Cation Dependence of Enniatin B/Membrane-Interactions Assessed Using Surface-Enhanced Infrared Absorption (SEIRA) Spectroscopy.

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 (Mz+=Na+, K+, Cs+, Li+, Mg2+, Ca2+) on the mechanism of enniatin B (EB) incorporation into membranes and identify the functionally relevant EBn : Mz+ complexes formed. We find that Na+ promotes a cooperative incorporation, modelled via an autocatalytic mechanism and mediated by a distorted 2 : 1-EB2 : Na+ complex. K+ (and Cs+) leads to a direct but less efficient insertion into membranes due to the adoption of "ideal" EB2 : K+ sandwich complexes. In contrast, the presence of Li+, Mg2+, and Ca2+ causes a (partial) extraction of EB from the membrane via the formation of "belted" 1 : 1-EB : Mz+ 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.

Trapping of a phenoxyl radical at a non-haem high-spin iron(II) centre.

The activation of dioxygen at haem and non-haem metal centres, and subsequent functionalization of unactivated C‒H bonds, has been a focal point of much research. In iron-mediated oxidation reactions, O2 binding at an iron(II) centre is often accompanied by an oxidation of the iron centre. Here we demonstrate dioxygen activation by sodium tetraphenylborate and protons in the presence of an iron(II) complex to form a reactive radical species, whereby the iron oxidation state remains unaltered in the presence of a highly oxidizing phenoxyl radical and O2. This complex, containing an unusual iron(II)-phenoxyl radical motif, represents an elusive example of a spectroscopically characterized oxygen-derived iron(II)-reactive intermediate during chemical and biological dioxygen activation at haem and non-haem iron active centres. The present report opens up strategies for the stabilization of a phenoxyl radical cofactor, with its full oxidizing capabilities, to act as an independent redox centre next to an iron(II) site during substrate oxidation reactions.

A high-spin alkylperoxo-iron(iii) complex with cis-anionic ligands: implications for the superoxide reductase mechanism.

The N3O macrocycle of the 12-TMCO ligand stabilizes a high spin (S = 5/2) [FeIII(12-TMCO)(OOtBu)Cl]+ (3-Cl) species in the reaction of [FeII(12-TMCO)(OTf)2] (1-(OTf)2) with tert-butylhydroperoxide (tBuOOH) in the presence of tetraethylammonium chloride (NEt4Cl) in acetonitrile at -20 °C. In the absence of NEt4Cl the oxo-iron(iv) complex 2 [FeIV(12-TMCO)(O)(CH3CN)]2+ is formed, which can be further converted to 3-Cl by adding NEt4Cl and tBuOOH. The role of the cis-chloride ligand in the stabilization of the FeIII-OOtBu moiety can be extended to other anions including the thiolate ligand relevant to the enzyme superoxide reductase (SOR). The present study underlines the importance of subtle electronic changes and secondary interactions in the stability of the biologically relevant metal-dioxygen intermediates. It also provides some rationale for the dramatically different outcomes of the chemistry of iron(iii)peroxy intermediates formed in the catalytic cycles of SOR (Fe-O cleavage) and cytochrome P450 (O-O bond lysis) in similar N4S coordination environments.

On the Role of a Conserved Tryptophan in the Chromophore Pocket of Cyanobacteriochrome.

The cyanobacteriochrome Slr1393 can be photoconverted between a red (Pr) and green absorbing form (Pg). The recently determined crystal structures of both states suggest a major movement of Trp496 from a stacking interaction with ring D of the phycocyanobilin (PCB) chromophore in Pr to a position outside the chromophore pocket in Pg. Here, we investigated the role of this amino acid during photoconversion in solution using engineered protein variants in which Trp496 was substituted by natural and non-natural amino acids. These variants and the native protein were studied by various spectroscopic techniques (UV-vis absorption, fluorescence, IR, NIR and UV resonance Raman) complemented by theoretical approaches. Trp496 is shown to affect the electronic transition of PCB and to be essential for the thermal equilibrium between Pr and an intermediate state O600. However, Trp496 is not required to stabilize the tilted orientation of ring D in Pr, and does not play a role in the secondary structure changes of Slr1393 during the Pr/Pg transition. The present results confirm the re-orientation of Trp496 upon Pr â†’ Pg conversion, but do not provide evidence of a major change in the microenvironment of this residue. Structural models indicate the penetration of water molecules into the chromophore pocket in both Pr and Pg states and thus water-Trp contacts, which can readily account for the subtle spectral changes between Pr and Pg. Thus, we conclude that reorientation of Trp496 during the Pr-to-Pg photoconversion in solution is not associated with a major change in the dielectric environment in the two states.

