Surface-enhanced Resonance Raman Spectroscopy Research Articles

Did the first two Argentine flags of Macha and Ayohuma wave together?

Historical sources claim that the Argentine ensigns commonly referred to as Macha and Ayohuma flags have the same origin. The difference in their designs is remarkable, with the former being white, blue, and white in horizontal bands, while the latter has the coloured stripes at its ends. According to a historical account, these two flags were used by the patriot and creator of the Argentine flag, one of the parents of the homeland, Manuel Belgrano, when he first raised them on the banks of the Paraná River on February 27, 1812. Both flags were found by Father Martín Castro in 1883 behind a painting of Santa Cecilia in the church of Titiri near the town of Macha in Bolivia at an altitude of 4350 m above sea level. The composition of the Ayohuma flag was analysed in this paper, and the results were compared with those obtained and already published for the study conducted on the flag of Macha. It was determined by ATR-FTIR spectroscopy that the Ayohuma flag is made of silk, with the diameter of the individual silk filament of 12.5 μm, the dye used to colour the silk is indigotin, according to its resonance Raman spectrum, and methylene blue was detected by Surface-Enhanced Resonance Raman Spectroscopy, presumably utilized for the restoration of the coloured part of the flag.

Identification of transgenic crops by CRISPR/Cas12a-assisted magnetic surface-enhanced resonance Raman spectroscopy

Rapid and reliable identification of genetically modified (GM) and non-GM crops is in urgent demand and remains a significant challenge in food safety. Here, a new and reliable Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-mediated magnetic surface-enhanced resonance Raman scattering (SERRS) biosensing is reported to identify specific gene sequences of transgenic crops (CaMV 35 S promoter and Nos terminator). This assay mainly combines the gene-specific recognition ability of CRISPR, the ultra-high sensitivity due to the Raman resonance effect, and the simple separation property of magnetic nanomaterials. A multifunctional SERRS-active nanoprobe was designed by decorating a gold-coated magnetic bead with the G-triplet deoxyribonucleic acid (DNA) sequences that enable a high loading of Raman reporters (methylene blue) inserted in G-triplet DNAs. The assay is achieved by the recognition of the CRISPR/Cas12a protein for bound target DNAs and the activation of the unlimited trans-cleavage action on the G-triplet DNAs, which causes a release of methylene blue above the nanoprobes and a reduction of the SERRS signal. Gel electrophoresis experiments imply that the designed crRNAs have good recognition and cleavage function for target DNAs and verify the feasibility of the experimental scheme. This SERRS assay exhibits a linear response of 4.15 × 10−12 M to 4.15 × 10−9 M with a pM detection level. Tests of the GM and non-GM crops of actual crop samples have been realized, which implies that this CRISPR-mediated magnetic SERRS method is available for assessing natural samples and working for GM crop regulation.

In Situ Monitoring of Membrane Protein Electron Transfer via Surface-Enhanced Resonance Raman Spectroscopy.

In situ analysis of membrane protein-ligand interactions under physiological conditions is of significance for both fundamental and applied science, but it is still a big challenge due to the limits in sensitivity and selectivity. Here, we demonstrate the potential of surface-enhanced resonance Raman spectroscopy (SERRS) for the investigation of membrane protein-protein interactions. Lipid biolayers are successfully coated on silver nanoparticles through electrostatic interactions, and a highly sensitive and biomimetic membrane platform is obtained in vitro. Self-assembly and immobilization of the reduced cytochrome b5 on the coated membrane are achieved and protein native biological functions are preserved. Owing to resonance effect, the Raman fingerprint of the immobilized cytochrome b5 redox center is selectively enhanced, allowing for in situ and real-time monitoring of the electron transfer process between cytochrome b5 and their partners, cytochrome c and myoglobin. This study provides a sensitive analytical approach for membrane proteins and paves the way for in situ exploration of their structural basis and functions.

