Electrochemical Comparison of Heme Proteins by Insulated Electrode Voltammetry

Abstract

Abstract. Relatively simple electrochemical probes of redox active proteins can yield a wealth of diverse information concerning their kinetic and thermodynamic reactivity, charge state, and transport rates. In this report, the electrochemical properties of three heme-containing proteins are probed using Au electrodes derivatized with self-assembled monolayers of co-hydroxyalkanethiols. Cytochrome c and b s (wild type from horse and rat, respectively) display low reorganization energies (0.58 eV and 0.44 eV, respectively) and electronic coupling terms which are quite comparable to small redox molecule models. In contrast, metmyoglobin reduction is characterized by a somewhat higher reorganization energy (0.76 eV) and an order of magnitude reduction in its electronic coupling to the electrode. Upon binding oxygen, the reduced oxymyoglobin becomes electroinactive. This radical decrease in the oxymyoglobin redox reactivity is associated with structural and electronic differences caused by the spin-state change induced by oxygen binding. The reactivity differences between these heme-containing proteins are discussed in light of their biochemical function, evolutionary pressures, and structural differences.

Impact of self‐assembly composition on the alternate interfacial electron transfer for electrostatically immobilized cytochrome c

“…3 On the contrary, immobilization of CytC at unicomponent carboxyl terminated SAMs under conventional electrochemical conditions (up to 60 mM electrolyte) normally is strong and irreversible. 9,10,12,13 Previous rather scarce studies indicate that, at least for relatively thick SAMs of mixed assemblies, standard rate constants are notably higher than unicomponent SAMs with ''active'' carboxyl groups. 32,33 A possible explanation is the closer approach of the reactant centers of CytC to the electrode in the case of mixed SAMs, possibly as a consequence of the partial immersion into the interfacial region, due to the more flexible nature of solvent-exposed fragments of SAM ''active'' chains.…”

Electrochemical and Spectroscopic Investigations of Immobilized De Novo Designed Heme Proteins on Metal Electrodes

“…This finding is consistent with the fact that the electron transfer of 5cHS hemes is much slower than that of 6cLS hemes, which is mainly due to the substantially higher reorganization energies. [29] On the basis of this complex coupling of redox and conformational transitions and the quite different timescales of the individual reaction steps, the kinetic and thermodynamic data provided by CV and SERRS can be rationalized. The voltammograms reveal a nonideal behavior, which indicates a complex redox mechanism.…”

High‐Pressure Probing of a Changeover in the Charge‐Transfer Mechanism for Intact Cytochrome c at Gold/Self‐Assembled Monolayer Junctions

“…[7,8] Previous work that exploited SAM-implicated bioelectrochemical systems revealed kinetic behavior and a distinct change in mechanism with the CT distance for cases with CytC irreversibly attached (probably heavily altered in the native structure) to SAM terminal groups. [15][16][17][18] Only fragmentary work has been performed for totally intact CytC freely diffusing to electrode/SAM junctions, [8,19,20] which includes our pioneering HP kinetic study of CytC at "primitively" modified Au electrodes. [6] In the latter work, however, it was demonstrated for the first time that the application of hydrostatic pressure allows a variation of the internal viscosity (conformational mobility) of the protein without significant variation of the properties of the aqueous medium.…”

“…This method has been proven to be fully adequate for both the determination of the physical condition (structural accomplishment) of composite systems and the accurate determi-nation of dynamic characteristics, such as heterogeneous kinetic constants and diffusion coefficients, under variable experimental conditions. [6,8,30,31] The methods for preparation of the SAM-modified electrodes, [19,20] the instrumentation, [6] and the HP setup [2,6] were similar to those reported elsewhere, and are also briefly described in the Supporting Information.…”

“…[54] To this extent, within the framework of the generalized theoretical model adopted here, the HP kinetic results fully conform with two types of intrinsic mechanisms of CT, namely, nonadiabatic (long-range) and adiabatic (shortrange) processes, in good accordance with two other experimental manifestations fully exhibited by our experiments. The individual components that contribute to the experimental volumes of activation (see Supporting Information) can be estimated on the basis of Equations (6) and (7) by assuming that the overall value of l = 0.6-0.8 eV [8,[17][18][19][20] (representing the Franck-Condon term) is roughly composed of about 50 % "outer-sphere" (solution) component, which can contribute about half of the activation volume, and about 50 % "innersphere" protein reorganization component that is negligibly altered by pressure (resulting in a zero activation volume contribution). [42] Moreover, the reorganizational component due to the SAM interior seems to be negligible (l SAM % 0), [47] which also yields the zero activation volume increment.…”