In Situ Raman Spectroscopy Reveals Cytochrome c Redox-Controlled Modulation of Mitochondrial Membrane Permeabilization That Triggers Apoptosis.

The selective interaction of cytochrome c (Cyt c) with cardiolipin (CL) is involved in mitochondrial membrane permeabilization, an essential step for the release of apoptosis activators. The structural basis and modulatory mechanism are, however, poorly understood. Here, we report that Cyt c can induce CL peroxidation independent of reactive oxygen species, which is controlled by its redox states. The structural basis of the Cyt c-CL binding was unveiled by comprehensive spectroscopic investigation and mass spectrometry. The Cyt c-induced permeabilization and its effect on membrane collapse, pore formation, and budding are observed by confocal microscopy. Moreover, cytochrome c oxidase dysfunction is found to be associated with the initiation of Cyt c redox-controlled membrane permeabilization. These results verify the significance of a redox-dependent modulation mechanism at the early stage of apoptosis, which can be exploited for the design of cytochrome c oxidase-targeted apoptotic inducers in cancer therapy.

Experimental Assessment of the Electronic and Geometrical Structure of a Near-Infrared Absorbing and Highly Fluorescent Microbial Rhodopsin.

The recently discovered Neorhodopsin (NeoR) exhibits absorption and emission maxima in the near-infrared spectral region, which together with the high fluorescence quantum yield makes it an attractive retinal protein for optogenetic applications. The unique optical properties can be rationalized by a theoretical model that predicts a high charge transfer character in the electronic ground state (S0) which is otherwise typical of the excited state S1 in canonical retinal proteins. The present study sets out to assess the electronic structure of the NeoR chromophore by resonance Raman (RR) spectroscopy since frequencies and relative intensities of RR bands are controlled by the ground and excited state's properties. The RR spectra of NeoR differ dramatically from those of canonical rhodopsins but can be reliably reproduced by the calculations carried out within two different structural models. The remarkable agreement between the experimental and calculated spectra confirms the consistency and robustness of the theoretical approach.

Vibrational Spectroscopy of Phytochromes.

Phytochromes are biological photoswitches that translate light into physiological functions. Spectroscopic techniques are essential tools for molecular research into these photoreceptors. This review is directed at summarizing how resonance Raman and IR spectroscopy contributed to an understanding of the structure, dynamics, and reaction mechanism of phytochromes, outlining the substantial experimental and theoretical challenges and describing the strategies to master them. It is shown that the potential of the various vibrational spectroscopic techniques can be most efficiently exploited using integral approaches via a combination of theoretical methods as well as other experimental techniques.

Orthogonal translation with 5-cyanotryptophan as an infrared probe for local structural information, electrostatics, and hydrogen bonding.

Orthogonal translation is an efficient tool that provides many valuable spectral probes capable of covering different parts of the electromagnetic spectrum and thus enabling parameterization of various structural and dynamic phenomena in proteins. In this context, nitrile-containing tryptophan analogs are very useful probes to study local electrostatics and hydrogen bonding in both rigid and dynamic environments. Here, we report a semi-rational approach to engineer a tyrosyl-tRNA synthetase (TyrRS) variant of Methanocaldococcus jannaschii capable of incorporating 5-cyanotryptophan (5CNW) via orthogonal translation. We combined one round of the well-established positive selection system with saturation mutagenesis at preselected TyrRS positions, resulting in a novel 5CNW-specific enzyme that also exhibits high substrate tolerance to other aromatic noncanonical amino acids. We demonstrated the utility of our orthogonal pair by inserting 5CNW into the cyanobacteriochrome Slr1393g3, a bilin-binding photosensor of the phytochrome superfamily. The nitrile (CN) group of the inserted 5CNW provides non-invasive labeling in the local structural context while yielding information on local electrostatics and hydrogen bonding by IR spectroscopy. 5CNW is a versatile probe that can be used for both static and dynamic measurements.

Parameterization of a single H-bond in Orange Carotenoid Protein by atomic mutation reveals principles of evolutionary design of complex chemical photosystems.