Tunable Tamm plasmon cavity as a scalable biosensing platform for surface enhanced resonance Raman spectroscopy

Surface enhanced Resonance Raman spectroscopy (SERRS) is a powerful technique for enhancing Raman spectra by matching the laser excitation wavelength with the plasmonic resonance and the absorption peak of biomolecules. Here, we propose a tunable Tamm plasmon polariton (TPP) cavity based on a metal on distributed Bragg reflector (DBR) as a scalable sensing platform for SERRS. We develop a gold film-coated ultralow-loss phase change material (Sb2S3) based DBR, which exhibits continuously tunable TPP resonances in the optical wavelengths. We demonstrate SERRS by matching the TPP resonance with the absorption peak of the chromophore molecule at 785 nm wavelength. We use this platform to detect cardiac Troponin I protein (cTnI), a biomarker for early diagnosis of cardiovascular disease, achieving a detection limit of 380 fM. This scalable substrate shows great promise as a next-generation tunable biosensing platform for detecting disease biomarkers in body fluids for routine real-time clinical diagnosis.

In Situ and Real-Time Monitoring of Mitochondria-Endoplasmic Reticulum Crosstalk in Apoptosis via Surface-Enhanced Resonance Raman Spectroscopy.

The crosstalk between mitochondria and endoplasmic reticula plays a crucial role in apoptotic pathways in which reactive oxygen species (ROS) produced by microsomal monooxygenase (MMO) are believed to accelerate cytochrome c release. Herein, we successfully demonstrate the potential of surface-enhanced resonance Raman spectroscopy (SERRS) for monitoring MMO-derived ROS formation and ROS-mediated cytochrome c release. Silver nanoparticles coated with nickel shells are used as both Raman signal enhancers and electron donors for cytochrome c. SERRS of cytochrome c is found to be sensitive to ROS, allowing for in situ probing of ROS formation with a cell death inducer. Label-free evaluation of ROS-induced apoptosis is achieved by SERRS-based monitoring of cytochrome c release in living cells. This study verifies the capability of SERRS for label-free, in situ, and real-time monitoring of the mitochondria-endoplasmic reticulum crosstalk in apoptosis and provides a novel strategy for the rational design and screening of ROS-inducing drugs for cancer treatment.

Ultra-trace detection of diverse analyte molecules using femtosecond laser structured Ag–Au alloy substrates and SERRS

Choosing a wavelength close to the electronic excitation of the analyte molecules is known to enhance the Surface Enhanced Raman Spectroscopy signal further. This technique is often referred to as Surface Enhanced Resonance Raman Spectroscopy (SERRS). Here we use SERRS for sensing different classes of molecules and demonstrate how it can be used for ultra-trace detection. Diverse SERS substrates were fabricated by femtosecond laser ablation of Ag–Au alloy (1:1) in the air by changing the angle of incidence. Further, we have examined the SERRS performance of each substrate. Often in laser ablation in air, the surface debris is considered undesirable. Here we have shown that this debris of nanoparticles can be used as an advantage for ultra-trace detection of different molecules. Further, with the advantage of having randomly stacked nanoparticles in debris and periodic substrates without debris from a single experiment, we have examined the much-debated relationship between enhancement and reproducibility in SERS and found that they are inversely correlated. The sensitivity of the substrate for rhodamine 6G, crystal violet, picric acid, and cysteine was found to be 10 fM, 100 fM, 100 nM, and 100 nM, respectively, with good reproducibility.

Silver nanostructure-modified graphite electrode for in-operando SERRS investigation of iron porphyrins during high-potential electrocatalysis.

