Milan Fedurco

Redox reactions of heme-containing metalloproteins: dynamic effects of self-assembled monolayers on thermodynamics and kinetics of cytochrome c electron-transfer reactions

Abstract The ability of metalloproteins to recognize their biological redox partners, as well as kinetics of inter-protein electron-transfer reactions may be affected by chemical modification of amino acid residues located on the protein surface. Similarly, the rate of heterogeneous electron transfer can be tuned either via protein or electrode surface modification. Both of these methods allow one to modulate the extent of protein–protein or protein–electrode association, called also adsorptivity. On the other hand, the use of chemical coupling agents allows for a covalent attachment of metalloproteins to electrode surfaces and results in corresponding monolayers, or robust multilayers, with more or less preserved catalytic function of the redox protein. The latter approach is of prime importance in the construction of biosensors, immunosensors, optoelectronic devices, and other applications. The main goal of the present review is to summarize multidisciplinary efforts in bioelectrochemistry (during the last decade or so), attempting to comprehend kinetics of redox reactions associated with metalloproteins at the electrode/solution interface. Experimentally obtained kinetic data for the reduction of cytochrome c on chemically modified electrodes are correlated with its electronic structure, heme solvation and the protein contribution to the total reorganization energy. Two modes of charge transfer between the electrode and cytochrome c are discussed, namely, the heme edge and axial ligand-assisted electron tunneling. Rates of electrode reactions associated with corresponding redox process are shown to depend not only on the thickness of a self-assembled thiol monolayer, but also on its polarizability and ability to undergo ionization reactions. Acid–base equilibria existing on ω-mercaptoalkanoic acid-covered gold electrodes exposed to aqueous solutions of varying pH, seem to be directly responsible for strong electrostatic binding of this positively charged metalloprotein to carboxyl-terminated interface. In this respect, the latter type of self-assembled monolayers serves as very interesting model systems for protein binding to biological membranes. Furthermore, the possibility of variation in the reaction driving force at the electrode/long-chain thiol/solution interface offers one the possibility of measuring reorganization energy λ for a given metalloprotein. Both dynamics of cytochrome c rotational movements in self-assembled thiol monolayer, as well as the amount of electronic coupling between the heme and metal electrode, are shown to affect kinetics of non-adiabatic charge-transfer process. Dramatic differences in kinetics of cytochrome c reduction on gold electrodes modified by aliphatic and aromatic thiols are pointed out. This phenomenon is assigned as due to differing facility of establishment of hydrogen-bonding contacts between the protein and corresponding thiolate monolayer. On the other hand, a mixed hydrophilic thiol monolayer (i.e. containing OH/COOH, NH 3 + /COO − terminal groups), and the hydrophobic–hydrophilic thiol films (CH 3 /OH, CH 3 /COOH, CH 3 /4-mercaptopyridine), show either acceleration effects on the redox reactions of cytochrome c , or lead to their diminution, depending on the thiol mole fraction in the solution during the self-assembly process. Future directions in the area of metalloprotein electrochemistry are briefly outlined.

Effects of urea and acetic acid on the heme axial ligation structure of ferric myoglobin at very acidic pH.

The heme iron coordination of ferric myoglobin (Mb) in the presence of 9.0M urea and 8.0M acetic acid at acidic pH values has been probed by electronic absorption, magnetic circular dichroism and resonance Raman spectroscopic techniques. Unlike Mb at pH 2.0, where heme is not released from the protein despite the acid denaturation and the loss of the axial ligand, upon increasing the concentration of either urea or acetic acid, a spin state change is observed, and a novel, non-native six-coordinated high-spin species prevails, where heme is released from the protein.

Electrochemical Reduction of Cyanides at Metallic Cathodes: A Comparison with Biological HCN Reduction

The electrochemical reduction of cyanides has been studied at a number of cathodes both in near-neutral and in alkaline solutions. Nickel appears as the most effective cathode material, promoting cyanide reduction with current efficiencies close to 70%, even in moderately alkaline solutions. In all cases, the eletroreduction of cyanides leads to a mixture of 4e (methylamine) and 6e products (methane and ammonia). The use of Nafion films loaded with Ni microparticles enabled us to markedly increase the effective current densities for cyanide reduction. The electrochemical reduction of HCN/CN is shown to present interesting similarities and differences with the biological cyanide reduction process.

INTERACTION OF PYRIDINE WITH PHOTOELECTRONS EMITTED AT THE SILVER/AQUEOUS SOLUTION INTERFACE

Pyridine added to a solution containing scavengers of photoelectrons emitted from silver (N2O, CO2) causes a strong decrease in the measured photocurrent or even its complete suppression for concentrations of pyridine exceeding 0.1 M. Such behaviour, which remains in apparent contradiction to the effect of deposited pyridine on photoemission from silver in vacuum, is due to the fact that pyridine itself acts as an acceptor of the emitted photoelectrons. As the anion radical formed undergoes reoxidation (back electron transfer) at the silver surface, this process leads to a decrease in the net photocurrent which is dependent on the concentration ratio of the two competing acceptors (i.e. pyridine and N2O). The observation that pyridine (adsorbed on silver and in solution) sustains a reversible electron transfer, along with the previous observation that photoemission from a roughened Ag electrode into an aqueous solution extends to at least 650 nm, provides a possible interpretation of the surface-enhanced Raman spectroscopy effect for pyridine, which involves both electromagnetic and charge-transfer mechanisms.