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

Resonance Raman Spectroscopy on [NiFe] Hydrogenase Provides Structural Insights into Catalytic Intermediates and Reactions

[NiFe] hydrogenases catalyze the reversible cleavage of hydrogen and, thus, represent model systems for the investigation and exploitation of emission-free energy conversion processes. Valuable information on the underlying molecular mechanisms can be obtained by spectroscopic techniques that monitor individual catalytic intermediates. Here, we employed resonance Raman spectroscopy and extended it to the entire binuclear active site of an oxygen-tolerant [NiFe] hydrogenase by probing the metal–ligand modes of both the Fe and, for the first time, the Ni ion. Supported by theoretical methods, this approach allowed for monitoring H-transfer from the active site and revealed novel insights into the so far unknown structure and electronic configuration of the hydrogen-binding intermediate of the catalytic cycle, thereby providing key information about catalytic intermediates and reactions of biological hydrogen activation

Reversible Active Site Sulfoxygenation Can Explain the Oxygen Tolerance of a NAD⁺‑Reducing [NiFe] Hydrogenase and Its Unusual Infrared Spectroscopic Properties

Oxygen-tolerant [NiFe] hydrogenases are metalloenzymes that represent valuable model systems for sustainable H₂ oxidation and production. The soluble NAD⁺-reducing [NiFe] hydrogenase (SH) from Ralstonia eutropha couples the reversible cleavage of H₂ with the reduction of NAD⁺ and displays a unique O₂ tolerance. Here we performed IR spectroscopic investigations on purified SH in various redox states in combination with density functional theory to provide structural insights into the catalytic [NiFe] center. These studies revealed a standard-like coordination of the active site with diatomic CO and cyanide ligands. The long-lasting discrepancy between spectroscopic data obtained <i>in vitro</i> and <i>in vivo</i> could be solved on the basis of reversible cysteine oxygenation in the fully oxidized state of the [NiFe] site. The data are consistent with a model in which the SH detoxifies O₂ catalytically by means of an NADH-dependent (per)­oxidase reaction involving the intermediary formation of stable cysteine sulfenates. The occurrence of two catalytic activities, hydrogen conversion and oxygen reduction, at the same cofactor may inspire the design of novel biomimetic catalysts performing H₂-conversion even in the presence of O₂

Magnetic Silver Hybrid Nanoparticles for Surface-Enhanced Resonance Raman Spectroscopic Detection and Decontamination of Small Toxic Molecules

Magnetic hybrid assemblies of Ag and Fe₃O₄ nanoparticles with biocompatibly immobilized myoglobin (Mb) were designed to detect and capture toxic targets (NO₂^–, CN^–, and H₂O₂). Mb was covalently attached to chitosan-coated magnetic silver hybrid nanoparticles (M-Ag-C) <i>via</i> glutaraldehyde that serves as a linker for the amine groups of Mb and chitosan. As verified by surface-enhanced resonance Raman (SERR) spectroscopy, this immobilization strategy preserves the native structure of the bound Mb as well as the binding affinity for small molecules. On the basis of characteristic spectral markers, binding of NO₂^–, CN^–, and H₂O₂ could be monitored and quantified, demonstrating the high sensitivity of this approach with detection limits of 1 nM for nitrite, 0.2 μM for cyanide, and 10 nM for H₂O₂. Owing to the magnetic properties, these particles were collected by an external magnet to achieve an efficient decontamination of the solutions as demonstrated by SERR spectroscopy. Thus, the present approach combines the highly sensitive analytical potential of SERR spectroscopy with an easy approach for decontamination of aqueous solutions with potential applications in food and in environmental and medical safety control

Complex Formation with the Activator RACo Affects the Corrinoid Structure of CoFeSP

Activation of the corrinoid [Fe-S] protein (CoFeSP), involved in reductive CO₂ conversion, requires the reduction of the Co­(II) center by the [Fe-S] protein RACo, which according to the reduction potentials of the two proteins would correspond to an uphill electron transfer. In our resonance Raman spectroscopic work, we demonstrate that, as a conformational gate for the corrinoid reduction, complex formation of Co­(II)­FeSP and RACo specifically alters the structure of the corrinoid cofactor by modifying the interactions of the Co­(II) center with the axial ligand. On the basis of various deletion mutants, the potential interaction domains on the partner proteins can be predicted

Monitoring the Orientational Changes of Alamethicin during Incorporation into Bilayer Lipid Membranes

