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.

Systematic cyclic voltammetry (CV) studies of glucose oxidase (GOx) and its cofactor, flavine adenine dinucleotide (FAD), almost similarly captured by the matrix of single-walled carbon nanotube and polymer complex, in turn, deposited on GC electrodes have been performed. The comparative analysis of kinetic data obtained for the FAD and GOx specimens treated through the same Marcus theory-based algorithmic procedure strongly suggests that the GOx species, notwithstanding the deeply buried position of FAD, mechanistically behave virtually in the same manner as isolated FADs (the operationally capable, nearly intact structure of GOx was confirmed by the catalytic activity vs glucose), strongly suggesting that FADs inside GOx are directly wired to the GC electrode, presumably, via almost direct contact of nanotubes with both FADs residing inside each GOx biomolecule (as basically suggested by Guiseppi-Elie, A.; et al. Nanotechnology 2002, 13, 559−564, and shortly supported by a number of valued researchers). Furthermore, the nonadiabatic, quasi-simultaneous two-proton-coupled two-electron transfer/exchange mechanism was concluded from further cross-analysis based on a generalized Marcus theory for the proton-coupled electron transfer (PCET), extra furnished by the first-time temperature-dependent CV studies and a subsequent Arrhenius treatment.

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