Fred M. Hawkridge

The Direct Electron Transfer Reactions of Cytochrome c at Electrode Surfaces

Abstract Examples of facile electron transfer between electrode surfaces and electron transfer proteins were first reported nearly twenty years ago. Substantial progress has been achieved in understanding the fundamental requirements that must be met in order to observe such reactions. Despite the large body of work that has now been published on a host of examples of such reactions, there continues to be a lack of understanding over just what conditions must be met in order to use direct electrochemical methods to characterize the electron transfer thermodynamics and kinetics of electron transfer proteins. Part of the problem lies in the inherent difficulties associated with using solid electrodes. Equally as important is the wide range of attention that has been paid to the purity of the electron transfer protein solutions being studied. The goal here is to bring together those aspects of this problem that enjoy some degree of agreement among scientists in this field and to reflect upon those issues tha...

Immobilization of cytochrome c oxidase into electrode-supported lipid bilayer membranes for in vitro cytochrome c sensing

Blood and tissue biochemical oxidation-reduction (redox) reactions are ubiquitous and are reflective of many important biological processes in the body, ranging from the state of cellular oxygenation to the overall antioxidant status. It is likely that, similar to acid-base balance, the body optimally operates within a narrow redox potential range made possible by various homeostatic mechanisms, and that deviation from this range will occur in tissue damage. A means to monitor the redox potential of blood or tissue would be valuable in both the diagnosis and treatment of diseases or conditions that adversely affect the bodys redox potential. The biosensor described herein involves the immobilization of bovine cytochrome c oxidase (CCO) into electrode-supported lipid bilayer membranes. As a first proof of concept, the biosensor was used to potentiometrically monitor the concentration ratio of a redox pair (oxidized and reduced cytochrome c) in an in vitro system. The response of this modified electrode is reproducible and exhibits Nernstian behavior consistent with the four-electron reduction of the CCO. The oxidase-modified electrode can also operate as an amperometric biosensor for the detection of solution-resident ferrocytochrome c at concentrations as low as 0.1 /spl mu/M. Because this biosensor uses an electron sensor native to the body, it may be of future value to explore the biosensor as a point of care test to measure blood redox potential or perhaps as an implantable sensor to measure tissue redox potential in many settings.

The effects of temperature and electrolyte at acidic and alkaline pH on the electron transfer reactions of cytochrome c at In2O3 electrodes

Abstract Spectroelectrochemical and electrochemical methods were used to investigate the characteristics of heterogeneous electron transfer between cytochrome c and indium oxide electrodes. A linear temperature dependence of the formal potential of cytochrome c was observed from 5 to 75 °C in acidic media. This behavior is attributed to a linear variation in the conformation of ferricytochrome c that results in an increase in solvent exposure of the solvent-exposed heme edge. A break in the linear temperature dependence of the formal potential occurred at 40 °C in alkaline media. This reflects a distinct conformational change that accompanies the onset of thermal denaturation of ferricytochrome c. The small change in reaction center entropy, ΔS°rc, of ca. −54 J K−1 mol−1 in neutral and acidic media (5 to ⩾ 55 °C) and in alkaline media (below 40 °C) is consistent with a small shift to a more stable conformation of cytochrome c that occurs upon reduction. Adsorption of reactant and product was detected. The strength and type of adsorption were found to be temperature- and pH-dependent. The characteristics of electron transfer between cytochrome c and an electrode depend on bulk solvent properties and electrode surface characteristics.

Computer decomposition of the ultraviolet-visible absorption spectrum of the methyl viologen cation radical and its dimer in solution

Abstract The first reduction of 1,1′-dimethyl-r,r′-bipyridinium dichloride (methyl viologen, MV +- results in the formation of both a monomeric, MV ± , and a dimeric (MV +- 2 product. The molar absorptivities and the wavelengths of maximum absorption for both reduction products can be determined by computer decomposition of the u.v.-visible absorption spectra resulting from the chemical and in situ electrochemical reduction of MV 2+ . The FORTRAN computer program, SPECSOLV, is used to establish the spectral parameters of MV± and (MV±) 2 .

An electrochemical thin-layer cell for spectroscopic studies of photosynthetic electron-transport components

Abstract An electrochemical thin-layer cell, 0.5 ml in volume and 0.25 mm in pathlength, employing an optically transparent gold minigrid electrode has been constructed and used in redox studies of photosynthetic electron-transport components. Light-induced absorption changes accompanying P700 photo-oxidation in photosystem-I subchloroplasts were measured at a series of electrochemically poised potentials at 88°K. The fluorescence-yield changes in photosystem-II subchloroplasts were measured as the poised potential was varied electrochemically. The utility of the cell was further demonstrated by a redox titration of soluble (spinach) ferredoxin using circular dichroism to monitor changes in the redox state. One important advantage of electrochemical vs chemical titration of photosynthetic electron-transport components is that it is possible to attain much more negative potentials than is thermodynamically possible in chemical titrations. This permits the exhaustive titration of certain components at physiologically reasonable pH values not possible chemically.