Antimicrobial peptides (AMPs) are the first line of defense after contact of an infectious invader, for example, bacterium or virus, with a host and an integral part of the innate immune system of humans. Their broad spectrum of biological functions ranges from cell membrane disruption over facilitation of chemotaxis to interaction with membrane-bound or intracellular receptors, thus providing novel strategies to overcome bacterial resistances. Especially, the clarification of the mechanisms and dynamics of AMP incorporation into bacterial membranes is of high interest, and different mechanistic models are still under discussion. In this work, we studied the incorporation of the peptaibol alamethicin (ALM) into tethered bilayer lipid membranes on electrodes in combination with surface-enhanced infrared absorption (SEIRA) spectroscopy. This approach allows monitoring the spontaneous and potential-induced ion channel formation of ALM in situ. The complex incorporation kinetics revealed a multistep mechanism that points to peptide–peptide interactions prior to penetrating the membrane and adopting the transmembrane configuration. On the basis of the anisotropy of the backbone amide I and II infrared absorptions determined by density functional theory calculations, we employed a mathematical model to evaluate ALM reorientations monitored by SEIRA spectroscopy. Accordingly, ALM was found to adopt inclination angles of ca. 69°–78° and 21° in its interfacially adsorbed and transmembrane incorporated states, respectively. These orientations can be stabilized efficiently by the dipolar interaction with lipid head groups or by the application of a potential gradient. The presented potential-controlled mechanistic study suggests an N-terminal integration of ALM into membranes as monomers or parallel oligomers to form ion channels composed of parallel-oriented helices, whereas antiparallel oligomers are barred from intrusion

Effect of the Protonation Degree of a Self-Assembled Monolayer on the Immobilization Dynamics of a [NiFe] Hydrogenase

Understanding the interaction and immobilization of [NiFe] hydrogenases on functionalized surfaces is important in the field of biotechnology and, in particular, for the development of biofuel cells. In this study, we investigated the adsorption behavior of the standard [NiFe] hydrogenase of Desulfovibrio gigas on amino-terminated alkanethiol self-assembled monolayers (SAMs) with different levels of protonation. Classical all-atom molecular dynamics (MD) simulations revealed a strong correlation between the adsorption behavior and the level of ionization of the chemically modified electrode surface. While the hydrogenase undergoes a weak but stable initial adsorption process on SAMs with a low degree of protonation, a stronger immobilization is observable on highly ionized SAMs, affecting protein reorientation and conformation. These results were validated by complementary surface-enhanced infrared absorption (SEIRA) measurements on the comparable [NiFe] standard hydrogenases from Desulfovibrio vulgaris Miyazaki F and allowed in this way for a detailed insight into the adsorption mechanism at the atomic level

Monitoring the Transmembrane Proton Gradient Generated by Cytochrome <i>bo</i>₃ in Tethered Bilayer Lipid Membranes Using SEIRA Spectroscopy

Membrane proteins act as biocatalysts or ion/proton pumps to convert and store energy from ubiquitous environmental sources. Interfacing these proteins to electrodes allows utilizing the energy for enzymatic biofuel cells or other auspicious biotechnological applications. To optimize the efficiency of these devices, appropriate membrane models are required that ensure structural and functional integrity of the embedded enzymes and provide structural insight. We present a spectroelectrochemical surface-enhanced infrared absorption (SEIRA) and electrical impedance spectroscopy (EIS) study of the bacterial respiratory ubiquinol/cytochrome <i>bo</i>₃ (cyt <i>bo</i>₃) couple incorporated into a tethered bilayer lipid membrane (tBLM). Here, we employed a new lipid tether (WK3SH, dihydrocholesteryl (2-(2-(2-ethoxy)­ethoxy)­ethanethiol), which was synthesized using a three-step procedure with very good yield and allowed measuring IR spectra without significant spectral interference of the tBLM. The functional integrity of the incorporated cyt <i>bo</i>₃ was demonstrated by monitoring the enzymatic O₂ reduction current and the formation of the transmembrane proton gradient. Based on a SEIRA-spectroscopic redox titration, a shift of the pH-dependent redox potential of the ubiquinones under turnover conditions was correlated with an alkalinization of the submembrane reservoir by +0.8 pH units. This study demonstrates the high potential of tBLMs and the SEIRA spectroscopic approach to study bioenergetic processes

Characterization of wild-type mAOX1 by UV-VIS absorption spectroscopy.

<p>Spectra of 7 µM of the air-oxidized mAOX1 in 50 mM Tris, 1 mM EDTA, pH 7.5, under anaerobic conditions.</p

Purification of recombinant mAOX1 after expression in <i>E. coli</i> TP1000 cells.

a<p>Total protein was quantified with the Bradford assay.</p>b<p>The activity was measured by monitoring the decrease in absorption at 600 nm in the presence of 500 µM benzaldehyde and 100 µM DCPIP.</p>c<p>Specific enzyme activity (units/mg) is defined as the oxidation of 1 µM benzaldehyde per min and mg of enzyme under the assay conditions.</p