The role of protein dynamics and thermal fluctuations in regulating cytochrome c/cytochrome c oxidase electron transfer

In this overview we present recent combined electrochemical, spectroelectrochemical, spectroscopic and computational studies from our group on the electron transfer reactions of cytochrome c and of the primary electron acceptor of cytochrome c oxidase, the Cu_A site, in biomimetic complexes. Based on these results, we discuss how protein dynamics and thermal fluctuations may impact on protein ET reactions, comment on the possible physiological relevance of these results, and finally propose a regulatory mechanism that may operate in the Cyt/CcO electron transfer reaction in vivo. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Quantitative Comparison of the Signals of an Electrochemical Bioactivity Sensor During the Cultivation of Different Microorganisms

The microbial activity of different microorganisms was determined by means of an electrochemical bioactivity sensor (BAS). The BAS is based on a biofuel cell and was used for analytical purposes. Online determination of microbial activity using the BAS demonstrated that when different microorganisms with different metabolic pathways were cultivated, a distinct activity signal was detectable with all organisms applied. Furthermore, the results permitted a quantitative comparison of the BAS signals. Among other findings it was shown that the quotient of the BAS signal and the utilized glucose varied from 0.16–29.08 mV g⁻¹, the quotient of the maximum BAS signal and the released energy of the reaction exhibited a lower variation of 0.07–0.19 mV kJ⁻¹. Furthermore it was demonstrated that the highest BAS signals could be measured during anaerobic E. coli fermentations, the reason being the formation of electroactive fermentation products, such as formic acid and H₂.     

Thermodynamic and kinetic properties of the outer membrane cytochrome OmcF,a key protein for extracellular electron transfer in Geobacter sulfurreducens

Gene knock-out studies on Geobacter sulfurreducens have shown that the monoheme c-type cytochrome OmcF is essential for the extracellular electron transfer pathways involved in the reduction of iron and uranium oxy-hydroxides, as well as, on electricity production in microbial fuel cells. A detailed electrochemical characterization of OmcF was performed for the first time, allowing attaining kinetics and thermodynamic data. The heterogeneous electron transfer rate constant was determined at pH?7 (0.16?±?0.01?cm?s^?1) indicating that the protein displays high electron transfer efficiency compared to other monoheme cytochromes. The pH dependence of the redox potential indicates that the protein has an important redox-Bohr effect in the physiological pH range for G. sulfurreducens growth. The analysis of the structures of OmcF allowed us to assign the redox-Bohr centre to the side chain of His⁴⁷ residue and its pK_a values in the reduced and oxidized states were determined (pK_ox?=?6.73; pK_red?=?7.55). The enthalpy, entropy and Gibbs free energy associated with the redox transaction were calculated, pointing the reduced form of the cytochrome as the most favourable. The data obtained indicate that G. sulfurreducens cells evolved to warrant a down-hill electron transfer from the periplasm to the outer-membrane associated cytochrome OmcF.     

Probing copper/tau protein interactions electrochemically

Alzheimer’s disease (AD) is a neurodegenerative disorder that is characterized by peptide and protein misfolding and aggregation, in part due to the presence of excess metal ions such as copper(II) [Cu(II)]. Recently, the brain levels of Cu(II) complexes in vivo were linked to the oxidative stress in neurodegenerative disorders, including AD. Amyloid β-peptide (Aβ), found outside neuronal cells, has been investigated extensively in connection with Cu(II) ion toxicity; however, the effects of metallation on tau are less known. Normal tau protein binds and stabilizes the microtubules in neurons, but in diseased cells tau hyperphosphorylation and aggregation are evident and compromise tau function. There is increasing evidence that the Cu(II) ion may play an important role in tau biochemistry. Here, we present an electrochemical study of the interactions between full-length tau-410 and Cu(II) ions. The coordination of Cu(II) ions to tau immobilized on gold surfaces induces an electrochemical signal at approximately 140 ± 5 mV versus Ag/AgCl due to the Cu(II)/Cu(I) redox couple. Redox potentials and current intensities of Cu(II)-containing nonphosphorylated tau (nTau) and phosphorylated tau (pTau) films were determined at different pH conditions. Greater Cu(II) uptake by pTau over nTau films was observed at low pH. Competitive zinc(II) [Zn(II)] ion binding studies revealed significant Cu(II) ion displacement in pTau films. X-ray photoelectron spectroscopy analysis indicated the presence of Cu 2p and Zn 2p binding energies in protein samples, further supporting metal ion coordination to protein films. The surface-based electrochemical technique requires a minimal protein amount (a few microliters) and allows monitoring the bound Cu(II) ions and the redox activities of the resulting metalloprotein films.     

Bioelectronome. Integrated Approach to Receptor Chemistry,Radicals, Electrochemistry,Cell Signaling,and Physiological Effects Based on Electron Transfer

Bioelectronome refers to the host of electron transfer (ET) reactions that occur in living systems. This review presents an integrated approach to receptor chemistry based on electron transfer, radicals, electrochemistry, cell signaling, and end result. First, receptor activity is addressed from the unifying standpoint of redox transformations in which various receptors are discussed. After a listing of receptor-binding modes, receptor chemistry is treated with focus on generation of reactive oxygen species (ROS), activation by ROS, and subsequent cell signaling involving ROS. A general electrostatic mechanism is proposed for receptor-ligand action with supporting evidence. Cell-signaling processes appear to entail electron transfer, ROS, redox chains, and relays. The widespread involvement of phosphate from phosphorylation may be rationalized electrostatically by analogy with DNA phosphate. Extensive evidence supports important participation of ET functionalities in the mechanism of drugs and toxins. The integrated approach is applied to the main ET classes, namely, quinones, metal complexes, iminium species, and aromatic nitro compounds.     

