Heterogeneous Electron Transfer of Cytochrome c on Coated Silver Electrodes. Electric Field Effects on Structure and Redox Potential

Abstract

Cytochrome c (Cyt-c) was electrostatically bound to self-assembled monolayers (SAM) of ω-carboxylalkanethiols that were covalently attached to Ag electrodes. Employing surface-enhanced resonance Raman (SERR) spectroscopy, the redox equilibria and the structural changes of the adsorbed Cyt-c were analyzed quantitatively for SAMs of different chain lengths ranging from 2-mercaptoacetic acid (C 2 -SAM) to 16mercaptohexadecanoic acid (C 16 -SAM). In the presence of Cyt-c in the bulk solution, the SERR spectra of the adsorbed Cyt-c display the characteristic vibrational band pattern of the native protein conformation denoted as state B1. The enhancement of the SERR signals decreases with increasing chain length, but even at distances as large as 24 Å (C 16 -SAM), SERR spectra of high quality could be obtained. Conversely, no SERR signals could be detected for SAMs including hydroxyl instead of carboxylate headgroups, implying that Cyt-c is adsorbed via electrostatic interactions. On the basis of potential-dependent SERR experiments, the redox equilibria of the adsorbed Cyt-c (B1) were analyzed, revealing ideal Nernstian behavior (n = 1). However, the redox potentials exhibit negative shifts compared to that of Cyt-c in solution, which increase with the chain length of the SAMs. In the absence of excess Cyt-c in solution (i.e., 0.2 µM), a new conformational state B2 of the adsorbed Cyt-c is observed. This state B2, which differs from the native state B1 by the heme pocket structure, includes three substates of different spin and coordination configurations. The distribution among these substates as well as the total contribution of state B2 varies with the chain length of the SAM such that the latter decreases from 73% at C 2 -SAM to 0% at C 11 -and C 16 -SAMs. These results imply that the formation of B2 is induced by the electric field at the binding site, generated by the potential drop across the electrode/SAM interface. When an electrostatic model for the interfacial potential distribution for the electrode/ SAM/protein device is employed, both the redox potential shifts and the electric-field-induced structural changes can be consistently explained. The impact of these findings for the processes of Cyt-c at biological interfaces is discussed.

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

“…3, spectrum A). Essentially the same upshifts of the marker bands are observed for ferric cytochrome c in solution containing 6 M guanidinium hydrochloride [17,37] or after binding to negatively charged surfaces at high electric fields (i.e., state B2) [32,[37][38][39]. Under these conditions, the Met80 ligand is replaced by His33 (or His26) [17,37], and this replacement evidently also takes place for ycc immobilised on the SAM-coated electrode in the presence of 8 M urea.…”

Surface-Enhanced Raman Scattering (SERS) and Surface-Enhanced Resonance Raman Scattering (SERRS): A Review of Applications

“…A good example of this is the spectrum obtained from the enzyme cytochrome C. Using excitation that coincides with the electronic transitions of the heme group in the protein, only vibrations attributable to the heme are observed despite the fact that the heme is not directly attached to the surface and the number of non-heme bonds is far greater than the number of heme bonds. 16 Another example demonstrating the advantages of this selectivity is in the study of the interaction of NO gas (muscle relaxant factor in biology) with the liver heme enzyme P450 in the presence of oxygen 17 ( Fig. 2).…”

“…57 SERRS Applications. Most effective SERRS applications either use naturally occurring chromophores to investigate in situ changes to proteins and enzymes 16,17 or use specific labeling chemistry to tackle high-value problems such as detection of DNA fragments 38 or antibodies. 58 In forensics, the technique can be effectively applied to determine the dyes in inks on paper 59 and in fibers.…”

Novel Approaches for SER Spectroscopic Analysis of Protein Cofactors

“…For Ag-S-Au systems this leads to a value of log(REF) = 0.11 nm -1 (Figure 7 A) which fits nicely to the theory. For SAM coated Ag electrodes a much stronger dependence on SAM thickness (log(EF) = 0.53 nm -1 ) and a less linear characteristic was found (Murgida & Hildebrandt, 2001). Variation of the thickness of the inner dielectric layer in Ag-S-Au systems reveals a different behaviour: Here a very weak distance dependence of log(EF) = 0.02 nm -1 is determined from measurements with SiO 2 coatings of different thickness (Figure 6 B).…”

“…Further increase of the ionic strength leads to an increase in redox rate but does not alter the midpoint potential. Shifts in midpoint potential in respect to its value in solution can be associated with the formation of a strongly bound SAM/protein complex (Murgida & Hildebrandt, 2001). Hence we can say that for ionic strength > 150 mM direct electrostatic attraction between the Cyt b5 domain and the SAM surface has been mostly eliminated.…”