STUDY OF ELECTRONIC TRANSPORT MECHANISMS IN BIOLOGICAL NANOOBJECTS
Let us see the adaptation of methods for the study of charge transfer for biosystems and a demonstration of their application on the specific example of one nanoobject. The problem of studying the mechanisms of electron transport in biological systems was first formulated in 1941 (Gralnick, & Newman, 2007). Until this moment, charge transfer was considered in living systems from the point of view of ion currents on which the work of excitable tissues - nerves, myocardium - is built. In the post-war period, a series of important works was carried out that determined the development of ideas about electronic transport in proteins. In 1956, R. Marcus (Richter et al., 2009) proposed his theory of external sphere electron transfer for simple ions, the coordination sphere of which did not undergo significant changes as a result of the redox reaction. It was quickly noticed that Markus ions are complexing agents of many prosthetic groups of proteins involved in electron transport in the respiratory chain of mitochondria, chloroplasts, and bacteria, such as heme iron and copper, iron-sulfur clusters, and so on. The experiments of Chance (Gorby, & Yanina. 2006), Nishimura, de Volta (Pirbadian et al., 2014; Subramanian, Pirbadian, El-Naggar, & Jensen, 2018), Vredenberg and Duisens (Reardon, & Mueller, 2013), performed in the late 50s-mid 60s, by estimating the electron transfer rate between c-type cytochrome and the photocenter in purple bacteria showed that starting from 100K and lower, the reaction rate ceases to depend on temperature. A possible explanation for this process is sub-barrier electron transfer.
In the following decades, it became clear that tunnel charge transfer is a very common phenomenon for many biological systems containing gems and iron-sulfur clusters. The theory of localized states for amorphous semiconductors allows us to consider a protein as a system in which there is a set of loci, for example, aromatic conjugated heme systems, in which part of the valence electrons are socialized throughout the entire volume of the conjugated system, that is, are quasi-free (Malvankar et al„ 2011). The creation and study of doped aromatic semiconductors, such as polyaniline and polyacetylene, and subsequently organic superconductors, led to the understanding that large classes of organic compounds, including metabolites synthesized in biological systems (Mostert et al., 2012), theoretically can under certain conditions organize in conducting phases. Apparently, they did not deal with a directed search for such structures until the discovery of the phenomenon of long-range (by the standards of microbiology) charge transport in some bacterial colonies. Initially, a positive correlation was established between the number of filaments in cells, growing on the anode, and the amount of current generated by them. A wave of research on the conductivity of protein nanofilaments, hereinafter referred to as “pili” or ■'nanowires” (nanowires can be extended over long distances from cells to restore soluble iron oxide, turning it into a crystalline structure), began in 2000, after the discovery of facultative or obligate anaerobic species capable of restoring various organic and inorganic compounds to ensure the respiratory cycle in the body (Breuer, Rosso, Blumberger, & Butt, 2015).
Shewanella oneidensis MR-1 is a facultative anaerobe that respires using a variety of inorganic and organic compounds. MR-1 is also capable of utilizing extracellular solid materials, including anodes in microbial fuel cells, for electricity generation. Therefore MR-1 has been extensively studied to identify the molecular systems that are involved in electricity generation in microbial fuel cells. Kouzuma, Kasai, Hirose, and Watanabe (2015) have demonstrated the importance of extracellular electron-transfer (EET) pathways that electrically connect the quinone pool in the cytoplasmic membrane to extracellular electron acceptors. Figure 3.10 shows EET pathways (Mtr pathway) in 5. oneidensis MR-1 for transferring electrons from intracellular electron carriers (e.g., NADH and quinones), across the inner membrane (IM) and outer membrane (OM), to extracellularly located insoluble electron acceptors.
