Novel Approaches for SER Spectroscopic Analysis of Protein Cofactors

Biomimetic systems employed for biotechnological applications i.e. as biosensors or bio fuel cells, require initial formation of conducting support/protein complexes with controlled properties. The specific interaction of the protein with the support determines important qualities of the device such as electrical communication, long-term stability and catalytic efficiency. In this respect the system parameters have to be chosen in a way that high protein loading on the support is achieved while protein denaturation upon adsorption is prevented. The conditions on the surface have to be adjusted in such a way that the desired surface reaction of the protein i.e. electron transfer to either the electrode or a second redox partner, is still guaranteed. Hence the choice of support, its functionlisation as well as the right adjustment of solution parameters play a crucial role in the rational design of these support/protein constructs

Calculating average surface enhancement factors of randomly nanostructured electrodes by a combination of SERS and impedance spectroscopy

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.Polyhedron Ag nanostructures were created on top of a polished Au electrode via step-wise electrodeposition and tested as substrates for SERS spectroscopy. Average Raman enhancement factors were derived by combining SERS measurements with electrochemical impedance spectroscopy (EIS), which is able to determine the electroactive surface area of a randomly nanostructured surface. Depending on the deposition step an alternating increase and decrease of surface area was observed while the SERS intensity showed a clear maximum for the first deposition cycle. SEM pictures reveal the formation of Ag polyhedrons that are randomly dispersed on the Au surface. Furthermore the presence of a sub nanostructure on top of the polyhedron after the first deposition cycle is observed which becomes smoother after subsequent deposition cycles. Correlating the SEM pictures with SERS and EIS measurements it is concluded that the coral-like sub nanostructure is dominating the enhancement factor while the polyhedron structure itself only plays a minor role for electromagnetic field enhancement.DFG, EXC 314, Unifying Concepts in CatalysisDFG, GSC 1013, School of Analytical Sciences Adlershof (SALSA

Tailored silica coated Ag nanoparticles for non-invasive surface enhanced Raman spectroscopy of biomolecular targets

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.Silica coated Ag nanoparticles with defined surface plasmon resonances are used to selectively detect and analyze protein cofactors in solution and on interfaces via surface enhanced resonance Raman spectroscopy. The silica coating has a surprisingly small effect on optical amplification but minimizes unwanted interactions between the protein and the nanoparticle.DFG, EXC 314, Unifying Concepts in Catalysi

Insights from surface enhanced resonance Raman spectroscopy and QM/MM calculations

Understanding the coupling between heme reduction and proton translocation in cytochrome c oxidase (CcO) is still an open problem. The propionic acids of heme a3 have been proposed to act as a proton loading site (PLS) in the proton pumping pathway, yet this proposal could not be verified by experimental data so far. We have set up an experiment where the redox states of the two hemes in CcO can be controlled via external electrical potential. Surface enhanced resonance Raman (SERR) spectroscopy was applied to simultaneously monitor the redox state of the hemes and the protonation state of the heme propionates. Simulated spectra based on QM/MM calculations were used to assign the resonant enhanced CH2 twisting modes of the propionates to the protonation state of the individual heme a and heme a3 propionates respectively. The comparison between calculated and measured H2OD2O difference spectra allowed a sound band assignment. In the fully reduced enzyme at least three of the four heme propionates were found to be protonated whereas in the presence of a reduced heme a and an oxidized heme a3 only protonation of one heme a3 propionates was observed. Our data supports the postulated scenario where the heme a3 propionates are involved in the proton pathway

Functionalized Ag nanoparticles with tunable optical properties for selective protein analysis

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.We present a preparation procedure for small sized biocompatibly coated Ag nanoparticles with tunable surface plasmon resonances. The conditions were optimised with respect to the resonance Raman signal enhancement of heme proteins and to the preservation of the native protein structure.DFG, EXC 314, Unifying Concepts in Catalysi

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 638nm. 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 a(3) to heme a

Cobalt-based Co3Mo3N/Co4N/Co Metallic Heterostructure as a Highly Active Electrocatalyst for Alkaline Overall Water Splitting

