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The Met80Ala variant of yeast iso-1-cytochrome c, immobilized on a gold electrode, is found to exchange electrons efficiently with it in nondenaturing conditions and to provide robust and persistent catalytic currents for O[sub2] and nitrite ion reduction from pH 3 to 11. Direct covalent protein linkage to gold yields the best electrochemical and electrocatalytic performances without drastically affecting the structural properties of the bound protein compared to the freely diffusing species. Therefore, this biocatalytic interface can be of use for the amperometric detection of the above species, which are of great environmental, industrial, and clinical interest, with particular reference to the exploitation in nanostructured biosensing devices. This work shows that the use of a small engineered electron transfer (ET) protein, featuring an axial heme iron coordination position available for the binding of exogenous ligands, in place of a large heme enzyme is a viable strategy for the improvement of the heterogeneous ET rate and the stability and efficiency of sensing gold-protein interfaces over a wide range of T and pH. [ABSTRACT FROM AUTHOR]

Cytochrome c functions as an electron carrier in the mitochondrial electron-transport chain using the Fe(II)-Fe(III) redox couple of a covalently attached heme prosthetic group, and it has served as a paradigm for both biological redox activity and protein folding. On the basis of a wide variety of biophysical techniques, it has been suggested that the protein is more flexible in the oxidized state than in the reduced state, which has led to speculation that it is the dynamics of the protein that has been evolved to control the cofactor's redox properties. To test this hypothesis, we incorporated carbon-deuterium bonds throughout cytochrome c and characterized their absorption frequencies and line widths using IR spectroscopy. The absorption frequencies of several residues on the proximal side of the heme show redox-dependent changes, but none show changes in line width, implying that the flexibility of the oxidized and reduced proteins is not different. However, the spectra demonstrate that folded protein is in equilibrium with a surprisingly large amount of locally unfolded protein, which increases with oxidation for residues localized to the proximal side of the heme. The data suggest that while the oxidized protein is not more flexible than the reduced protein, it is more locally unfolded. Local unfolding of cytochrome c might be one mechanism whereby the protein evolved to control electron transfer. [ABSTRACT FROM AUTHOR]

Cyclic voltammetry experiments were carried out on native Saccharomyces cerevisiae iso-1-cytochrome c and its C102T/N62C variant immobilized on bare polycrystalline gold electrode through the S-Au bond formed by a surface cysteine. Experiments were carried out at different temperatures (5-65 °C) and pH values (1.5-7). The E°' value at pH 7 (+370 mV vs SHE) is approximately 100 mV higher than that for the protein in solution. This difference is enthalpic in origin and is proposed to be the result of the electrostatic repulsion among the densely packed molecules onto the electrode surface. Two additional electrochemical waves are observed upon lowering the pH below 5 (E°' = +182 my) and 3 (E°' = +71 mV), which are attributed to two conformers (referred to as "intermediate" and "acidic", respectively) featuring an altered heme axial ligation. This is the first determination of the reduction potential for low-pH conformers of cytochrome c in the absence of denaturants. Since the native form of cytochrome c can be restored, bringing back the pH to neutrality, the possibility offered by this transition to reversibly modulate the redox potential of cytochrome c is appealing for bioelectronic applications. The immobilized C102T/N62C variant, which differs from the native protein in the orientation of the heme group with respect to the electrode, shows very similar reduction thermodynamics. For both species, the rate constant for electron transfer between the heme and the electrode increases for the acidic conformer, which is also found to act as a biocatalytic interface for dioxygen reduction. [ABSTRACT FROM AUTHOR]

An electrode surface is presented that enables the characterization of redox-active membrane enzymes in a native-like environment. An ubiquinol oxidase from Escherichia coli, cytochrome bo3 (cbo3), has been co-immobilized into tethered bilayer lipid membranes (tBLMs). The tBLM is formed on gold surfaces functionalized with cholesterol tethers which insert into the lower leaflet of the membrane. The planar membrane architecture is formed by self-assembly of proteoliposomes, and its structure is characterized by surface plasmon resonance (SPR), electrochemical impedance spectroscopy (EIS), and tapping-mode atomic force microscopy (TM-AFM). The functionality of cbo3 is investigated by cyclic voltammetry (CV) and is confirmed by the catalytic reduction of oxygen. Interfacial electron transfer to cbo3 is mediated by the membrane-localized ubiquinol-8, the physiological electron donor of cbo3. Enzyme coverages observed with TM-AFM and CV coincide (2-8.5 fmol·cm-2), indicating that most-if not all-cbo3 on the surface is catalytically active and thus retains its integrity during immobilization. [ABSTRACT FROM AUTHOR]

A light-activated electron-transfer chain was assembled using solubilized cyanobacterialphotosystem I as photoactive enzyme, cytochrome 06 (also from cyanobacteria) as electron donor, andmethyl viologen as electron acceptor. The photocatalytic activity of the ensemble was measured by directand reversible electrochemistry of cytochrome 06 at a surface-modified gold electrode. Analysis of theelectrochemical response with an appropriate model for the reaction mechanism allowed the relation of the overall catalytic reaction rate to the individual steps of the catalytic cycle. Second-order rate constants were determined for the first time under steady-state conditions. The results validate this approach as an efficient method for the study of electron transfer between photoactive enzymes and their redox partners. [ABSTRACT FROM AUTHOR]

Studies the protein docking and gated electron-transfer reactions between zinc cytochrome c and the plastocyanin from the fern Dryopteris crassirhizoma. State of the zinc porphyrin produced by a laser flash; Intracomplex processes caused by different protein configurations; Surface regions of fern plastocyanin where intracomplex reactions occur.

Performs a computational simulation of the dynamic association between plastocyanin and cytochrome f. Consequences for the electron-transfer reaction; Calculation of the total energy of association with a thermodynamic cycle; Analysis of the electronic coupling between the copper and heme sites.

The article reports on the Met axial ligand in determining the redox potential in CuA site, the electron entry port in cytochrome c oxidases. The critical value of the protein environment in balancing the metal ions for electron transfer is clear but the details for optimal control is unknown. The study analyzed a series of axial Met ligand mutants of the Thermus thermophilus' soluble subunit II and discovered the important changes in the redox potential of the dimetallic center.

The article focuses on the protein film voltammetry (PFV) of the tetraheme cytochrome c554 (cyt c554) from Nitrosomonas europaea. Cyt c554 is the natural redox partner of the enzyme hydroxylamine oxidoreductase (HAO), which oxidizes hydroxylamine to nitrite. The study examines the redox reactions of cyt c554. The authors find evidence for previously unobserved cooperativity and redox directionality.

The article addresses the problem of low external power conversion efficiency after immobilization of the reaction center (RC) on electrodes. This paper discusses the possibility for the construction of a multiprotein electron transfer chain on an electrode and the use of cytochrome for improving the electrical connection between photosynthetic RC and the electrode.

This article focuses on thermal unfolding profiles. An electron-transfer protein, soluble monoheme cytochrome c (cyt c) is among the essential redox proteins sustaining biological activity. Another fundamental way to elucidate the redox function of cyt c is to characterize its thermo-dynamic property of both oxidized and reduced forms because the redox potential is derived from the difference between these two redox states. Conventionally, there are two protein thermal-unfolding experiments. First, differential scanning calorimetric (DSC) measurements can be carried out at more than 100 degree celcius.

Studies relationship between Redox function and protein stability of cytochromes c. Denaturation temperatures and thermodynamic parameters of Redox reaction for cytochromes c; Electrochemiocal measurement to determine the relationship betwen Redox function and protein stability of cytochromes c; Experimental observation of the analysis.