pharmaceutical ingredients determines the efficacy of their usage. Diclofenac (DCL), which is a widely used nonsteroidal anti-inflammatory drug, is characterized by extremely good membrane permeability, but low water solubility limiting drug effectiveness. The present research focuses on the fundamental explanation of this limitation using the combination of ab initio and classical molecular dynamics simulations of different ionic forms of DCL in water, namely, ionized, un-ionized and the mixture of them both. The analysis of diclofenac solvation in an aqueous environment is used to understand the origin of drug precipitation, especially in gastric pH. The used computational approach reveals the formation of micelle-like self-associated aggregates of diclofenac in water as the result of intermolecular π-π interactions and C-Hπ hydrogen bonds. The DCL aggregation in water is shown to depend mostly on drug concentration, protonation and temperature of the aqueous environment. The detected self-association properties of the drug in water are likely to be of great importance during the development of new drug formulations and fabrication of drug adsorbents for wastewater.