The Inhibition Study of Cytochrome bd Oxidase Using the Enzyme-Based Electrochemical Sensor

Membrane proteins that participate in multiple vital functions of every living organism such as transport, signaling and respiration, provide 80 to 90% of the relevant targets for the pharmaceutical industries. The family of cytochrome bd oxidase enzymes is of great interest for the development of future antibiotics as they are found only in the respiratory chain of the prokaryotes and they are believed to be involved in bacterial adaptability mechanisms. They catalyze the reduction of molecular oxygen in water and oxidation of quinols and contribute to the proton motive force required for ATP synthesis. Due to their hydrophobic nature, membrane proteins are more difficult to handle than soluble proteins. Protein film voltammetry is a very convenient technique, because it allows for working at a very low concentration and for optimizing the electrode surface to the nature of the enzyme. Here, we have developed a biosensor for the study of terminal oxidases based on their immobilization on gold nanoparticles modified with a self-assembled monolayer of thiols. The stability of the protein films can be optimized by varying the nature of thiols and amount of lipids. This enzyme-based electrochemical sensor was successfully used for the inhibition screening of a target-focused library of 34 compounds which belong to the families of quinones, naphthoquinones, phenols, quinolones, coumarins and flavonoids against cytochrome bd oxidase. Moreover, the developed device was applied for the study of the catalytic reaction of the enzyme with small gaseous signaling molecules.

A Multiple-Technique Approach to Inferring Bio-electrochemical Reaction Parameters

Uncovering the secrets of the biological Faradaic reactions, essential to the understanding of complex metalloenzymes, requires an information recovery process that is robust, rapid and replicable. This paper is a description of the workflow we have developed over the course of inferring chemical reaction parameters for a simple protein system, a bacterial cytochrome domain from \textit{Cellvibrio japonicus}. This was a challenging task, as the signal-to-noise ratio in such protein-film voltammetry experiments is significantly lowered relative to the voltammetric data generated by simple chemicals. We have overcome these challenges by using a multiple-technique approach, which compensates for the difficulties inherent to analysis of the individual voltammetry experiments. We have shown that the parameters inferred are robust across multiple experiments performed for different preperations of the protein. This is an important proof-of-concept result for analysis of more complex metalloenzymes, which incorporate catalytic processes and multiple internal electron-transfer sites. <br>

A Multiple-Technique Approach to Inferring Bio-electrochemical Reaction Parameters

Uncovering the secrets of the biological Faradaic reactions, essential to the understanding of complex metalloenzymes, requires an information recovery process that is robust, rapid and replicable. This paper is a description of the workflow we have developed over the course of inferring chemical reaction parameters for a simple protein system, a bacterial cytochrome domain from \textit{Cellvibrio japonicus}. This was a challenging task, as the signal-to-noise ratio in such protein-film voltammetry experiments is significantly lowered relative to the voltammetric data generated by simple chemicals. We have overcome these challenges by using a multiple-technique approach, which compensates for the difficulties inherent to analysis of the individual voltammetry experiments. We have shown that the parameters inferred are robust across multiple experiments performed for different preperations of the protein. This is an important proof-of-concept result for analysis of more complex metalloenzymes, which incorporate catalytic processes and multiple internal electron-transfer sites. <br>

Application of voltammetry in biomedicine - Recent achievements in enzymatic voltammetry

Protein-film voltammetry (PFV) is considered the simplest methodology to study the electrochemistry of lipophilic redox enzymes in an aqueous environment. By anchoring particular redox enzymes on the working electrode surface, it is possible to get an insight into the mechanism of enzyme action. The PFV methodology enables access to the relevant thermodynamic and kinetic parameters of the enzyme-electrode reaction and enzyme-substrate interactions, which is important to better understand many metabolic pathways in living systems and to delineate the physiological role of enzymes. PFV additionally provides important information which is useful for designing specific biosensors, simple medical devices and bio-fuel cells. In the current review, we focus on some recent achievements of PFV, while presenting some novel protocols that contribute to a better communication between redox enzymes and the working electrode. Insights to several new theoretical models that provide a simple strategy for studying electrode reactions of immobilized enzymes and that enable both kinetic and thermodynamic characterization of enzyme-substrate interactions are also provided. In addition, we give a short overview to several novel voltammetric techniques, derived from the perspective of square-wave voltammetry, which seem to be promising tools for application in PFV.

Electroanalytical and electrocatalytical characteristics of cytochrome P450 3A4 using electrodes modified with nanocomposite carbon nanomaterials

The electroanalytical characteristics of recombinant cytochrome P450 3A4 (P450 3A4) immobilized on the surface of screen-printed graphite electrodes modified with multi-walled carbon nanotubes have been studied. The role and the influence of graphite working electrode modification with carbon nanotubes on electroanalytical characteristics of cytochrome P450 3A4 have been demonstrated. The conditions for the immobilization of cytochrome P450 3A4 on the obtained screen-printed graphite electrodes modified with carbon multi-walled nanotubes have been optimized. The electrochemical parameters of the oxidation and reduction of the heme iron of the enzyme have been estimated. The midpoint potential E0′ was -0.35±0.01 V vs Ag/AgCl; the calculated heterogeneous electron transfer rate constant ks, was 0.57±0.04 s-1; the amount of electroactive cytochrome P450 3A4 on the electrode Г0, was determined as (2.6±0.6)⋅10-10 mol/cm2. The functioning mechanism of P450 3A4-based electrochemical sensor followed the “protein film voltammetry”. In order to develop electrochemical analysis of drugs being substrates of that hemoprotein and respective medical biosensors the voltammetric study of catalytic activity of immobilized cytochrome P450 3A4 was carried out. Electrocatalytic properties of cytochrome P450 3A4, immobilized on modified screen-printed graphite electrodes, has been investigated using erythromycin (macrolide antibiotics). It has been shown that the modification of electrodes plays a decisive role for the study of the properties of cytochromes P450 in electrochemical investigations. Smart electrodes can serve as sensors for analytical purposes, as well as electrocatalysts for the study of biotransformation processes and metabolic processes. Electrodes modified with carbon nanomaterials are applicable for analytical purposes in the registration of hemoproteins. Electrodes modified with synthetic membrane-like compounds (e.g. didodecyldimethylammonium bromide) are effective in enzyme-dependent electrocatalysis.