In-operando spectroscopic observation of the intermediates formed during various electrocatalytic oxidation and reduction reactions is crucial to propose the mechanism of the corresponding reaction. Surface-enhanced resonance Raman spectroscopy coupled to rotating disk electrochemistry (SERRS-RDE), developed about a decade ago, proved to be an excellent spectroscopic tool to investigate the mechanism of heterogeneous oxygen reduction reaction (ORR) catalyzed by synthetic iron porphyrin complexes under steady-state conditions in water. The information about the formation of the intermediates accumulated during the course of the reaction at the electrode interface helped to develop better ORR catalysts with second sphere residues in the porphyrin rings. To date, the application of this SERRS-RDE setup is limited to ORR only because the thiol self-assembled monolayer (SAM)-modified Ag electrode, used as the working electrode in these experiments, suffers from stability issues at more cathodic and anodic potential, where H2O oxidation, CO2 reduction, and H+ reduction reactions occur. The current investigation shows the development of a second-generation SERRS-RDE setup consisting of an Ag nanostructure (AgNS)-modified graphite electrode as the working electrode. These electrodes show higher stability (compared to the conventional thiol SAM-modified Ag electrode) upon exposure to very high cathodic and anodic potential with a good signal-to-noise ratio in the Raman spectra. The behavior of this modified electrode toward ORR is found to be the same as the SAM-modified Ag electrode, and the same ORR intermediates are observed during electrochemical ORR. At higher cathodic potential, the signatures of Fe(0) porphyrin, an important intermediate in H+ and CO2 reduction reactions, was observed at the electrode-water interface.

Emergence of Raman Spectroscopy as a Probing Tool for Theranostics.

Although medical advances have increased our grasp of the amazing morphological, genetic, and phenotypic diversity of diseases, there are still significant technological barriers to understanding their complex and dynamic character. Specifically, the complexities of the biological systems throw a diverse set of challenges in developing efficient theranostic tools and methodologies that can probe and treat pathologies. Among several emerging theranostic techniques such as photodynamic therapy, photothermal therapy, magnetic resonance imaging, and computed tomography, Raman spectroscopy (RS) is emerging as a promising tool that is a label-free, cost-effective, and non-destructive technique. It can also provide real-time diagnostic information and can employ multimodal probes for detection and therapy. These attributes make it a perfect candidate for the analytical counterpart of the existing theranostic probes. The use of biocompatible nanomaterials for the fabrication of Raman probes provides rich structural information about the biological molecules, cells, and tissues and highly sensitive information down to single-molecule levels when integrated with advanced RS tools. This review discusses the fundamentals of Raman spectroscopic tools such as surface-enhanced Raman spectroscopy and Resonance Raman spectroscopy, their variants, and the associated theranostic applications. Besides the advantages, the current limitations, and future challenges of using RS in disease diagnosis and therapy have also been discussed.

Antibiotic Susceptibility Testing with Raman Biosensing.

Antibiotics guard us against bacterial infections and are among the most commonly used medicines. The immediate consequence of their large-scale production and prescription is the development of antibiotic resistance. Therefore, rapid detection of antibiotic susceptibility is required for efficient antimicrobial therapy. One of the promising methods for rapid antibiotic susceptibility testing is Raman spectroscopy. Raman spectroscopy combines fast and contactless acquisition of spectra with good selectivity towards bacterial cells. The antibiotic-induced changes in bacterial cell physiology are detected as distinct features in Raman spectra and can be associated with antibiotic susceptibility. Therefore, the Raman-based approach may be beneficial in designing therapy against multidrug-resistant infections. The surface-enhanced Raman spectroscopy (SERS) and resonance Raman spectroscopy (RRS) additionally provide excellent sensitivity. In this review, we present an analysis of the Raman spectroscopy-based optical biosensing approaches aimed at antibiotic susceptibility testing.

Second-Sphere Hydrogen-Bond Donors and Acceptors Affect the Rate and Selectivity of Electrochemical Oxygen Reduction by Iron Porphyrins Differently.

