Redox Potentials Of Proteins Research Articles

Fluctuation Relations to Calculate Protein Redox Potentials from Molecular Dynamics Simulations.

The tunable design of protein redox potentials promises to open a range of applications in biotechnology and catalysis. Here, we introduce a method to calculate redox potential changes by combining fluctuation relations with molecular dynamics simulations. It involves the simulation of reduced and oxidized states, followed by the instantaneous conversion between them. Energy differences introduced by the perturbations are obtained using the Kubo-Onsager approach. Using a detailed fluctuation relation coupled with Bayesian inference, these are postprocessed into estimates for the redox potentials in an efficient manner. This new method, denoted MD + CB, is tested on a de novo four-helix bundle heme protein (the m4D2 "maquette") and five designed mutants, including some mutants characterized experimentally in this work. The MD + CB approach is found to perform reliably, giving redox potential shifts with reasonably good correlation (0.85) to the experimental values for the mutants. The MD + CB approach also compares well with redox potential shift predictions using a continuum electrostatic method. The estimation method employed within the MD + CB approach is straightforwardly transferable to standard equilibrium MD simulations and holds promise for redox protein engineering and design applications.

Virtual Model Compound Approach for Calculating Redox Potentials of [Fe2S2]-Cys4 Centers in Proteins - Structure Quality Matters.

The midpoint potential of the [Fe2S2]-Cys4-cluster in proteins is known to vary between -200 and -450 mV. This variation is caused by the different electrostatic environment of the cluster in the respective proteins. Continuum electrostatics can quantify the impact of the protein environment on the redox potential. Thus, if the redox potential of a [Fe2S2]-Cys4-cluster model compound in aqueous solution would be known, then redox potentials in various protein complexes could be calculated. However, [Fe2S2]-Cys4-cluster models are not water-soluble, and thus, their redox potential can not be measured in aqueous solution. To overcome this problem, we introduce a method that we call Virtual Model Compound Approach (VMCA) to extrapolate the model redox potential from known redox potentials of proteins. We carefully selected high-resolution structures for our analysis and divide them into a fit set, for fitting the model redox potential, and an independent test set, to check the validity of the model redox potential. However, from our analysis, we realized that the some structures can not be used as downloaded from the PDB but had to be re-refined in order to calculate reliable redox potentials. Because of the re-refinement, we were able to significantly reduce the standard deviation of our derived model redox potential for the [Fe2S2]-Cys4-cluster from 31 mV to 10 mV. As the model redox potential, we obtained -184 mV. This model redox potential can be used to analyze the redox behavior of [Fe2S2]-Cys4-clusters in larger protein complexes.

Effect of Electro-Oil Acclimation of an Indigenous Strain on the Performance of Sediment Microbial Fuel Cells (SMFC)

Sediment microbial fuel cell (SMFC) is a type of MFC without a proton exchange membrane. However, SMFC have had problems with low-power production performance. In this paper, the effects of native bacteria (K1) in oily sludge and their electro-oil-induced domestication on the power generation and oil removal performance of SMFC were studied. The results showed that K1 belonged to Ochrobactrum intermedium. During the domestication process, an upward trend was shown in the OD600 and ORP values in the culture medium, and it grown best at 0.7 V. Ochrobactrum intermedium K1 significantly increased the average output voltage, electromotive force, and maximum power density of SMFC and reduced the apparent internal resistance of the battery. The maximum power density was 169.43 mW/m3, which was 8.59 times higher than that of the control group. Ochrobactrum intermedium K1 improved the degradation of crude oil by SMFC. Ochrobactrum intermedium K1 enhanced the degradation of high-carbon alkanes and even-carbon alkanes in n-alkanes. Cyclic voltammetry and chronoamperometry tests showed that after acclimation, Ochrobactrum intermedium K1 improved the extracellular electron transfer efficiency (EET) mediated by c-Cyts and flavin by increasing the surface protein redox potential.

