Electronic coupling to electrodes, Γ, as well as that across the examined molecules, H, is critical for solid-state electron transport (ETp) across proteins. Assessing the importance of each of these couplings helps to understand the mechanism of electron flow across molecules. We provide here experimental evidence for the importance of both couplings for solid-state ETp across the electron-mediating protein cytochrome c (CytC), measured in a monolayer configuration. Currents via CytC are temperature-independent between 30 and ∼130 K, consistent with tunneling by superexchange, and thermally activated at higher temperatures, ascribed to steady-state hopping. Covalent protein-electrode binding significantly increases Γ, as currents across CytC mutants, bound covalently to the electrode via a cysteine thiolate, are higher than those through electrostatically adsorbed CytC. Covalent binding also reduces the thermal activation energy, E a , of the ETp by more than a factor of two. The importance of H was examined by using a series of seven CytC mutants with cysteine residues at different surface positions, yielding distinct electrode-protein(-heme) orientations and separation distances. We find that, in general, mutants with electrode-proximal heme have lower E a values (from high-temperature data) and higher conductance at low temperatures (in the temperatureindependent regime) than those with a distal heme. We conclude that ETp across these mutants depends on the distance between the heme group and the top or bottom electrode, rather than on the total separation distance between electrodes (protein width).bioelectronics | temperature dependence | protein conduction

We investigate functional role of the P76GTKMIFA83 fragment of the primary structure of cytochrome c. Based on the data obtained by the analysis of informational structure (ANIS), we propose a model of functioning of cytochrome c. According to this model, conformational rearrangements of the P76GTKMIFA83 loop fragment have a significant effect on conformational mobility of the heme. It is suggested that the conformational mobility of cytochrome c heme is responsible for its optimal orientation with respect to electron donor and acceptor within ubiquinol–cytochrome c oxidoreductase (complex III) and cytochrome c oxidase (complex IV), respectively, thus, ensuring electron transfer from complex III to complex IV. To validate the model, we design several mutant variants of horse cytochrome c with multiple substitutions of amino acid residues in the P76GTKMIFA83 sequence that reduce its ability to undergo conformational rearrangements. With this, we study the succinate–cytochrome c reductase and cytochrome c oxidase activities of rat liver mitoplasts in the presence of mutant variants of cytochrome c. The electron transport activity of the mutant variants decreases to different extent. Resonance Raman spectroscopy (RRS) and surface-enhanced Raman spectroscopy (SERS) data demonstrate, that all mutant cytochromes possess heme with the higher degree of ruffling deformation, than that of the wild-type (WT) cytochrome c. The increase in the ruffled deformation of the heme of oxidized cytochromes correlated with the decrease in the electron transport rate of ubiquinol–cytochrome c reductase (complex III). Besides, all mutant cytochromes have lower mobility of the pyrrol rings and methine bridges, than WT cytochrome c. We show that a decrease in electron transport activity in the mutant variants correlates with conformational changes and reduced mobility of heme porphyrin. This points to a significant role of the P76GTKMIFA83 fragment in the electron transport function of cytochrome c.

We showed that the genetically engineered carrier-protein albebetin and its biologically active constructs with interferon-alpha(2) octapeptide LKEKKYSP or differentiation factor hexapeptide TGENHR are inherently highly amyloidogenic at physiological pH. The kinetics of fibrillation were monitored by thioflavine-T (ThT) binding and the morphological changes by atomic force microscopy. Fibrillation proceeds via multiple pathways and includes a hierarchy of amyloid structures ranging from oligomers to protofilaments and fibrils. Comparative height and volume microscopic measurements allowed us to identify two distinct types of oligomeric intermediates: pivotal oligomers ca. 1.2 nm in height comprised of 10-12 monomers and on-pathway amyloid-competent oligomers ca. 2 nm in height constituted of 26-30 molecules. The former assemble into chains and rings with "bead-on-string morphology", in which a "bead" corresponds to an individual oligomer. Once formed, the rings and chains remain in solution simultaneously with fibrils. The latter give rise to protofilaments and fibrils, and their formation is concomitant with an increasing level of ThT binding. The amyloid nature of filamentous structures was confirmed by a pronounced ThT and Congo red binding and beta-sheet-rich far-UV circular dichroism. We suggest that transformation of the pivotal oligomers into the amyloid-prone ones is a limiting stage in amyloid assembly. Peptides, either fused to albebetin or added into solution, and an increased ionic strength promote fibrillation of albebetin (net charge of -12) by counterbalancing critical electrostatic repulsions. This finding demonstrates that the fibrillation of newly designed polypeptide-based products can produce multimeric amyloid species with a potentially "new" functionality, raising questions about their safety.