The factors that control the rate and selectivity of 4e-/4H+ O2 reduction are important for efficient energy transformation as well as for understanding the terminal step of respiration in aerobic organisms. Inspired by the design of naturally occurring enzymes which are efficient catalysts for O2 and H2O2 reduction, several artificial systems have been generated where different second-sphere residues have been installed to enhance the rate and efficiency of the 4e-/4H+ O2 reduction. These include hydrogen-bonding residues like amines, carboxylates, ethers, amides, phenols, etc. In some cases, improvements in the catalysis were recorded, whereas in some cases improvements were marginal or nonexistent. In this work, we use an iron porphyrin complex with pendant 1,10-phenanthroline residues which show a pH-dependent variation of the rate of the electrochemical O2 reduction reaction (ORR) over 2 orders of magnitude. In-situ surface-enhanced resonance Raman spectroscopy reveals the presence of different intermediates at different pH's reflecting different rate-determining steps at different pH's. These data in conjunction with density functional theory calculations reveal that when the distal 1,10-phenanthroline is neutral it acts as a hydrogen-bond acceptor which stabilizes H2O (product) binding to the active FeII state and retards the reaction. However, when the 1,10-phenanthroline is protonated, it acts as a hydrogen-bond donor which enhances O2 reduction by stabilizing FeIII-O2.- and FeIII-OOH intermediates and activating the O-O bond for cleavage. On the basis of these data, general guidelines for controlling the different possible rate-determining steps in the complex multistep 4e-/4H+ ORR are developed and a bioinspired principle-based design of an efficient electrochemical ORR is presented.

Aluminum-black silicon plasmonic nano-eggs structure for deep-UV surface-enhanced resonance Raman spectroscopy

By combining surface-enhanced Raman spectroscopy together with resonance Raman effects in the deep-UV region, ultra-sensitive and selective molecule detection can be achieved by deep-UV surface-enhanced resonance Raman spectroscopy (SERRS). Here, we report a deep-UV plasmonic nano-eggs structure consisting of elongated Al nanoparticles on black Si (BSi) for use in deep-UV SERRS characterization of biomolecules. The Al/BSi nano-eggs structure can be easily fabricated over a large area via conventional techniques including inductively coupled-plasma reactive ion etching on a Si substrate and Al sputtering without the need for accurate thickness control. A home-built deep-UV SERRS setup with the excitation wavelength of 266 nm is used to characterize adenine deposited on Al/BSi nano-eggs structures. High-intensity and reproducible Raman signals for adenine are obtained. A low-cost and easy-to-fabricate Al/BSi nano-eggs structure provides a convenient means to achieve deep-UV SERRS characterization, and it is thought to be beneficial for the development of ultra-sensitive molecule detection schemes.

Surface-Enhanced Resonance Raman Spectroscopy by Plasmon-Exciton Coupling for Large Area Mos2 Monolayers Characterization

Surface-Enhanced Resonance Raman Spectroscopy by Plasmon-Exciton Coupling for Large Area Mos2 Monolayers Characterization

Demonstration of a Superior Deep-UV Surface-Enhanced Resonance Raman Scattering (SERRS) Substrate and Single-Base Mutation Detection in Oligonucleotides.

In life science, rapid mutation detection in oligonucleotides is in a great demand for genomic and medical screening. To satisfy this demand, surface-enhanced resonance Raman spectroscopy (SERRS) in the deep-UV (DUV) regime offers a promising solution due to its merits of label-free nature, strong electromagnetic confinement, and charge transfer effect. Here, we demonstrate an epitaxial aluminum (Al) DUV-SERRS substrate that resonates effectively with the incident Raman laser and the ss-DNA at 266 nm, yielding significant SERRS signals of the detected analytes. For the first time, to the best of our knowledge, we obtaine SERRS spectra for all bases of oligonucleotides, not only revealing maximum characteristic Raman peaks but also recording the highest enhancement factor of up to 106 for a 1 nm thick adenine monomer. Moreover, our epitaxial Al DUV-SERRS substrate is able to enhance the Raman signal of all four bases of 12-mer ss-DNA and to further linearly quantify the single-base mutation in the 12-mer ss-DNA.

Supramolecular Reorientation During Deposition Onto Metal Surfaces of Quasi-Two-Dimensional Langmuir Monolayers Composed of Bifunctional Amphiphilic, Twisted Perylenes.