Introduction: Dissecting the intricate networks of covalent and non-covalent interactions that stabilize complex protein structures is notoriously difficult and requires subtle atomic-level exchanges to precisely affect local chemical functionality. The function of the Orange Carotenoid Protein (OCP), a light-driven photoswitch involved in cyanobacterial photoprotection, depends strongly on two H-bonds between the 4-ketolated xanthophyll cofactor and two highly conserved residues in the C-terminal domain (Trp288 and Tyr201). Method: By orthogonal translation, we replaced Trp288 in Synechocystis OCP with 3-benzothienyl-L-alanine (BTA), thereby exchanging the imino nitrogen for a sulphur atom. Results: Although the high-resolution (1.8 Å) crystal structure of the fully photoactive OCP-W288_BTA protein showed perfect isomorphism to the native structure, the spectroscopic and kinetic properties changed distinctly. We accurately parameterized the effects of the absence of a single H-bond on the spectroscopic and thermodynamic properties of OCP photoconversion and reveal general principles underlying the design of photoreceptors by natural evolution. Discussion: Such "molecular surgery" is superior over trial-and-error methods in hypothesis-driven research of complex chemical systems.

Stepwise assembly of the active site of [NiFe]-hydrogenase.

[NiFe]-hydrogenases are biotechnologically relevant enzymes catalyzing the reversible splitting of H2 into 2e- and 2H+ under ambient conditions. Catalysis takes place at the heterobimetallic NiFe(CN)2(CO) center, whose multistep biosynthesis involves careful handling of two transition metals as well as potentially harmful CO and CN- molecules. Here, we investigated the sequential assembly of the [NiFe] cofactor, previously based on primarily indirect evidence, using four different purified maturation intermediates of the catalytic subunit, HoxG, of the O2-tolerant membrane-bound hydrogenase from Cupriavidus necator. These included the cofactor-free apo-HoxG, a nickel-free version carrying only the Fe(CN)2(CO) fragment, a precursor that contained all cofactor components but remained redox inactive and the fully mature HoxG. Through biochemical analyses combined with comprehensive spectroscopic investigation using infrared, electronic paramagnetic resonance, Mössbauer, X-ray absorption and nuclear resonance vibrational spectroscopies, we obtained detailed insight into the sophisticated maturation process of [NiFe]-hydrogenase.

Substrate-Dependent Conformational Switch of the Noncubane [4Fe-4S] Cluster in Heterodisulfide Reductase HdrB.

The noncubane [4Fe-4S] cluster identified in the active site of heterodisulfide reductase (HdrB) displays a unique geometry among Fe-S cofactors found in metalloproteins. Here we employ resonance Raman (RR) spectroscopy and density functional theory (DFT) calculations to probe structural, electronic, and vibrational properties of the noncubane cluster in HdrB from a non-methanogenic Desulfovibrio vulgaris (Dv) Hildenborough organism. The immediate protein environment of the two neighboring clusters in DvHdrB is predicted using homology modeling. We demonstrate that in the absence of substrate, the oxidized [4Fe-4S]3+ cluster adopts a "closed" conformation. Upon substrate coordination at the "special" iron center, the cluster core translates to an "open" structure, facilitated by the "supernumerary" cysteine ligand switch from iron-bridging to iron-terminal mode. The observed RR fingerprint of the noncubane cluster, supported by Fe-S vibrational mode analysis, will advance future studies of enzymes containing this unusual cofactor.

Evidence of Sulfur Non-Innocence in [CoII (dithiacyclam)]2+ -Mediated Catalytic Oxygen Reduction Reactions.

In many metalloenzymes, sulfur-containing ligands participate in catalytic processes, mainly via the involvement in electron transfer reactions. In a biomimetic approach, we now demonstrate the implication of S-ligation in cobalt mediated oxygen reduction reactions (ORR). A comparative study between the catalytic ORR capabilities of the four-nitrogen bound [Co(cyclam)]2+ (1; cyclam=1,5,8,11-tetraaza-cyclotetradecane) and the S-containing analog [Co(S2 N2 -cyclam)]2+ (2; S2 N2 -cyclam=1,8-dithia-5,11-diaza-cyclotetradecane) reveals improved catalytic performance once the chalcogen is introduced in the Co coordination sphere. Trapping and characterization of the intermediates formed upon dioxygen activation at the CoII centers in 1 and 2 point to the involvement of sulfur in the O2 reduction process as the key for the improved catalytic ORR capabilities of 2.

A New Thiolate-Bound Dimanganese Cluster as a Structural and Functional Model for Class Ib Ribonucleotide Reductases.