Scanning Optical Spectroelectrochemistry: Applications in Protein Redox Potential Measurements

AbstractThe accurate measurement of redox potentials of small molecules is a relatively straightforward task using electrochemical methods such as cyclic voltammetry. However, proteins, in most cases, are not amenable to the same approach due to slow heterogeneous electron transfer and the possibility of denaturing at the electrode surface. This necessitates the use of small molecular weight redox mediators to facilitate electron transfer. This leads to spectroelectrochemical techniques where the applied electrochemical potential is coupled to a spectroscopic signal of the protein. Traditionally this is done at different applied (fixed) potentials akin to an electrochemical titration, but the time required for electrochemical equilibrium to be established, and its consistent application, are major sources of experimental error. Here we have utilised a continuously scanning potential synchronised with time‐resolved UV‐vis spectroscopy to provide an automated approach that can be used to measure protein redox potentials accurately in an expedient manner. The test cases are the heme proteins cytochrome c and myoglobin. The scope and limitations of the method are discussed.

Machine Learning for Efficient Prediction of Protein Redox Potential: The Flavoproteins Case.

Determining the redox potentials of protein cofactors and how they are influenced by their molecular neighborhoods is essential for basic research and many biotechnological applications, from biosensors and biocatalysis to bioremediation and bioelectronics. The laborious determination of redox potential with current experimental technologies pushes forward the need for computational approaches that can reliably predict it. Although current computational approaches based on quantum and molecular mechanics are accurate, their large computational costs hinder their usage. In this work, we explored the possibility of using more efficient QSPR models based on machine learning (ML) for the prediction of protein redox potential, as an alternative to classical approaches. As a proof of concept, we focused on flavoproteins, one of the most important families of enzymes directly involved in redox processes. To train and test different ML models, we retrieved a dataset of flavoproteins with a known midpoint redox potential (Em) and 3D structure. The features of interest, accounting for both short- and long-range effects of the protein matrix on the flavin cofactor, have been automatically extracted from each protein PDB file. Our best ML model (XGB) has a performance error below 1 kcal/mol (∼36 mV), comparing favorably to more sophisticated computational approaches. We also provided indications on the features that mostly affect the Em value, and when possible, we rationalized them on the basis of previous studies.

A Theoretical Study on Redox Potential and pKa of [2Fe-2S] Cluster Model from Iron-Sulfur Proteins

Abstract Iron-sulfur proteins are essential in several biological processes, such as electron transfers in photosystems and respiratory chains. The range of redox potentials of these proteins are so wide that many substrates can interact with them. Thus, the redox potentials of the iron-sulfur proteins are crucial to facilitate the process. This study aims to investigate the redox potential of iron-sulfur proteins with [2Fe-2S] core by using two typical exchange-correlation functionals in density functional theory (DFT), i.e., B3LYP and M06, with and without the diffuse function. Since it involves transition metals, several types of spin approximation were also explored, such as high spin (HS), low spin (LS), approximated spin projection (AP), and J coupling parameter (JC) methods. We found that the diffuse function contributes to improving the accuracy. We also found that M06 functional produced more accurate results than B3LYP. The best result is obtained for AP UM06/6-31++G(d,p) with the absolute error of 0.01 V.

Exploring Oxidation State Dependent Conformational Changes of Cytochrome C on Cardiolipin Containing Liposomes

¬A combination of circular dichroism, absorption and fluorescence spectroscopies were used to study the binding of oxidized and reduced cytochrome c to cardiolipin (CL) containing liposomes with different CL content as a function of cardiolipin concentration. Recently, our group provided evidence for a two-step binding process of the oxidized (ferricytochrome) state to CL containing liposomes. The second step of binding converts a native-like to a more unfolded structure in which M80 is replaced either by a lysine of a histidine as axial ligand. The conversion into this more unfolded conformation was found to be partially inhibited in the presence of NaCl. Currently we are investigating whether the initial binding and/or the equilibrium between the two membrane bound conformers depends on pH. Our work with the reduced (ferrocytochrome) state has revealed a dependence on NaCl for stability. In the absence of NaCl and at aerobic environment, the protein becomes oxidized upon binding to CL containing liposomes. When NaCl is added to the solution, it stabilizes the reduced state on the liposome surface and inhibits the auto-oxidation. At anaerobic conditions and in the absence of NaCl, the protein undergoes a notable conformational change, but clearly maintains its reduced state. This shows that the obtained oxidation of the protein involves the reduction of molecular oxygen. We are in the process of exploring the pH dependence of the underlying change of the protein's redox potential.

