Gold slit nanoantenna arrays with length-dependent geometries were engineered as lithographically defined plasmonic platforms for dual-mode vibrational detection of the membrane protein cytochrome bd-I oxidase. Disordered slit arrays were fabricated in thin Au films on CaF2 substrates with constant slit width of 300 nm, thickness, and comparable surface coverage, while systematically varying slit length between 0.7 and 1.9 mu m. The intrinsic infrared response exhibited a continuous red shift with increasing slit length, enabling spectral positioning of the plasmonic resonance across the mid-infrared protein fingerprint region. Following site-specific immobilization of cytochrome bd-I via Ni-NTA coordination, geometry-dependent signal enhancement was observed in both surface-enhanced infrared absorption spectroscopy (SEIRAS) and surface-enhanced Raman spectroscopy (SERS). In SEIRAS, the amide I band intensity increased progressively with slit length, leading to increased signal intensity compared to the bare CaF2 and AuNPs references. The deconvolution of the amide I band confirmed preservation of the predominantly alpha-helical secondary structure across all samples, indicating that the immobilization and the plasmonic amplification does not compromise structural integrity. In SERS, slit nanoantennas produced systematic amplification of both heme-specific and aliphatic CH2/CH3 vibrational markers, with enhancement factors exceeding 1 order of magnitude for the longest slits. These results establish slit length as an independently controllable structural parameter and demonstrate that gold slit nanoantennas provide a reproducible, geometry-dependent plasmonic response platform for surface-enhanced vibrational detection of structurally intact membrane proteins.

Cytochrome bd is a distinctive family of terminal oxidases present in the respiratory chains of many prokaryotes. Despite their biological importance, the redox chemistry of these proteins remains poorly understood, largely due to the presence of two b-type hemes and one d-type heme. Here, we report the first computational study of interheme electron transfer in the cytochrome bd family. We performed 10 μs of molecular dynamics simulations of E. coli cytochrome bd-I embedded in realistic membranes, combined with quantum chemical calculations to estimate the thermodynamic parameters of electron transfer from heme b595 to heme d within the framework of Marcus theory. We further identify the respective contributions of the hemes, protein scaffold, lipid bilayer, water, and counterions to the driving force and reorganization energy. The interheme electronic coupling was calculated using the Projected Orbital Diabatization (POD) method in a hybrid Quantum Mechanics/Molecular Mechanics scheme and rationalized through electron transfer pathway analysis. This study provides fundamental insights into how electron transfer steps are orchestrated in the catalytic cycle of E. coli cytochrome bd-I.

Graphene nanostructures are capable of supporting plasmonic resonances in the visible and infrared parts of the spectrum. Thus, they can be exploited as platforms for Surface-Enhanced Infrared Absorption Spectroscopy (SEIRAS) and Surface-Enhanced Raman Spectroscopy (SERS) studies. One application of SEIRAS and SERS is the study of proteins at very low concentrations, down to the picomolar range. Among the different forms of graphene, graphene nanodots are ideal nanostructures that can be produced on a large scale using established protocols relying on sonication-assisted exfoliation under specific experimental conditions. Their rich surface chemistry facilitates stable and nondenaturing adsorption of membrane proteins, ensuring preservation of their native secondary structure upon immobilization. In this study, we exploited graphene nanodots deposited by drop casting or spray coating onto a silicon wafer as a substrate to study the cytochrome bd-I oxidase fromE. coli, a membrane protein that is present in the respiratory chains of bacteria. The amide I signal was examined to confirm the structural integrity of the protein once immobilized onto the graphene nanodots. SEIRAS and SERS experiments revealed reproducible enhancement of the protein signal, approximately by a factor of 2 and 6-10 compared to other substrates, respectively, enabling analyte detection with a sensitivity down to the nanomolar range. Furthermore, our tailored substrate exhibited high stability of the protein exceeding 6 days, thus underscoring its high potential for biosensing.

Selective chemical modifications of biomolecules are in high demand for the development and implementation of chemical biology strategies. Methods involving radicals have emerged as powerful options yet potentially generate side reactivities when involving harsh reaction conditions. We report the development of a mild catalytic method for the selective functionalization of tryptophane residues in peptides with a trifluoromethyl group using a water-soluble copper complex with redox-active ligands. This method displays both inter- and intramolecular selectivity for tryptophane residues and its synthetic relevance is demonstrated by the selective conversion of a 13-residue peptidic sequence and an endomorphine analogue in good yields.

The reduction of oxygen to water is crucial to life under aerobic conditions. Cytochrome bd oxidases perform this reaction with a very high oxygen affinity. Members of this protein family are solely found in prokaryotes and some archaea playing an important role in bacterial virulence and antibiotic resistance. Here, we combine mutagenesis, electrocatalysis, nitric oxide binding and release experiments as well as FTIR spectroscopy to demonstrate that proton delivery to the active site is essentially rate limiting in Cyt bd-I electrocatalysis. D58 and D105 of subunit CydB are crucial residues in this proton path and communicate via a hydrogen bond network. Oxygen reduction depends on proton delivery to the active site, which also influences NO release.