Heterogeneous electron-transfer kinetic parameters of metalloproteins as studied by channel-flow hydrodynamic voltammetry

Abstract Channel-flow hydrodynamic voltammetry was used to study the direct electron-transfer reactions of two metalloproteins, myoglobin and cytochrome c, under steady-state conditions at methyl viologen modified (MVM) gold foil electrodes. Utilization of a dual working electrode cell with this technique permitted determination of the heterogeneous electron-transfer kinetics for both the reduction and oxidation of myoglobin. The formal heterogeneous electron-transfer rate constants in pH 7.00 phosphate buffered solutions were found to be 8.9 (±1.5)×10−5 cm s−1 for the reduction, and 7.7 (±1.2)×10−5 cm s−1 for the reoxidation of myoglobin. The transfer coefficient values obtained were 0.21 (±0.01) for the reductive (α) and 0.82 (±0.01) for the oxidative (1−α) electrode reactions. Ionic strength and pH dependences were observed in these direct electron-transfer reactions. Collective current efficiency measurements in the myoglobin experiments indicated that an overall simple charge-transfer process occurred at the respective electrode interfaces. A formal rate constant of 3.4 (±0.2)×10−5 cm s−1 with a transfer coefficient of 0.25 (±0.01) for the reduction (α) of cytochrome c was obtained by this hydrodynamic technique. The use of channel-flow hydrodynamic voltammetry in characterizing an electrode reaction as well as an interpretation of the data presented are discussed.

The electrochemical properties of three dipyridinium salts as mediators

Abstract The electrochemical properties of three dipyridinium salts were studied in order to assess their behavior for use as mediators in titrations of biological molecules. The compounds studied were 1,1′-trimethylene-2,2′-dipyridinium dibromide, 5,5′-dimethyl-1,1′-trimethylene-2,2′-dipyridinium dibromide and 4,4′-dimethyl-1,1′-trimethylene-2,2′-dipyridinium mide. Differential pulse polarographic analysis showed that each of these compounds undergoes two successive one-electron reductions, and that the formal potentials of these reduction steps are independent of pH. Cyclic voltammetry at the hanging mercury drop electrode (HMDE) indicated that in all cases the first reduction step is reversible. However, the sample potentials measured during controlled potential electrolysis of these compounds were not as negative as theoretically expected, indicating decomposition of the electrolysis products. This study concludes, however, that these mediators can be used in the indirect coulometric titration of large biological redox molecules if there is sufficient separation between the formal potential, E 1 0′ , of the mediator and the E 0′ of the biological molecules. This condition prevents the accumulation of the product of the electrode reaction, the cation radical and therefore the decomposition of the mediator.

Spectroelectrochemical and electrochemical determination of ligand binding and electron transfer properties of myoglobin, cyanomyoglobin, and imidazolemyoglobin

Abstract Electrochemistry and spectroelectrochemistry were used to study solutions of horse myoglobin, both by itself and complexed with cyanide or imidazole, at indium-tin oxide electrodes. In the absence of ligands other than water, myoglobin exhibited slow quasi-reversible heterogeneous electron transfer kinetics, with a formal heterogeneous electron transfer rate constant ( k °′) value of 1.6(±0.4)×10 −5 cm s −1 . Differences from simple simulations in the reverse sweep (anodic) cyclic voltammograms are proposed to be due to residual amounts of dioxygen in solution and/or in the protein molecule. Those differences were not seen in the anodic waveforms of simultaneously acquired derivative cyclic voltabsorptometry (DCVA) experiments. Cyanomyoglobin exhibited faster heterogeneous electron transfer kinetics, with a k °′ value of 6.0(±1.0)×10 −4 cm s −1 . Values for the homogeneous rate constants for dissociation ( k f ) and association ( k b ) of cyanide from and to the electrochemically reduced protein were determined to be 0.08(±0.03) s −1 and 0.06(±0.03) M −1 s −1 , respectively. Imidazolemyoglobin was found to transfer electrons faster than cyanomyoglobin, with a k °′ value of 2.0(±0.5)×10 −3 cm s −1 . The values for the homogeneous rate constants k f and k b were 0.40(±0.1 5) s −1 and 1.1(±0.1) M −1 s −1 , respectively.

Thermodynamic and kinetic studies of cytochrome c from different species

Abstract The direct heterogeneous electron transfer reactions of cytochrome c from different vertebrate species are compared in this report. A kinetic study revealed a biphasic temperature dependence at neutral pH for the formal heterogeneous electron transfer rate constants of these cytochromes. The properties of the electrode-solution interface are believed to cause this biphasic kinetic effect. This effect does not correlate with the conformational transitions evident in both formal potential and circular dichroism (CD) measurements as a function of temperature. The conformational transitions seen in the formal potential and CD measurements arise from the alkaline isomerization and heme crevice changes. These are subtle effects compared with the structural changes associated with unfolding of the protein that are evident in differential scanning calorimetric experiments. The conformational stabilities of cytochrome c between species relate to the differences in their amino acid sequences. The amino acid interactions which stabilize the structure of these different cytochrome c molecules have been evaluated and will be discussed.

Electron transfer reactions of cytochrome c at metal electrodes

The heterogeneous electron transfer reactions of cytochromec occurring at platinum, gold and mercury electrodes are shown to be quasi-reversible. In each case the electrodes have not been modified and the cytochromec samples are native. This work extends previous work and demonstrates that biological molecule electron transfer reactions can be studied at clean metal surfaces to gain fundamental knowledge of the mechanisms of these reactions.