Catalytic Activity and Proton Translocation of Reconstituted Respiratory Complex I Monitored by Surface-Enhanced Infrared Absorption Spectroscopy.

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 ].

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.

Common Structural Elements in the Chromophore Binding Pocket of the Pfr State of Bathy Phytochromes.

Phytochromes are bimodal photoreceptors which, upon light absorption by the tetrapyrrole chromophore, can be converted between a red-absorbing state (Pr) and far-red-absorbing state (Pfr). In bacterial phytochromes, either Pr or Pfr are the thermally stable states, thereby constituting the classes of prototypical and bathy phytochromes, respectively. In this work, we have employed vibrational spectroscopies to elucidate the origin of the thermal stability of the Pfr states in bathy phytochromes. Here, we present the first detailed spectroscopic analysis of RpBphP6 (Rhodopseudomas palustris), which together with results obtained for Agp2 (Agrobacterium tumefaciens) and PaBphP (Pseudomonas aeruginosa) allows identifying common structural properties of the Pfr state of bathy phytochromes, which are (1) a homogenous chromophore structure, (2) the protonated ring C propionic side chain of the chromophore and (3) a retarded H/D exchange at the ring D nitrogen. These properties are related to the unique strength of the hydrogen bonding interactions between the ring D N-H group with the side chain of the conserved Asp194 (PaBphP numbering). As revealed by a comparative analysis of homology models and available crystal structures of Pfr states, these interactions are strengthened by an Arg residue (Arg453) only in bathy but not in prototypical phytochromes.

Substrate-Protein Interactions of Type II NADH:Quinone Oxidoreductase from Escherichia coli.

Type II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins involved in respiratory chains and responsible for the maintenance of NADH/NAD(+) balance in cells. NDH-2s are the only enzymes with NADH dehydrogenase activity present in the respiratory chain of many pathogens, and thus, they were proposed as suitable targets for antimicrobial therapies. In addition, NDH-2s were also considered key players for the treatment of complex I-related neurodegenerative disorders. In this work, we explored substrate-protein interaction in NDH-2 from Escherichia coli (EcNDH-2) combining surface-enhanced infrared absorption spectroscopic studies with electrochemical experiments, fluorescence spectroscopy assays, and quantum chemical calculations. Because of the specific stabilization of substrate complexes of EcNDH-2 immobilized on electrodes, it was possible to demonstrate the presence of two distinct substrate binding sites for NADH and the quinone and to identify a bound semiprotonated quinol as a catalytic intermediate.

A protonation-coupled feedback mechanism controls the signalling process in bathy phytochromes.

Phytochromes are bimodal photoswitches composed of a photosensor and an output module. Photoactivation of the sensor is initiated by a double bond isomerization of the tetrapyrrole chromophore and eventually leads to protein conformational changes. Recently determined structural models of phytochromes identify differences between the inactive and the signalling state but do not reveal the mechanism of photosensor activation or deactivation. Here, we report a vibrational spectroscopic study on bathy phytochromes that demonstrates that the formation of the photoactivated state and thus (de)activation of the output module is based on proton translocations in the chromophore pocket coupling chromophore and protein structural changes. These proton transfer steps, involving the tetrapyrrole and a nearby histidine, also enable thermal back-isomerization of the chromophore via keto-enol tautomerization to afford the initial dark state. Thus, the same proton re-arrangements inducing the (de)activation of the output module simultaneously initiate the reversal of this process, corresponding to a negative feedback mechanism.

Structure of the biliverdin cofactor in the Pfr state of bathy and prototypical phytochromes.

Phytochromes act as photoswitches between the red- and far-red absorbing parent states of phytochromes (Pr and Pfr). Plant phytochromes display an additional thermal conversion route from the physiologically active Pfr to Pr. The same reaction pattern is found in prototypical biliverdin-binding bacteriophytochromes in contrast to the reverse thermal transformation in bathy bacteriophytochromes. However, the molecular origin of the different thermal stabilities of the Pfr states in prototypical and bathy bacteriophytochromes is not known. We analyzed the structures of the chromophore binding pockets in the Pfr states of various bathy and prototypical biliverdin-binding phytochromes using a combined spectroscopic-theoretical approach. For the Pfr state of the bathy phytochrome from Pseudomonas aeruginosa, the very good agreement between calculated and experimental Raman spectra of the biliverdin cofactor is in line with important conclusions of previous crystallographic analyses, particularly the ZZEssa configuration of the chromophore and its mode of covalent attachment to the protein. The highly homogeneous chromophore conformation seems to be a unique property of the Pfr states of bathy phytochromes. This is in sharp contrast to the Pfr states of prototypical phytochromes that display conformational equilibria between two sub-states exhibiting small structural differences at the terminal methine bridges A-B and C-D. These differences may mainly root in the interactions of the cofactor with the highly conserved Asp-194 that occur via its carboxylate function in bathy phytochromes. The weaker interactions via the carbonyl function in prototypical phytochromes may lead to a higher structural flexibility of the chromophore pocket opening a reaction channel for the thermal (ZZE → ZZZ) Pfr to Pr back-conversion.

