Redox and Conformational Equilibria and Dynamics of Cytochrome c at High Electric Fields

Abstract

Cytochrome c (Cyt-c) adsorbed in the electrical double layer of the Ag electrode/electrolyte interface has been studied by stationary and time-resolved surface-enhanced resonance Raman spectroscopy to analyse the effect of strong electric fields on structure and reaction equilibria and dynamics of the protein. In the potential range between +0.1 and -0.55 V (versus saturated calomel electrode), the adsorbed Cyt-c forms a potential-dependent reversible equilibrium between the native state B1 and a conformational state B2. The redox potentials of the bis-histidine-coordinated six-coordinated low-spin and five-coordinated high-spin substates of B2 were determined to be -0.425 and -0.385 V, respectively, whereas the additional six-coordinated aquo-histidine-coordinated high-spin substate was found to be redox-inactive. The redox potential for the conformational state B1 was found to be the same as in solution in agreement with the structural identity of the adsorbed B1 and the native Cyt-c. For all three redox-active species, the formal heterogeneous electron transfer rate constants are small and of the same order of magnitude (3-13 s-1), which implies that the rate-limiting step is largely independent of the redox-site structure. These findings, as well as the slow and potential-dependent transitions between the various conformational (sub-)states, can be rationalized in terms of an electric field-induced increase of the activation energy for proton-transfer steps linked to protein structural reorganisation. Further increasing the electric field strength by shifting the electrode potential above +0.1 V leads to irreversible structural changes that are attributed to an unfolding of the polypeptide chain.

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.…”

“…The solution contained 5 mM sodium perchlorate and 5 mM phosphate buffer (pH 7, T = 278 K). The spectra were obtained with 413-nm excitation which exhibits the same haem pocket structure as B1 [38], and for cytochrome c in urea-containing solution [36,41,43,44] and is consistent with a change of the ligand from S-Met to N-His [3,35,36,47]. The difference in the reduction potential, DE°0, between these two states is 0.031 V more positive for ycc than for K72AK73AK79A ( Table 2).…”

“…The view that the local structural change in the haem pocket including the ligand exchange is the main determinant for the large negative shift of the reduction potential is further supported by the quite similar reduction potentials of the state B2, in which the same haem pocket structural change is induced by electrostatic interactions rather than denaturants [38].…”

“…For the sake of simplicity, we adopt this nomenclature and assign wave II to the U[6cLS] state. The appearance of the anodic counterpart of wave II only for high scan rates is probably related to the instability of the reduced bishistidine form which transforms rapidly into the corresponding His-Met form, denoted as the B1 state [38]. This is not surprising since the reduced form of cytochrome c in solution retains its His-Met ligation up to 9 M urea [41,42].…”

Evidence for heme release in layer-by-layer assemblies of myoglobin and polystyrenesulfonate on pyrolitic graphite

“…On gold, silver and indium oxide electrodes spectroscopic techniques such as surface-enhanced resonance Raman spectroscopy (SERRS) [5] and surface-enhanced infrared difference adsorption spectroscopy (SEIDAS) can be employed [6]. With these techniques it has, e.g., been shown that cytochrome c retains a near-native conformation on carboxylic acid terminated self-assembled monolayers [7], whereas it undergoes significant conformational changes on bare metal electrodes [5,8,9]. On ''dark'' materials such as graphite spectroscopic measurements are difficult and therefore one has to rely largely on indirect electrochemical evidence.…”