Extending the essential dynamics analysis to investigate molecular properties: application to the redox potential of proteins

Here, a methodology is proposed to investigate the collective fluctuation modes of an arbitrary set of observables, maximally contributing to the fluctuation of another functionally relevant observable. The methodology, based on the analysis of fully classical molecular dynamics (MD) simulations, exploits the essential dynamics (ED) method, originally developed to analyse the collective motions in proteins. We apply this methodology to identify the residues that are more relevant for determining the reduction potential (E(0)) of a redox-active protein. To this aim, the fluctuation modes of the single-residue electrostatic potentials mostly contributing to the fluctuations of the total electrostatic potential (the main determinant of E(0)) are investigated for wild-type azurin and two of its mutants with a higher E(0). By comparing the results here obtained with a previous study on the same systems [Zanetti-Polzi et al., Org. Biomol. Chem., 2015, 13, 11003] we show that the proposed methodology is able to identify the key sites that determine E(0). This information can be used for a general deeper understanding of the molecular mechanisms on the basis of the redox properties of the proteins under investigation, as well as for the rational design of mutants with a higher or lower E(0). From the results of the present analysis we propose a new azurin mutant that, according to our calculations, shows a further increase of E(0).

Analytical expression of transient and steady-state catalytic current of mediated bioelectrocatalysis

Mediated bioelectrocatalysis is very useful to build bioreactors, biofuel cells and it can also be employed for measuring enzyme kinetics and protein redox potentials. Mathematical modeling of a mediated bioelectrocatalysis has been discussed. An analytical expression of the concentration of mediated bioelectrocatalysis for the steady- and non-steady-state conditions have been derived using Danckwerts’ expression and new approach to homotopy perturbation method. Here the concentration of substrate is sufficiently higher than the Michaelis constant and the redox reaction of a mediator is obeying the Nernst equation at an electrode surface. The analytical expressions of steady- and non-steady-state catalytic current are also reported. Our analytical results are compared with previous limiting case results and satisfactory agreement is noted.

A High Redox Potential Form of Cytochrome c550 in Photosystem II from Thermosynechococcus elongatus

Cytochrome c(550) (cyt c(550)) is a component of photosystem II (PSII) from cyanobacteria, red algae, and some other eukaryotic algae. Its physiological role remains unclear. In the present work, measurements of the midpoint redox potential (E(m)) were performed using intact PSII core complexes preparations from a histidine-tagged PSII mutant strain of the thermophilic cyanobacterium Thermosynechococcus (T.) elongatus. When redox titrations were done in the absence of redox mediators, an E(m) value of +200 mV was obtained for cyt c(550). This value is ∼300 mV more positive than that previously measured in the presence of mediators (E(m) = -80 mV). The shift from the high potential form (E(m) = +200 mV) to the low potential form (E(m) = -80 mV) of cyt c(550) is attributed to conformational changes, triggered by the reduction of a component of PSII that is sequestered and out of equilibrium with the medium, most likely the Mn(4)Ca cluster. This reduction can occur when reduced low potential redox mediators are present or under highly reducing conditions even in the absence of mediators. Based on these observations, it is suggested that the E(m) of +200 mV obtained without mediators could be the physiological redox potential of the cyt c(550) in PSII. This value opens the possibility of a redox function for cyt c(550) in PSII.

Properties of the Thioredoxin Fold Superfamily Are Modulated by a Single Amino Acid Residue

The ubiquitous thioredoxin fold proteins catalyze oxidation, reduction, or disulfide exchange reactions depending on their redox properties. They also play vital roles in protein folding, redox control, and disease. Here, we have shown that a single residue strongly modifies both the redox properties of thioredoxin fold proteins and their ability to interact with substrates. This residue is adjacent in three-dimensional space to the characteristic CXXC active site motif of thioredoxin fold proteins but distant in sequence. This residue is just N-terminal to the conservative cis-proline. It is isoleucine 75 in the case of thioredoxin. Our findings support the conclusion that a very small percentage of the amino acid residues of thioredoxin-related proteins are capable of dictating the functions of these proteins.