Vibrational stark effect of the electric-field reporter 4-mercaptobenzonitrile as a tool for investigating electrostatics at electrode/SAM/solution interfaces.

4-mercaptobenzonitrile (MBN) in self-assembled monolayers (SAMs) on Au and Ag electrodes was studied by surface enhanced infrared absorption and Raman spectroscopy, to correlate the nitrile stretching frequency with the local electric field exploiting the vibrational Stark effect (VSE). Using MBN SAMs in different metal/SAM interfaces, we sorted out the main factors controlling the nitrile stretching frequency, which comprise, in addition to external electric fields, the metal-MBN bond, the surface potential, and hydrogen bond interactions. On the basis of the linear relationships between the nitrile stretching and the electrode potential, an electrostatic description of the interfacial potential distribution is presented that allows for determining the electric field strengths on the SAM surface, as well as the effective potential of zero-charge of the SAM-coated metal. Comparing this latter quantity with calculated values derived from literature data, we note a very good agreement for Au/MBN but distinct deviations for Ag/MBN which may reflect either the approximations and simplifications of the model or the uncertainty in reported structural parameters for Ag/MBN. The present electrostatic model consistently explains the electric field strengths for MBN SAMs on Ag and Au as well as for thiophenol and mercaptohexanoic acid SAMs with MBN incorporated as a VSE reporter.

Role of Met80 and Tyr67 in the low-pH conformational equilibria of cytochrome c.

The low-pH conformational equilibria of ferric yeast iso-1 cytochrome c (ycc) and its M80A, M80A/Y67H, and M80A/Y67A variants were studied from pH 7 to 2 at low ionic strength through electronic absorption, magnetic circular dichroism, and resonance Raman spectroscopies. For wild-type ycc, the protein structure, axial heme ligands, and spin state of the iron atom convert from the native folded His/Met low-spin (LS) form to a molten globule His/H(2)O high-spin (HS) form and a totally unfolded bis-aquo HS state, in a single cooperative transition with an apparent pK(a) of ~3.0. An analogous cooperative transition occurs for the M80A and M80A/Y67H variants. This is preceded by protonation of heme propionate-7, with a pK(a) of ~4.2, and by an equilibrium between a His/OH(-)-ligated LS and a His/H(2)O-ligated HS conformer, with a pK(a) of ~5.9. In the M80A/Y67A variant, the cooperative low-pH transition is split into two distinct processes because of an increased stability of the molten globule state that is formed at higher pH values than the other species. These data show that removal of the axial methionine ligand does not significantly alter the mechanism of acidic unfolding and the ranges of stability of low-pH conformers. Instead, removal of a hydrogen bonding partner at position 67 increases the stability of the molten globule and renders cytochrome c more susceptible to acid unfolding. This underlines the key role played by Tyr67 in stabilizing the three-dimensional structure of cytochrome c by means of the hydrogen bonding network connecting the Ω loops formed by residues 71-85 and 40-57.

Human sulfite oxidase electrochemistry on gold nanoparticles modified electrode.

The present study reports a facile approach for sulfite biosensing, based on enhanced direct electron transfer of a human sulfite oxidase (hSO) immobilized on a gold nanoparticles modified electrode. The spherical core shell AuNPs were prepared via a new method by reduction of HAuCl(4) with branched poly(ethyleneimine) in an ionic liquids resulting particles with a diameter less than 10nm. These nanoparticles were covalently attached to a mercaptoundecanoic acid modified Au-electrode where then hSO was adsorbed and an enhanced interfacial electron transfer and electrocatalysis was achieved. UV/Vis and resonance Raman spectroscopy, in combination with direct protein voltammetry, are employed for the characterization of the system and reveal no perturbation of the structural integrity of the redox protein. The proposed biosensor exhibited a quick steady-state current response, within 2 s, a linear detection range between 0.5 and 5.4 µM with a high sensitivity (1.85 nA µM(-1)). The investigated system provides remarkable advantages in the possibility to work at low applied potential and at very high ionic strength. Therefore these properties could make the proposed system useful in the development of bioelectronic devices and its application in real samples.

Mapping local electric fields in proteins at biomimetic interfaces.

We present a novel approach for determining the strength of the electric field experienced by proteins immobilised on membrane models. It is based on the vibrational Stark effect of a nitrile label introduced at different positions on engineered proteins and monitored by surface enhanced infrared absorption spectroscopy.