Long-Range Electron Transfer with Myoglobin Immobilized at Au/Mixed-SAM Junctions: Mechanistic Impact of the Strong Protein Confinement

Abstract

Horse muscle myoglobin (Mb) was tightly immobilized at Au-deposited ~15-Å-thick mixed-type (1:1) alkanethiol SAMs, HS-(CH₂)₁₁-COOH/HS-(CH₂)₁₁-OH, and placed in contact with buffered H₂O or D₂O solutions. Fast-scan cyclic voltammetry (CV) and a Marcus-equation-based analysis were applied to determine unimolecular standard rate constants and reorganization free energies for electron transfer (ET), under variable-temperature (15-55 °C) and -pressure (0.01-150 MPa) conditions. The CV signal was surprisingly stable and reproducible even after multiple temperature and pressure cycles. The data analysis revealed the following values: standard rate constant, 33 s⁻¹ (25 °C, 0.01 MPa, H₂O); reorganization free energy, 0.5 ± 0.1 eV (throughout); activation enthalpy, 12 ± 3 kJ mol⁻¹; activation volume, -3.1 ± 0.2 cm³ mol⁻¹; and pH-dependent solvent kinetic isotope effect (k(H)⁰/k(D)⁰), 0.7-1.4. Furthermore, the values for the rate constant and reorganization free energy are very similar to those previously found for cytochrome c electrostatically immobilized at the monocomponent Au/HS-(CH₂)₁₁-COOH junction. In vivo, Mb apparently forms a natural electrostatic complex with cytochrome b₅ (cyt-b₅) through the "dynamic" (loose) docking pattern, allowing for a slow ET that is intrinsically coupled to the water's removal from the "defective" heme iron (altogether shaping the biological repair mechanism for Mb's "met" form). In contrary, our experiments rather mimic the case of a "simple" (tight) docking of the redesigned (mutant) Mb with cyt-b₅ (Nocek et al. J. Am. Chem. Soc. 2010, 132, 6165-6175). According to our analysis, in this configuration, Mb's distal pocket (linked to the "ligand channel") seems to be arrested within the restricted configuration, allowing the rate-determining reversible ET process to be coupled only to the inner-sphere reorganization (minimal elongation/shortening of an Fe-OH₂ bond) rather than the pronounced detachment (rebinding) of water and, hence, to be much faster.

Electron transfer with myoglobin in free and strongly confined regimes: disclosing diverse mechanistic role of the Fe-coordinated water by temperature- and pressure-assisted voltammetric studies

“…The measurement and data processing procedures applied in this work have been described in previous work [17][18][19][20][21][22]. Figure 1, panels (a) and (b), depicts representative cyclic voltammograms recorded for Mb involved in the electron exchange with electrode, either in a freely diffusing (at Au-deposited HS-(CH 2 ) 3 -OH SAM films) or immobilized (at Au-deposited mixed-type (1 : 1) HS-(CH 2 ) 11 -COOH and HS-(CH 2 ) 11 -OH SAM films) regimes, respectively.…”

“…Furthermore, it has been well established that Mb, in general, is a rather flexible protein that is required for the realization of its function − ligand binding/release through the "gating" motion of the protein matrix throughout the region of the "ligand channel" [32][33][34][44][45][46][47][48]. However, strong confinement of proteins due to the solution glass-forming [35][36][37][38] or tight electrostatic immobilization involving terminal groups -OH and -COOH of 1 : 1 mixed SAM [22,83], seemingly, restricts the opening motions of the ligand channel and locks the Fe-coordinated ligand (water, in this particular case) in the bound condition. In general, it becomes obvious that there is some correlation between the stabilizing impact of Mb's environment and the ET efficiency.…”

“…It was well documented that high-pressure kinetic studies of electron exchange at bare and SAM-modified electrodes involving model metallocomplexes (dissolved in non-aqueous solutions) and redox-active proteins (dissolved in aqueous solutions) provide the most reliable information regarding the intrinsic physical mechanism (non-adiabatic versus frictional) of ET. Specifically, the long-range ET processes associated with non-adiabatic mechanistic regime, equation (12), exhibited negative volume of activation, whereas those occurring in the frictional (dynamically controlled) regime, equation (13), displayed positive values [17][18][19][20][21][22]70] (for similar manifestations in nonbiological electrode processes, see [5,53,54,71,77]). In general, this interplay rigorously correlates with the biphasic character for the logarithmic dependence of the standard rate constant of electron exchange on the number of methylene units, n, within the alkanethiol chain, HS-(CH 2 ) n -ω, constituting SAM, where ω is the SAM's terminal group (here: -OH and/or -COOH), see figure 4.…”

“…The terms were aligned to possess no energy gaps, according to the experimental condition for the zero overvoltage (see also Ref. [22]). …”

“…In particular, among the first high-pressure biomolecular kinetic studies, those encompassing electron-transfer (ET) processes involving small (model) proteins with heme groups, containing iron ions as redox-active cores, such as cytochromes [10][11][12][13] and myoglobin (Mb) [14][15][16], were explored. Later, the high-pressure biomolecular ET studies were extended to nanoscale interfacial systems/devices including heme and non-heme metalloproteins functionalized at SAM-coated Au electrodes (SAM = self-assembled monolayer), acting either in the freely diffusing [17][18][19] or diversely immobilized regimes [20][21][22]. These studies allowed for direct verification of the latest theoretical mechanistic conjectures [23][24][25][26][27][28][29].…”