Transition metal complexes as mediator-titrants in protein redox potentiometry

A selection of nine macrocyclic Fe(III/II) and Co(III/II) transition metal complexes has been chosen to serve as a universal set of mediator-titrants in redox potentiometry of protein samples. The potential range spanned by these mediators is approximately from +300 to -700 mV vs the normal hydrogen electrode, which covers the range of most protein redox potentials accessible in aqueous solution. The complexes employed exhibit stability in both their oxidized and their reduced forms as well as pH-independent redox potentials within the range 6 < pH < 9. The mediators were also chosen on the basis of their very weak visible absorption maxima in both oxidation states, which will enable (for the first time) optical redox potentiometric titrations of proteins with relatively low extinction coefficients. This has previously been impractical with organic mediators, such as indoles, viologens and quinones, whose optical spectra interfere strongly with those of the protein.

A novel electrochemical approach to the characterization of oxidoreductase reactions.

Electrochemical methods based on enzyme-electrochemical reactions have been developed for studying oxidoreductase reactions. The methods measure a current resulting from an oxidoreductase reaction with an electrode serving as a final electron acceptor (or donor) in the reaction. A theoretical equation for the enzyme-electrochemical reaction, called bioelectrocatalysis, is derived, which enables kinetic analysis of the reaction. In combination with spectrophotometry, the electrochemical method provides a method for determining the redox potentials of proteins and enzymes. An alternative method based on bulk electrolysis in a quartz cell for UV-vis spectroscopy has been developed for the measurements of protein redox potentials on a conventional spectrophotometer. The electrochemical methods are applied to kinetic and thermodynamic analyses for the reactions of a variety of enzymes including a newly discovered enzyme, quinohemoprotein amine dehydrogenase (QH-AmDH), and bilirubin oxidase (BOD) [EC 1.3.3.5, from Myrothecium verrucaria], a copper-containing enzyme useful for bioelectrocatalytic O(2) reduction in biofuel cells. The electrochemical method for kinetic analysis has been successfully applied to the analysis of oxidoreductase reactions in vivo, as demonstrated by the reaction of glucose dehydrogenase in Escherichia coli. The advantages of the electrochemical methods are discussed.

Protein dynamics and cytochrome c: correlations between ligand vibrations and redox activity.

A recently developed method to directly observe specific protein vibrations, based on deuteration, has been employed to examine the redox-dependent structural and fluctional properties of cytochrome c. The dynamics of the protein-based methionine heme ligand were examined by selectively deuterating the ligand's methyl group. The frequency and line width of the C-D bonds were easily observable and shown to be sensitive to mutation-induced changes in the protein redox potential. However, of seven mutants examined, the C-D line widths were independent of the redox-state of the protein. Therefore, although the ligand dynamics depend on the protein's redox state, there are no detected differences in protein dynamics of the oxidized and reduced proteins.

Redox Potentials of Proteins and Other Compounds of Bioelectrochemical Interest in Aqueous Solutions.

Redox Potentials of Proteins and Other Compounds of Bioelectrochemical Interest in Aqueous Solutions.

An Electrochemical Approach to the Studies of Biological Redox Reactions and Their Applications to Biosensors, Bioreactors, and Biofuel Cells.

Enzymatic redox reactions for the oxidation (or reduction) of the substrates use organic dyes or metal complexes as electron acceptors (or donors), which can be regenerated by electrochemical reactions of the compounds at an electrode surface. This type of coupling of the enzymatic reactions with the electrochemical reactions is called bioelectrocatalysis and allows us to measure the enzymatic reactions by an electrochemical method. The enzyme-electrochemical method provides a new technique for investigating a variety of biological redox reactions and for applying the reactions to biosensors, bioreactors, and biofuel cells. The bioelectrocatalysis-based research works are described here. First, a new method of protein redox potential measurements and a novel electrochemical kinetic analysis of oxidoreductase reactions are described. Second, a new methodology of characterizing microbial catalytic activities is presented. In the last, applications to the constructions of biosensors, bioreactors, and biofuel cells are mentioned.

