Surface-Enhanced Resonance Raman Spectroscopic and Electrochemical Study of Cytochrome c Bound on Electrodes through Coordination with Pyridinyl-Terminated Self-Assembled Monolayers

Cytochrome c (Cyt-c) is immobilized on Ag and Au electrodes coated with self-assembled monolayers (SAM), comprised of pyridine-terminated alkanethiols and a shorter chain diluent thiol. Surface-enhanced resonance Raman (SERR) spectroscopy of coated Ag electrodes reveals that the adsorbed Cyt-c forms a potential-dependent coordination equilibrium with a predominant five-coordinated high-spin (5cHS) state in the reduced form and six-coordinated low-spin (6cLS) state prevailing in the oxidized form. In the oxidized species, the native Met-80 ligand of the heme is replaced by a pyridinyl residue of the bifunctional thiols that according to earlier scanning tunneling microscopy form islands in the hydrophobic monolayer. The redox potentials derived from the SERR band intensities are estimated to be -0.24 and -0.18 V (vs AgCl) for the 6cLS and 5cHS states, respectively, and lie in the range of the midpoint potential determined for Cyt-c on coated Au electrodes by cyclic voltammetry (CV). Whereas in the latter case, a nearly ideal Nernstian behavior for a one-electron couple was observed, the SERR spectroscopic analysis yields about 0.4 for the number of transferred electrons for each spin state. This discrepancy is mainly attributed to a distribution of substates of the immobilized protein in both the 6cLS and 5cHS forms, as indicated by substantial band broadening in the SERR spectra. These substates may arise from different orientations and heme pocket structures and exhibit different redox properties. Whereas SERR spectroscopy probes all adsorbed Cyt-c species including those that are largely redox inactive, CV measurements reflect only the substates that are electrochemically active

Surface enhanced vibrational spectroscopic evidence for an alternative DNA-independent redox activation of endonuclease III

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.Surface enhanced vibrational spectro-electrochemistry of endonuclease III provides direct evidence that the [4Fe-4S] cluster is responsible for the enzyme redox activity, and that this process is not exclusively DNA-mediated, as currently proposed. We report the first surface enhanced resonance Raman spectrum of a [4Fe-4S](2+) cluster containing enzyme.DFG, EXC 314, Unifying Concepts in Catalysi

Surface enhanced resonance Raman detection of a catalytic intermediate of DyP-type peroxidase

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 report herein the vibrational spectroscopic characterisation of a catalytic intermediate formed by the reaction of H2O2 with DyP-type peroxidase immobilised on a biocompatible coated metal support. The SERR spectroscopic approach is of general applicability to other peroxidases which form relatively stable catalytic intermediates.DFG, EXC 314, Unifying Concepts in Catalysi

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

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

The impact of urea-induced unfolding on the redox process of immobilised cytochrome c

We have studied the effect of urea-induced unfolding on the electron transfer process of yeast iso-1-cytochrome c and its mutant K72AK73AK79A adsorbed on electrodes coated by mixed 11-mercapto-1-undecanoic acid/11-mercapto-1-undecanol self-assembled monolayers. Electrochemical measurements, complemented by surface enhanced resonance Raman studies, indicate two distinct states of the adsorbed proteins that mainly differ with respect to the ligation pattern of the haem. The native state, in which the haem is axially coordinated by Met80 and His18, displays a reduction potential that slightly shifts to negative values with increasing urea concentration. At urea concentrations higher than 6 M, a second state prevails in which the Met80 ligand is replaced by an additional histidine residue. This structural change in the haem pocket is associated with an approximately 0.4 V shift of the reduction potential to negative values. These two states were found for both the wild-type protein and the mutant in which lysine residues 72, 73 and 79 had been substituted by alanines. The analysis of the reduction potentials, the reaction enthalpies and entropies as well as the rate constants indicates that these three lysine residues have an important effect on stabilising the protein structure in the adsorbed state and facilitating the electron transfer dynamics