Supramolecular dye structures, which are often ruled by π-π interactions between planar chromophores, crucially determine the optoelectronic properties of layers and interfaces. Here, we present the interfacial assembly of perylene monoanhydride and monoimide that do not feature a planar chromophore but contain chlorine substituents in the bay positions to yield twisted chromophores and hence modified π-stacking. The assembly of the twisted perylene monoanhydride and monoimide is driven by their amphiphilicity that ensures proper Langmuir layer formation. The shielding of the hydrophilic segment upon attaching an alkyl chain to the imide moiety yielded a more rigid Langmuir layer, even though the degrees of freedom were increased due to this modification. For the characterization of the Langmuir layer's supramolecular structure, the layers were deposited onto glass, silver, and gold substrates via Langmuir-Blodgett (LB) and Langmuir-Schaefer (LS) techniques and were investigated with atomic force microscopy and surface-enhanced resonance Raman spectroscopy (SERRS). From the similarity between all SERR spectra of the LS and LB layers, we concluded that the perylenes have changed their orientation upon LB deposition to bind to the silver surface of the SERRS substrate via sulfur atoms. In the Langmuir layer, the perylenes, which are π-stacked with half of the twisted chromophores, must already be inclined and cannot achieve full parallel alignment because of the twisting-induced steric hindrance. However, upon rotation, the energetically most favorable antiparallel aligned structures can be formed and bind to the SERRS substrate. Thus, we present, to the best of our knowledge, the first fabrication of quasi-two-dimensional films from twisted amphiphilic perylene monoimides and their reassembly during LB deposition. The relation between the molecular structure, supramolecular interfacial assembly, and its adoption during adsorption revealed here is crucial for the fabrication of defined functionalizations of metal surfaces, which is key to the development of organic (opto)electronic devices.

Rejigging Electron and Proton Transfer to Transition between Dioxygenase, Monooxygenase, Peroxygenase, and Oxygen Reduction Activity: Insights from Bioinspired Constructs of Heme Enzymes.

Nature has employed heme proteins to execute a diverse set of vital life processes. Years of research have been devoted to understanding the factors which bias these heme enzymes, with all having a heme cofactor, toward distinct catalytic activity. Among them, axial ligation, distal super structure, and substrate binding pockets are few very vividly recognized ones. Detailed mechanistic investigation of these heme enzymes suggested that several of these enzymes, while functionally divergent, use similar intermediates. Furthermore, the formation and decay of these intermediates depend on proton and electron transfer processes in the enzyme active site. Over the past decade, work in this group, using in situ surface enhanced resonance Raman spectroscopy of synthetic and biosynthetic analogues of heme enzymes, a general idea of how proton and electron transfer rates relate to the lifetime of different O2 derived intermediates has been developed. These findings suggest that the enzymatic activities of all these heme enzymes can be integrated into one general cycle which can be branched out to different catalytic pathways by regulating the lifetime and population of each of these intermediates. This regulation can further be achieved by tuning the electron and proton transfer steps. By strategically populating one of these intermediates during oxygen reduction, one can navigate through different catalytic processes to a desired direction by altering proton and electron transfer steps.

A Resonance Raman Marker Band Characterizes the Slow and Fast Form of Cytochrome c Oxidase.

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 a3 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.

Urea-induced denaturation of immobilized yeast iso-1 cytochrome c: Role of Met80 and Tyr67 in the thermodynamics of unfolding and promotion of pseudoperoxidase and nitrite reductase activities

The Met80Ala and Met80Ala/Tyr67Ala variants of S. cerevisiae iso-1 cytochrome c (ycc) immobilized on a decane-1-thiol coated gold electrode subjected to the denaturing action of urea were studied through variable temperature cyclic voltammetry and Surface-Enhanced Resonance Raman spectroscopy (SERRS). We found that the His/OH− axial heme iron coordination in both variants is unaffected by urea up to 7 M, although some conformational changes occur that increase exposure of the heme center to solvent. The thermodynamics of the unfolding process were determined with an unprecedented approach, which can be of general use for electroactive proteins. The free energy of unfolding for both variants includes relevant entropic contributions and is lower than that for the species carrying an intact Met/His coordination, consistent with the role of the axial Fe-(S)Met bond and the H-bond network involving Tyr67 in stabilizing the polypeptide matrix in the heme crevice. Their lower conformational stability results in a different interaction with the MUA/MU SAM compared to the His/Met ycc forms. Denaturation invariably slows down the heterogeneous electron transfer process, but its effect on the activation enthalpy and pre-exponential factor differs for the species with and without His/Met axial heme ligation. In particular, urea unfolding of the M80A and M80A/Y67A mutants lowers the structural restraint to the heterogeneous ET. Here we show that removal of the Met ligand and an increased accessibility of the heme center to solvent through partial protein unfolding– which mimic the molecular stress experienced by mammalian cytochromes c upon binding to cardiolipin in the early events of apoptosis - add up to transform cytochrome c into an efficient electrocatalyst toward the reduction of hydrogen peroxide and nitrite.