Okamoto. Hashimoto, and Nakamura (2012) investigated the role of c-type cytochromes (c-Cyts) in electronic conductivity using S. oneidensis MR-1 biofilms and the importance of electronic conductivity in generating biological current. Single and multilayer biofilms were formed on indium tin oxide (ITO) electrodes. Current-voltage dependences were obtained from c-Cyts, which were electrically connected to the surface of the electrode. The thickness of the multilayer biofilm is 16 pm. which indicates a redox peak with an 8-fold larger pendant area than a single-layer biofilm (about 0.5 pm thick), which indicates an excess of c-Cyts that are capable of performing the redox process. To determine whether this c-Cyts electron channel is involved in the generation of biological current, authors performed voltammetry with slow scanning of multilayer biofilms. The high anode current of c-Cyts, caused by the microbial oxidation of lactate, was observed during scanning of the slow potential, demonstrating the transfer of respiratory electrons through the sequential redox cycle of c-Cyts. Therefore, it is most likely that the redox cycle of c-Cyts in multilayer biofilms is a consequence of sequential electronic exchange through the physical cycle between OM c-Cyts (the outer membrane (OM) of the cell c-type cytochromes (c-Cyts)) (Figure 3.11a). As shown in the proposed model, if electrons formed as a result of metabolic oxidation of lactate along the surface of the cell are delivered to the farther electrode, the surface filling OM c-Cyts should fill the percolation threshold, which is estimated as 50% in two dimensions according to the network model with resistance. The authors suggest that the mobility of cells present in the biofilm helps establish flow conditions by increasing the apparent surface filling of c-Cyts in the biofilm matrix,
FIGURE 3.10 Proposed EET pathways (Mtr pathway) in S. oneidensis MR-l involved in direct EET (a) and mediated EET (b). OM. outer membrane; IM, inner membrane; MQIfi, reduced form of mena- quinone; MQ, oxidized form of menaquinone. Genetic and biochemical studies have identified five primary protein components, CymA, MtrA. MtrB. MtrC, and OmcA, comprising the EET pathway.
Reproduced from Kouzuma et al., 2015 under the terms of Creative Commons Attribution License.
leading to electronic conductivity through the multilayer biofilm (Figure 3.11b). Experiments with deletion mutants deficient in c-Cyts (OM) e-Cyts (AmtrC/AomcA, ApilD) and the biosynthetic protein of the capsular polysaccharide (AS03177) suggested that cell-surface-bound c-Cyts, those located on pili or extracellular polymeric substrates, play a predominant role in the long-range electron conduction in the biofilm of S. oneidensis MR-1.
Cytochrome c molecules with multiple homes transfer electrons from membrane-soluble quin- ones in the inner membrane (MQH2) to the periplasm (CymA). Electrons then move through the outer membrane (MtrA. MtrB protein) to the extracellular surface (MtrC and OmcA), where they can contact insoluble metals directly (pathway to the right) or be shuttled by flavins to metals at a farther distance from the cell surface (pathway to the left) (Fredrickson et al., 2008). In Schmid et al. (2010), pilli was studied using the methods atomic force and transmission electron microscopy, surface-enhanced Raman scattering, chromatography-mass spectrometric analysis, nuclear magnetic resonance, magnetic susceptibility and electron paramagnetic resonance, scanning probe microscopy, and IS. Kinetics is carried out by special filaments (saws) that some bacteria use to transfer electrons to the environment. The chemical composition and structural organization of filaments can have many differences. The common properties of such filaments are large spatial anisotropy (length = 10 pm at a cross section of = 10 nm), the presence of a large number of aromatic amino acids, and/or aromatic prosthetic groups (gems) in the protein components, as well as the ability to form branched networks that combine bacteria of one or several species constituting the colony. Protein structures are sensitive to physical and chemical effects - pits. Owing to these classical methods, photo and electronic lithography are not applicable in this case, as a result of widespread use in technological organic solvent lines, for example, commercially available and widely used electronic resistive polymethylmethacrylate dissolved in toluene. After application of the irradiation of the necessary areas of electrons, the manifestation occurs by means of a mixture of methyl isobutyltones and isopropyl alcohol (1:3 vol.). The most common
FIGURE 3.11 (a) Schematic illustrations for electron conduction in a multilayer biofilm of MR-1 cells
mediated via OM oCyts. Metabolically generated electrons are transferred from the upper and middle parts of the biofilm to a distant electrode, (b) Speculated model for biofilm-mediated electron conduction. Electron exchange along the cell-surface c-Cyts developed a percolation pathway of electrons across multilayer biofilm by the assistance of cell motility to increase the apparent surface coverage of OM c-Cyts.