In class Ib ribonucleotide reductases (RNRs) a dimanganese(II) cluster activates superoxide (O2 ⋅- ) rather than dioxygen (O2 ), to access a high valent MnIII -O2 -MnIV species, responsible for the oxidation of tyrosine to tyrosyl radical. In a biomimetic approach, we report the synthesis of a thiolate-bound dimanganese complex [MnII 2 (BPMT)(OAc)2 ](ClO)4 (BPMT=(2,6-bis{[bis(2-pyridylmethyl)amino]methyl}-4-methylthiophenolate) (1) and its reaction with O2 ⋅- to form a [(BPMT)MnO2 Mn]2+ complex 2. Resonance Raman investigation revealed the presence of an O-O bond in 2, while EPR analysis displayed a 16-line St =1/2 signal at g=2 typically associated with a MnIII MnIV core, as detected in class Ib RNRs. Unlike all other previously reported Mn-O2 -Mn complexes, generated by O2 ⋅- activation at Mn2 centers, 2 proved to be a capable electrophilic oxidant in aldehyde deformylation and phenol oxidation reactions, rendering it one of the best structural and functional models for class Ib RNRs.

Human endonuclease III/NTH1: focusing on the [4Fe-4S] cluster and the N-terminal domain.

Human Endonuclease III (EndoIII), hNTH1, is an FeS containing enzyme which repairs oxidation damaged bases in DNA. We report here the first comparative biophysical study of full-length and an N-terminally truncated hNTH1, with a domain architecture homologous to bacterial EndoIII. Vibrational spectroscopy, spectroelectrochemistry and SAXS experiments reveal distinct properties of the two enzyme forms, and indicate that the N-terminal domain is important for DNA binding at the onset of damage recognition.

Potential Distribution across Model Membranes.

Membrane models assembled on electrodes are widely used tools to study potential-dependent molecular processes at or in membranes. However, the relationship between the electrode potential and the potential across the membrane is not known. Here we studied lipid bilayers immobilized on mixed self-assembled monolayers (SAM) on Au electrodes. The mixed SAM was composed of thiol derivatives of different chain lengths such that between the islands of the short one, mercaptobenzonitrile (MBN), and the tethered lipid bilayer an aqueous compartment was formed. The nitrile function of MBN, which served as a reporter group for the vibrational Stark effect (VSE), was probed by surface-enhanced infrared absorption spectroscopy to determine the local electric field as a function of the electrode potential for pure MBN, mixed SAM, and the bilayer system. In parallel, we calculated electric fields at the VSE probe by molecular dynamics (MD) simulations for different charge densities on the metal, thereby mimicking electrode potential changes. The agreement with the experiments was very good for the calculations of the pure MBN SAM and only slightly worse for the mixed SAM. The comparison with the experiments also guided the design of the bilayer system in the MD setups, which were selected to calculate the electrode potential dependence of the transmembrane potential, a quantity that is not directly accessible by the experiments. The results agree very well with estimates in previous studies and thus demonstrate that the present combined experimental-theoretical approach is a promising tool for describing potential-dependent processes at biomimetic interfaces.

QuasAr Odyssey: the origin of fluorescence and its voltage sensitivity in microbial rhodopsins.

Rhodopsins had long been considered non-fluorescent until a peculiar voltage-sensitive fluorescence was reported for archaerhodopsin-3 (Arch3) derivatives. These proteins named QuasArs have been used for imaging membrane voltage changes in cell cultures and small animals, but they could not be applied in living rodents. To develop the next generation of sensors, it is indispensable to first understand the molecular basis of the fluorescence and its modulation by the membrane voltage. Based on spectroscopic studies of fluorescent Arch3 derivatives, we propose a unique photo-reaction scheme with extended excited-state lifetimes and inefficient photoisomerization. Molecular dynamics simulations of Arch3, of the Arch3 fluorescent derivative Archon1, and of several its mutants have revealed different voltage-dependent changes of the hydrogen-bonding networks including the protonated retinal Schiff-base and adjacent residues. Experimental observations suggest that under negative voltage, these changes modulate retinal Schiff base deprotonation and promote a decrease in the populations of fluorescent species. Finally, we identified molecular constraints that further improve fluorescence quantum yield and voltage sensitivity.

Infrared Spectroscopy Elucidates the Inhibitor Binding Sites in a Metal-Dependent Formate Dehydrogenase.

Biological carbon dioxide (CO2 ) 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 CO2 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 CO2 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.

Photoinduced reaction mechanisms in prototypical and bathy phytochromes.

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 XccBphP 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.