Aptamer-based surface-enhanced resonance Raman scattering assay on a paper fluidic platform for detection of cardiac troponin I.

.Significance: Cardiac troponin I (cTnI) is a primary biomarker for diagnosis of myocardial infarction (MI). In contrast to central laboratory tests for cTnI, point-of-care (POC) testing has the advantage of providing results when the patient is first encountered, which helps high-risk patients to be treated more rapidly and low-risk patients to be released in a timely fashion. A paper fluidic platform is good for POC testing because the paper is abundant, low cost, and disposable. However, current cTnI assays on paper platforms use antibodies as the recognition element, which has limitations due to the high cost of production and antibody stability issues at the POC.Aim: To develop an aptamer-based assay on a paper strip using surface-enhanced resonance Raman spectroscopy (SERRS) for detection of cTnI in the clinically relevant range at the POC.Approach: Gold nanoparticles (AuNPs) were functionalized with a Raman reporter molecule, malachite green isothiocyanate. The functionalized AuNPs were encapsulated in a silica shell and provided a SERRS signal using a handheld Raman system with a 638-nm excitation wavelength. A primary aptamer and a secondary aptamer of cTnI were used in a sandwich assay format to bind the cTnI on a test line of a paper fluidic platform. By measuring the SERRS signal from the test line, the concentration of cTnI was quantitatively determined.Results: The aptamer-based SERRS assay on a paper strip had a detection range of 0.016 to for cTnI, had good selectivity for cTnI compared to three other markers, had good stability over 10 days, and had good performance in the more complex serum sample matrix.Conclusions: The aptamer-based SERRS assay on a paper strip has the potential to provide a sensitive, selective, stable, repeatable, and cost-effective platform for the detection of cTnI toward eventual use in diagnosis of MI at the POC.

Adsorbing surface strongly influences the pseudoperoxidase and nitrite reductase activity of electrode-bound yeast cytochrome c. The effect of hydrophobic immobilization

The Met80Ala and Met80Ala/Tyr67Ala variants of S. cerevisiae iso-1 cytochrome c (ycc) and their adducts with cardiolipin immobilized onto a gold electrode coated with a hydrophobic self-assembled monolayer (SAM) of decane-1-thiol were studied through cyclic voltammetry and surface-enhanced resonance Raman spectroscopy (SERRS). The electroactive species - containing a six-coordinate His/His axially ligated heme and a five-coordinate His/- heme stable in the oxidized and reduced state, respectively - and the pseudoperoxidase activity match those found previously for the wt species and are only slightly affected by CL binding. Most importantly, the reduced His/- ligated form of these variants is able to catalytically reduce the nitrite ion, while electrode-immobilized wt ycc and other His/Met heme ligated variants under a variety of conditions are not. Besides the pseudoperoxidase and nitrite reductase functions, which are the most physiologically relevant abilities of these constructs, also axial heme ligation and the equilibria between conformers are strongly affected by the nature - hydrophobic vs. electrostatic - of the non-covalent interactions determining protein immobilization. Also affected are the catalytic activity changes induced by a given mutation as well as those due to partial unfolding due to CL binding. It follows that under the same solution conditions the structural and functional properties of immobilized ycc are surface-specific and therefore cannot be transferred from an immobilized system to another involving different interfacial protein-SAM interactions.

Characterisation of the Cyanate Inhibited State of Cytochrome c Oxidase

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 a3 to heme a.