methodology is use of Pt(acac)2 and W( CO)6 and focused gallium ion beam. Under the conditions of the experiment, this method allows the formation of contact longer than 10-15 pm.
Grebenko et al. (2018) demonstrate that the apparent conductivity of nanofibers is critical to moisture and contains several components, including those that have unusual behaviors assigned to electron transport. It was demonstrated that in the case of S. oneidensis MR-1 charge transfer, water is strongly mediated within these objects. Based on current analysis of conductivity data, it was concluded that the studied filaments of S. oneidensis MR-1 are capable of hybrid (conjugated) electron and ionic conductivity.
The shape and location of contacts are maximally reproduced case sample “Short circuit” - shorting two photolithographic gold contacts platinum or tungsten deposited with a potassium beam. Figure 3.12 shows nanofilaments with organized platinum nanocontacts marked as IBID Pt on image. “Dummy” is a section of silicon between ePt + iPt contacts that does not contain an anode. In some cases, “dummies” were described by a pure capacitance semicircle (blue vertical line on saw sample lay within the high-frequency range from 2 MHz to ~ 10 kHz, see Figure 3.13).
The test structure (see Figure 3.13a) can be described by the classical Warburg element (the linear part of the graph) at low frequencies (below 5 kHz), which usually corresponds to the diffusion of the reagent in the electrolyte solution. To determine the parameters of redox
FIGURE 3.12 Several nanofilaments with organized platinum nanocontacts fabricated by ion beam (marked as IBID Pt on image). The leakage area can be seen as a brighter area around the contact and is indicated by green arrow.
Reproduced from Grebenko et al. (2018) under the terms of ССЛ License.
FIGURE 3.13 Impedance Nyquist plots, (a) Comparison of the filament (red line) and empty circuit with almost identical nanocontact geometry (blue line); half circle that continued by straight line with the slope of ca. 45° represents reactant diffusion (Warburg element) in test structure, while nanofilament hodograph consists only of several half-circles, (b) Characteristic nanofilament hodograph: experimental data (red dots) and calculated line for equivalent scheme (blue line).
Reproduced from Grebenko ct al. (2018) under the terms of ССЛ License.
reactions and diffusion of reagents in the surrounding medium, the authors selected the impedance of the test circuit structure according to the ES scheme (empty structure) (see Figure 3.14). This scheme describes the sample with nanofilament (three bottom R-C blocks,
FIGURE 3.14 Equivalent schemes, (a) Scheme ES (empty structure), equivalent scheme for empty circuit test structure, which models redox reactions and diffusion of reactants in the surrounding medium. Technically, this scheme models electrolyte film impedance, (b) Scheme ES+NF (empty-structure + nanofilament).
Reproduced from Grebenko et al. (2018) under the terms of ССЛ License.
two for electrochemical reactions occurring on the left and right nanowire-contact interfaces and one for the nanofilament itself) in parallel with the electrolyte film impedance.
However, the straight-line feature for the nanofilament sample can be found in lower frequency region. Full frequency range for all measurements is 1 Hz -2 MHz. Both impedances were acquired at 80% relative humidity.
Finally, precise control of the medium during measurements revealed specific properties. The authors demonstrate that the use of IS in the study of charge transfer in 5. oneidensis MR-1 nanofibers provides more information on the conduction mechanisms in microbial nanofibers than traditional DC measurements. One of the types of nanofibers intensively produced by S. oneidensis MR-1 under the discussed conditions has a complex type of conjugated ion charge transfer.