Fundamentals and Practices of Mediated Bioelectrocatalysis

Recent progress in mediated bioelectrocatalysis is reviewed. In a section concerning fundamentals, special attention is paid to describing the property of a steady-state current in this system from thermodynamic and kinetic viewpoints. Practical applications of the system includes biosensors for enzyme substrates, amplified detection of mediators, bioreactors, biofuel cells, enzyme kinetic measurements, and protein redox potential measurements.

The effect of mutation at valine-45 on the stability and redox potentials of trypsin-cleaved cytochrome b5

In an attempt to elucidate the determinants of redox potential and protein stability in cytochrome b 5, three mutants at a highly conserved residue Val45, which is a member of heme hydrophobic pocket residues have been characterized. The V45Y mutant was designed to introduce a bulkier residue and a hydroxyl group to the heme pocket. The mutants V45H and V45E were constructed to test the effect of positive and negative charge on the stability and redox potential of proteins. The influence of these mutants on the protein stability towards thermal, urea, acid, ethanol and on the redox potential were studied. It is concluded that the decrease of hydrophobic free energy and the larger volume of the tyrosine make the phenylhydroxyl group of tyrosine still sitting inside the hydrophobic pocket, while the side chain of the mutant V45E and V45H shift away from the heme pocket. The redox potentials of mutants V45Y, V45H, V45E and wild-type of cytochrome b 5 are −35 mV, 8 mV, −26 mV and −3 mV, respectively. The bigger change of the V45Y on redox potential is due to the close contact between the hydroxyl group and the heme, while the changes of the V45E and V45H result from the alteration of charge density and distribution around the heme. Different relative stability of these mutants towards heat have been observed with the order: WT>V45Y∼V45H>V45E being both in the oxidized and reduced state. The relative stability induced by addition of urea decreases in the order: WT>V45Y>V45H>V45E. These results suggest that the difference in the hydrophobic free energy is a major factor contributing to the stability of the Val45 mutants. Also the loose of the helix III in the mutant V45E makes it more unstable. These results indicate that residue Val45 plays an important role in the stability and redox potential of the protein.

Protein redox potential measurements based on kinetic analysis with mediated continuous-flow column electrolytic spectroelectrochemical technique. Application to TTQ-containing methylamine dehydrogenase.

Kinetic determination of protein redox potentials with a mediated continuous-flow column electrolytic spectroelectrochemical technique (CFCESET) is described. In this method, the redox state of the mediator is completely regulated by the continuous-flow column electrolysis, and the homogeneous redox reaction between the mediator and a protein sample in the column is monitored spectroscopically at the downstream of the column. The protein/mediator reaction is in the pseudo-first-order kinetics, and then the rate equation is analytically solved. The kinetic analysis provides the protein redox potential as well as the homogeneous rate constant. In the kinetic measurements, equilibration of the system within the column is not required, which allows the use of increased kinds of mediators. This method was successfully applied to quinoprotein methylamine dehydrogenase containing tryptophan tryptophylquinone (TTQ) as a prosthetic group. The kinetic aspect is also valuable for the thermodynamic analysis with the mediated CFCESET. The half-life time of the kinetics can be utilized to optimize the system for the attainment of the equilibrated state within the column and can provide the assurance that the system is in equilibrium.

Mediator-Assisted Continuous-Flow Column Electrolytic Spectroelectrochemical Technique for the Measurement of Protein Redox Potentials. Application to Peroxidase

A mediator-assisted continuous-flow column electrolytic spectroelectrochemical method was designed to study redox properties of proteins. This method is based on a flow-through column electrolytic control of a redox buffer containing relatively high concentrations of mediators and an accelerated reaction of a protein with the mediators to reach equilibrium within the electrolysis time. A small amount of a protein sample is introduced into the electrochemically regulated redox buffer in the mode of flow injection analysis (FIA). The equilibrated redox state of the protein is evaluated by a highly sensitive flow-through photodiode array detector. Reproducible and stable background spectra and the employment of the FIA mode allow precise background subtraction in spectral analysis. This method was successfully applied to determining the redox potentials of horseradish peroxidase (HRP) using potassium hexachloroiridate as a mediator. The absorbance versus the electrode potential curves for the redox reactions among the ferric form, compound II, and compound I of HRP were well interpreted by a Nernstian equation based on the two-step one-electron-transfer model. The advantage and the kinetic aspects of this method are discussed in detailed.