Terminal oxidases are critical for aerobic respiratory chains of prokaryotes and eukaryotes, responsible for the final step in the electron transport chain. These enzymes catalyze the transfer of electrons from reduced electron carriers (such as cytochrome c or quinols) to the terminal electron acceptor, molecular oxygen (O₂), thereby reducing it to water. They play a pivotal role in aerobic respiration and energy metabolism, adapting to diverse environmental and physiological needs across different organisms. This review summarizes the electrochemical properties of terminal oxidases from different organisms and reveals their high degree of adaptivity with redox potentials spanning more than 500 mV. The electrocatalytic response in direct electrochemical approaches is described giving insight into the rich and complex electron and proton transfer catalysed by these essential enzymes.

IspH is the last enzyme of the methylerythritol phosphate pathway. It catalyzes the reductive dehydroxylation of (E)-4-hydroxy-3-methyl-but-2-en-1-yl diphosphate into isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are precursors for the biosynthesis of terpenoids, essential molecules for the survival of all living organisms. This pathway is absent in humans, making it a promising target for drug discovery. Escherichia coli IspH harbors an unusual [4Fe-4S]2+ cluster linked to three conserved cysteines with a unique iron site proposed to be coordinated to three water molecules. Here, the first resonance Raman spectroscopic study of the cluster of IspH in the 2+ oxidation state is reported. Using isotopic labeling with 2H2O and H2 18O, the bands of the cluster that are sensitive to water coordination or hydrogen bonding are identified. The change of geometry of the cluster upon binding of the substrate, an alkyne diphosphate inhibitor, and the two enzyme products is also analyzed. Distinct binding modes to the cluster may indeed be at the origin of the different distribution of IPP and DMAPP observed during catalysis.

Respiratory complexes, such as cytochrome oxidases, are cofactor-containing multi-subunit protein complexes that are critically important for energy metabolism in all domains of life. Their intricate assembly strictly depends on accessory proteins, which coordinate subunit associations and cofactor deliveries. The small membrane protein CcoS was previously identified as an essential assembly factor to produce an active cbb3-type cytochrome oxidase (cbb3-Cox) in Rhodobacter capsulatus, but its function remained unknown. Here we show that the ΔccoS strain assembles a heme b deficient cbb3-Cox, in which the CcoN-CcoO subunit association is impaired. Chemical crosslinking demonstrates that CcoS interacts with the CcoN and CcoP subunits of cbb3-Cox, and that it stabilizes the interaction of the Cu-chaperone SenC with cbb3-Cox. CcoS lacks heme- or Cu-binding motifs, and we did not find evidence for direct heme or Cu binding; rather our data indicate that CcoS, together with SenC, coordinates heme and Cu insertion into cbb3-Cox.

Biological membranes are potentially involved in the transport of metal ions, such as Ti(IV), and, sometimes, their associated ligands. Understanding the interactions of Ti(IV) ions and complexes with biological membranes provides a basis for elucidating the action mechanism of titanium anticancer drugs. Herein, we investigated the interactions of two neutral titanium(IV) complexes, viz. [H2Ti(Cat)3] (1) and [H2Ti(Napht)3] (2), incorporated into DOPC multi-bilayers using ATR-FTIR spectroscopy. Infrared results showed that complexes 1 and 2, when interacting with DOPC multi-bilayers, highly affected the hydration of the lipid phosphate group and its mobility, revealing that the phosphate group is the main group involved in the interactions of complexes 1 and 2 with DOPC phospholipids. NMR studies involving complex 1 and DOPC dissolved in deuterated DMSO solution were performed, and interactions between the Ti complex and DOPC phosphate group could be evidenced. DFT calculations of model complexes were in good agreement with experimental data, and the stability of three model complexes was estimated. On the basis of the obtained data, it can be suggested that the oxygen atom(s) of the phosphato group of the DOPC ligand acted as donor atoms for Ti.