Electrochemical investigation of the effect of some organic phosphates on haemoglobin

The effects of DPG, IHP, GTP, GDP and GMP on the structure and stability of haemoglobin were electrochemically investigated with an iodide-modified silver electrode in 0.01 M KNO₃ at pH 7.0. Anodic and cathodic peaks of haemoglobin were observed at 250 mV and 12 mV with a formal potential value of 133 mV vs. Ag/AgCl. The effects of different concentrations of DPG, IHP, GTP, GDP and GMP on the anaerobic redox reaction were determined. The results showed that DPG and IHP can lead to a positive shift in the reduction peak of haemoglobin, indicating that the oxidation peak shift of haemoglobin was small as a result of stabilization of the reduced state and destabilization of the R-like state of haemoglobin. GTP elicited a more positive shift in the cathodic and anodic peaks of haemoglobin at a higher concentration, signifying that it has a low-affinity binding site on haemoglobin. The positive shift of the cathodic and anodic peaks revealed a slight variation in the structure and indicated the unfolding of haemoglobin in the presence of high concentrations of GTP. Our study also showed that GDP and GMP did not cause significant shift the cathodic and anodic peaks of haemoglobin even at high concentrations, refuting the existence of specific GDP-and GMP-binding sites on the protein. Moreover, the iodide-modified silver electrode method proved to be easy and useful in investigating the effects of ligands or other effectors on haemoglobin in solution.     

KCTCCA,a peptide-based facilitator for bioelectrochemistry

Electrochemical and scanning tunnelling microscopy (STM) studies have been carried out to investigate the suitability of the hexapeptide KCTCCA as a facilitator for bioelectrochemistry. The stable, quasi-reversible electro-chemical response of cytochromeb ₅₆₂ on a KCTCCA modified gold electrode and the high degree of surface coverage of KCTCCA on gold (111), as observed by STM, indicate applicability of the molecule as an electro-chemical facilitator.     

Can proton pumping by SERCA enhance the regulatory role of phospholamban and sarcolipin?

The effect of the incorporation of phosphorylated phospholamban (pPLN) and sarcolipin (SLN) in mercury-supported self-assembled lipid monolayers and in lipid bilayers tethered to mercury via a hydrophilic spacer was investigated by voltammetric techniques and electrochemical impedance spectroscopy. It was shown that pPLN and SLN do not permeabilize lipid bilayers toward ions at physiological pH. However, they exert a permeabilizing action toward inorganic monovalent cations such as K⁺ and Tl⁺, but not toward divalent cations such as Ca²⁺ and Cd^2 +, following a small decrease in pH. This behavior can be associated with their regulatory action on the Ca-ATPase of the sarcoplasmic reticulum (SERCA). SERCA pumps two Ca²⁺ ions from the cytosol to the lumen of the sarcoplasmic reticulum (SR) and two protons in the opposite direction, causing a transient decrease of pH in the immediate vicinity of its cytoplasmic domain. This decrease is expected to activate the liberated pPLN molecules and SLN to make the SR membrane leakier toward K⁺ and Na⁺ and the SLN ion channel to translocate small inorganic anions, such as Cl⁻. The effect of pPLN and SLN, which becomes synergic when they are both present in the SR membrane, is expected to favor a rapid equilibration of ions on both sides of the membrane.     

An analytical model of memristors in plants

The memristor, a resistor with memory, was postulated by Chua in 1971 and the first solid-state memristor was built in 2008. Recently, we found memristors in vivo in plants. Here we propose a simple analytical model of 2 types of memristors that can be found within plants. The electrostimulation of plants by bipolar periodic waves induces electrical responses in the Aloe vera and Mimosa pudica with fingerprints of memristors. Memristive properties of the Aloe vera and Mimosa pudica are linked to the properties of voltage gated K⁺ ion channels. The potassium channel blocker TEACl transform plant memristors to conventional resistors. The analytical model of a memristor with a capacitor connected in parallel exhibits different characteristic behavior at low and high frequency of applied voltage, which is the same as experimental data obtained by cyclic voltammetry in vivo.     

生物电化学系统处理废气的研究进展

生物电化学系统(Bioelectrochemical systems,BESs)可将污染物的降解转化与电能紧密耦联,具有适用基质广泛、反应过程温和且效率高的特点,在环境污染治理中具有广阔的应用前景。近几年,BESs也逐渐被应用到废气处理。由于微生物和电化学过程的复合作用,BESs显示出较高的处理效率和良好的应用前景。本文在对废气类型、效果及反应器构型进行总结的基础上,还对BESs中的重要功能微生物和微生物电化学反应机理进行介绍和讨论,并对BESs在废气处理方面需要解决的问题和研究方向进行展望,以期为提高生物电化学系统的处理性能提供参考。