Alkaline water electrolysis holds promise for large-scale hydrogen production, yet it encounters challenges like high voltage and limited stability at higher current densities, primarily due to inefficient electron transport kinetics. Herein, a novel cobalt-based metallic heterostructure (Co3Mo3N/Co4N/Co) is designed for excellent water electrolysis. In operando Raman experiments reveal that the formation of the Co3Mo3N/Co4N heterointerface boosts the free water adsorption and dissociation, increasing the available protons for subsequent hydrogen production. Furthermore, the altered electronic structure of the Co3Mo3N/Co4N heterointerface optimizes ΔGH of the nitrogen atoms at the interface. This synergistic effect between interfacial nitrogen atoms and metal phase cobalt creates highly efficient active sites for the hydrogen evolution reaction (HER), thereby enhancing the overall HER performance. Additionally, the heterostructure exhibits a rapid OH− adsorption rate, coupled with great adsorption strength, leading to improved oxygen evolution reaction (OER) performance. Crucially, the metallic heterojunction accelerates electron transport, expediting the afore-mentioned reaction steps and enhancing water splitting efficiency. The Co3Mo3N/Co4N/Co electrocatalyst in the water electrolyzer delivers excellent performance, with a low 1.58 V cell voltage at 10 mA cm−2, and maintains 100 % retention over 100 hours at 200 mA cm−2, surpassing the Pt/CRuO2 electrolyzer

Hydrogen evolution by cobalt hangman porphyrins under operating conditions studied by vibrational spectro-electrochemistry

Cobalt hangman complexes are promising catalysts for dihydrogen production, yet their electrocatalytic performance in aqueous environment is still a topic of dispute. Surface-enhanced resonance Raman (SERR) spectro-electrochemistry has a great potential to give insight into the reaction mechanism of such molecular catalysts attached to electrodes under turnover conditions. However, the intrinsic catalytic activity of plasmonic supports and photoinduced side-reactions make the in situ analysis of their structures very challenging. In this work, the structure of hangman complexes attached to electrodes via dip-coating was investigated during catalytic turnover by electrochemistry and SERR spectroscopy. In order to explore the relevance of the hanging group for proton supply, complexes bearing a carboxylic acid and an ester hanging group were compared. For the former, SERR spectra recorded under turnover conditions indicate the reductive formation of a Co^(III)–H species, followed by laser-induced translocation of a proton to the carboxylic hanging group and the associated formation of the Co^I state. Due to the lack of a proton accepting group, hangman complexes with an ester group could not be trapped in the Co^I intermediate state and as a consequence SERR spectra solely reflected the (photo-enriched) Co^(II) resting state under turnover conditions. These results represent the first Raman spectroscopic insights into intermediates of dihydrogen evolution catalysed by cobalt hangman complexes on electrodes and support the direct involvement of the hanging group as a proton shuttle

Hydrogen evolution by cobalt hangman porphyrins under operating conditions studied by vibrational spectro-electrochemistry

Cobalt hangman complexes are promising catalysts for dihydrogen production, yet their electrocatalytic performance in aqueous environment is still a topic of dispute. Surface-enhanced resonance Raman (SERR) spectro-electrochemistry has a great potential to give insight into the reaction mechanism of such molecular catalysts attached to electrodes under turnover conditions. However, the intrinsic catalytic activity of plasmonic supports and photoinduced side-reactions make the in situ analysis of their structures very challenging. In this work, the structure of hangman complexes attached to electrodes via dip-coating was investigated during catalytic turnover by electrochemistry and SERR spectroscopy. In order to explore the relevance of the hanging group for proton supply, complexes bearing a carboxylic acid and an ester hanging group were compared. For the former, SERR spectra recorded under turnover conditions indicate the reductive formation of a Co^(III)–H species, followed by laser-induced translocation of a proton to the carboxylic hanging group and the associated formation of the Co^I state. Due to the lack of a proton accepting group, hangman complexes with an ester group could not be trapped in the Co^I intermediate state and as a consequence SERR spectra solely reflected the (photo-enriched) Co^(II) resting state under turnover conditions. These results represent the first Raman spectroscopic insights into intermediates of dihydrogen evolution catalysed by cobalt hangman complexes on electrodes and support the direct involvement of the hanging group as a proton shuttle