As ample-type protein monolayer,t hat can be as tepping stone to practical devices,c an behave as an electrically driven switch. This feat is achieved using ar edox protein, cytochrome C( CytC), with its heme shielded from direct contact with the solid-state electrodes.A binitio DFT calculations,c arried out on the CytC-Aus tructure,s howt hat the coupling of the heme,t he origin of the protein frontier orbitals,tothe electrodes is sufficiently weak to prevent Fermi level pinning.T hus,e xternal bias can bring these orbitals in and out of resonance with the electrode.Using acytochrome C mutant for direct SÀAu bonding,a pproximately 80 %o ft he Au-CytC-Au junctions showatgreater than 0.5 Vbias aclear conductance peak, consistent with resonant tunneling.The onoff change persists up to room temperature,d emonstrating reversible,b ias-controlled switching of ap rotein ensemble, which,with its built-in redundancy,provides arealistic path to protein-based bioelectronics.

A systematic comparison between electron-transfer rate constants measured electrochemically for different cysteine-bearing mutants of cytochrome c chemisorbed on gold surfaces in different orientations has been performed. Experimental data have been correlated with electronic coupling theoretical estimates obtained from two different empirical models for the kinetics of protein electron transfer, the tunneling pathway model and the average packing density model. The results indicate that both models also hold in the case of immobilized redox proteins, outlining their role in the rational design of optimized electron-transfer-based bioinorganic interfaces.

Three forms of horse heart cytochrome c with specific substitutions of heme cleft surface located amino acid residues involved in specific interactions with ubiquinol:cytochrome c reductase (complex III) and cytochrome c oxidase (complex IV) were constructed, and their reactions with superoxide radical produced by NADH:ubiquinone reductase (complex I) were studied. The proteins with six (K27E/E69K/K72E/K86E/K87E/E90K and K8E/E62K/E69K/K72E/K86E/K87E) and eight (K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K) substitutions were inactive in the cytochrome c oxidase reaction, and their reduction rates by complex III were significantly lower than that seen with acetylated cytochrome c. The reduction of these modified cytochromes c under conditions where complex I generates superoxide was almost completely (about 90%) inhibited by superoxide dismutase. The genetically modified cytochromes c are useful analytical reagents for studies on superoxide generation by the mitochondrial respiratory chain. Quantitative comparison of superoxide-mediated cytochrome c reduction with hydrogen peroxide-mediated Amplex Red oxidation suggests that complex I within its native environment (submitochondrial particles) produces both superoxide (~50%) and hydrogen peroxide (~50%).

Cytochrome c is one of the key proteins involved in the programmed cell death, and lysine 72 is known to be required for its apoptogenic activity. We have engineered a number of horse and murine cytochrome c single-point mutants with various substitutions at position 72 and compared quantitatively their proapoptotic activity in living cells. Apoptosis was activated by transferring exogenous cytochrome c into the cytoplasm of cells via a nontraumatic electroporation procedure. All mutant proteins studied exhibited significantly reduced proapoptotic activities in comparison with those for the wild type cytochromes. Relative activity of the horse (h(K72X)) and murine (m(K72W)) mutant proteins diminished in the order: h(K72R) > h(K72G) > h(K72A) > h(K72E) > h(K72L) >> h(K72W) > m(K72W). As estimated, the horse and murine K72W mutants were at least 200- and 500-fold less active than corresponding wild type proteins. Thus, the K72W-substituted cytochrome c can serve as an adequate candidate for knock-in studies of cytochrome c-mediated apoptosis. The proapoptotic activity of wild-type cytochrome c from different species in murine monocytic WEHI-3 cells reduced in the order: murine cytochrome c > human cytochrome c approximately horse cytochrome c, thus indicating that apoptotic effect of cytochrome c depends on the species compatibility.

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