Disease modifying therapies including interferon-β (IFNβ) effectively counteract the inflammatory component in relapsing-remitting multiple sclerosis (RRMS) but this action, generally associated with severe side effects, does not prevent axonal/neuronal damages. Hence, axonal neuroprotection, which is pivotal for MS effective treatment, remains a difficult clinical challenge. Growing evidence suggested as promising candidate for neuroprotection, Emapunil (AC-5216) or XBD173, a ligand of the mitochondrial translocator protein highly expressed in glial cells and neurons. Indeed, elegant studies previously showed that low and well tolerated doses of XBD173 efficiently improved clinical symptoms and neuropathological markers in MS mice. Here we combined clinical scoring in vivo with Fourier transform infrared spectroscopy of sera samples to investigate the hypothesis that the concomitant treatment of RRMS mice with low doses of IFNβ and XBD173 may increase their beneficial effects against MS symptoms and additionally decrease IFNβ-induced side effects. Our results show a significant alteration of the composition of serum protein and lipids in the spectra of the sera of RRMS mice. While the signature of proteins remains altered upon treatment, the signature of lipids is recovered comparatively well with 20 kIU IFNβ and upon concomitant treatment with a low dose of XBD173 (10 mg/kg) and IFNβ (10 kIU), but not with 10 kIU of IFNβ alone. The concomitant therapy with XBD173 (10 mg/kg) and IFNβ (10 kIU), devoid of side effects, exhibited at least equal or even better efficacy than IFNβ (20 kIU) treatment against RRMS symptoms.

EDITORIAL article Front. Chem., 05 March 2024Sec. Theoretical and Computational Chemistry Volume 12 - 2024 | https://doi.org/10.3389/fchem.2024.1384385

In this paper, silver 3D foams have been synthesized using dynamic hydrogen bubble template and thiocyanatebased deposition bath. The material was used as electrocatalyst for nitrous acid (HNO2) reduction. On this material, we found that overpotential of nitrous acid reduction is decreased by at least 300 mV and the in situ formed nitric oxide (NO) reduction becomes the predominant step when the potential is decreased even further, comparing to flat silver polycrystalline electrode. Another difference between this material and the silver polycrystalline electrode concerns the influence of the nature of supporting electrolyte on the preferred reaction. Precisely, while Ag polycrystalline is preferentially reducing HNO2 into NO in low concentration of perchlorate electrolyte, Ag foam preferentially reduces the in situ formed NO into N2O, in all tested electrolytes, if the same potential range is considered. The initial reduction of HNO2 into NO is better evidenced after the partial removal of thiocyanate species. Finally, the use of isotope 15N in the nitrite salt and of a flow thin layer cell enabled the detection of nitrogen among other gaseous products for both silver foam and flat silver electrode.

The lipid cis-trans isomerase (Cti) is a periplasmic heme-c enzyme found in several bacteria including Pseudomonas aeruginosa, a pathogen known for causing nosocomial infections. This metalloenzyme catalyzes the cis-trans isomerization of unsaturated fatty acids in order to rapidly modulate membrane fluidity in response to stresses that impede bacterial growth. As a consequence, breakthrough in the elucidation of the mechanism of this metalloenzyme might lead to new strategies to combat bacterial antibiotic resistance. We report the first comprehensive biochemical, electrochemical and spectroscopic characterization of a Cti enzyme. This has been possible by the successful purification of Cti from P. aeruginosa (Pa-Cti) in favorable yields with enzyme activity of 0.41 μmol/min/mg when tested with palmitoleic acid. Through a synergistic approach involving enzymology, site-directed mutagenesis, Raman spectroscopy, Mössbauer spectroscopy and electrochemistry, we identified the heme coordination and redox state, pinpointing Met163 as the sixth ligand of the FeII of heme-c in Pa-Cti. Significantly, the development of an innovative assay based on liposomes demonstrated for the first time that Cti catalyzes cis-trans isomerization directly using phospholipids as substrates without the need of protein partners, answering the important question about the substrate of Cti within the bacterial membrane.

Metal ion-catalyzed overproduction of reactive oxygen species (ROS) is believed to contribute significantly to oxidative stress and be involved in several biological processes, from immune defense to development of diseases. Among the essential metal ions, copper is one of the most efficient catalysts in ROS production in the presence of O2 and a physiological reducing agent such as ascorbate. To control this chemistry, Cu ions are tightly coordinated to biomolecules. Free or loosely bound Cu ions are generally avoided to prevent their toxicity. In the present report, we aim to find stable Cu-ligand complexes (Cu-L) that can efficiently catalyze the production of ROS in the presence of ascorbate under aerobic conditions. Thermodynamic stability would be needed to avoid dissociation in the biological environment, and high ROS catalysis is of interest for applications as antimicrobial or anticancer agents. A series of Cu complexes with the well-known tripodal and tetradentate ligands containing a central amine linked to three pyridyl-alkyl arms of different lengths were investigated. Two of them with mixed arm length showed a higher catalytic activity in the oxidation of ascorbate and subsequent ROS production than Cu salts in buffer, which is an unprecedented result. Despite these high catalytic activities, no increased antimicrobial activity toward Escherichia coli or cytotoxicity against eukaryotic AGS cells in culture related to Cu-L-based ROS production could be observed. The potential reasons for discrepancy between in vitro and in cell data are discussed. Graphical Abstract Structure activity relationship of tripodal, tetradentade Cu-ligands for ascorbate oxidation.