Dr James McEvoy

Introduction

- Redox biochemistry

Life depends on redox reactions – in other words, reactions that involve the movement of electrons. These reactions are central to bioenergetic processes such as cellular respiration and photosynthesis, in which the movement of electrons is associated with the movement and transformation of energy. The movement of ions across biological membranes is associated with both energy and information transduction. There are various diseases associated with malfunctioning redox biochemistry in humans, and so understanding these processes has medical significance. We are interested in various problems in the area of biological redox chemistry, using electrochemistry and various spectroscopic, synthetic and molecular biological techniques as required.

- Biological Electrochemistry

We use electrochemistry to probe biologically important redox reactions by making the solid electrode a partner in the redox process. A potentiostat is used to change the potential energy of the electrode which drives electrons onto, or removes electrons from, various biological redox centers. In this way we can learn important thermodynamic and kinetic information about the reactions associated with electron transfer. Because electrochemistry examines redox reactions at an interface, it is particularly suitable for investigating surfaces such as biological membranes and biofilms. Along with structural information from microscopy and various spectroscopies one can build a mechanistic picture of events at the molecular level.

Current projects

- Antibiotic resistance in biofilms

Bacterial antibiotic resistance is a major threat to public health, particularly in hospital-acquired infections such as MRSA. The pathogenic bacteria responsible for such infections commonly live in biofilms, using these polysaccharide matrices to stick to surfaces and protect themselves from viruses and host immune responses. Bacteria in biofilms are particularly resistant to antibiotics  and may exchange resistance-coding plasmids at high rates. In addition, because biofilms provide additional spatial structure, they may facilitate social interactions such as the production of enzymes that are common goods, providing shared benefits to groups of bacteria. Laboratory experiments have shown that bacteria expressing antibiotic-hydrolyzing enzymes (β-lactamases) can be social by removing antibiotics that are harmful to their neighbours. However, it is currently unclear whether this type of antibiotic resistance can be social in natural populations. An important first step is to explore how antibiotics diffuse in biofilms, whether the hydrolysis of β-lactam antibiotics is social in this context and how bacterial genotypes that are sensitive or resistant to antibiotics compete within biofilms.

This project, funded by the Charles Sykes Trust and run in collaboration with Dr Ben Raymond, aims to explore the molecular and evolutionary basis of antibiotic resistance in gram-negative bacterial biofilms. We will use a combination of microbiological, chemical and evolutionary ecology techniques to analyze the interaction of antibiotics with biofilms, both in vitro and in vivo. Our cross-disciplinary, quantitative approach will allow us to draw real-world conclusions concerning the evolution of antibiotic resistance in biofilms. Successful outcomes from this work may lead to new strategies in the fight against bacterial antibiotic resistance.

- ETF-ubiquinone oxidoreductase (ETF-QO)

This enzyme is involved in the mitochondrial ‘β-oxidation’ of fatty acids, as well as the oxidation of some amino acids. It is therefore a crucial player in cellular energy transduction, particularly in those organs, such as the heart and kidney, which rely on fatty acids for energy. Electrons flow from the fatty acids to various acyl-CoA dehydrogenases and from there to ETF, a soluble flavoprotein, which is in turn oxidized by ETF-QO. ETF-QO is an integral membrane protein and passes electrons to ubiquinone in the inner mitochondrial membrane; reduced ubiquinol enters the mitochondrial electron transport pathway and is oxidized by Complex III. These events are summarized in the figure below.

Electron transfer (curved arrows) from fatty acid derivatives to mitochondrial Complex III. X-ray crystal structures of proteins have been imaged by Accelrys Discovery Studio Visualizer and obtained from the RCSB-PDB via the following accession numbers: ETF, 1EFV; ETF-QO, 2GMH; Complex III, 1KYO.

Inherited defects in either ETF or ETF-QO lead to the inborn error of metabolism known either as glutaric acidemia type II (GAII) or multiple acyl-CoA dehydrogenase deficiency (MADD). The most severe forms present in early infancy and are frequently fatal. We aim to understand the redox chemistry of ETF and ETF-QO by direct electrochemical methods, thereby gaining insights that will be important in designing therapies for MADD. This will depend upon strategies for bringing these proteins into close contact with the electrode while avoiding denaturation. Gold electrodes, for instance, may be chemically modified by thiols (self-assembled monolayers, or SAMs) and amphipathic lipids, creating a favorable environment for a membrane protein such as ETF-QO.

- Reactive oxygen species

Reactive oxygen species (ROS) such as superoxide (O₂⁻) and hydrogen peroxide (H₂O₂) have been implicated in many biological processes. Medically-relevant pathologies that are associated with high levels of ROS include ageing and neurodegenerative diseases such as Parkinson’s, but ROS also play an important signaling role. ETF and ETF-QO (see above) are thought to contribute significantly to mitochondrial ROS production.

Most methods for the detection of ROS are indirect, relying on the detection (often by fluorescence) of certain oxidized products. Electrochemical ROS sensors have been designed that are direct, highly sensitive and selective. Our lab is interested in combining the electrochemical manipulation of a biomolecule with in situ ROS detection. We aim to probe the topology of physiologically-relevant ROS production on an electrode, shedding light on the molecular mechanisms which underlie pathological and signaling processes.

- Photosynthesis

We have a long-standing interest in the redox chemistry of certain photosynthetic proteins, most recently photosystem II. This enzyme uses sunlight to oxidize water, producing all of the atmosphere’s O2 and therefore making aerobic life possible. It is currently under scrutiny as a model for the water-splitting component of an artificial photosynthetic system, a next-generation solar energy device. We aim to investigate the redox mechanism of this enzyme by attaching a His-tagged mutant to a suitably-modified gold electrode and probing it electrochemically.

I teach and run the whole of the 1st year course BS1030, "Princliples of Molecular Biosciences". This course runs over the first two terms of the year and comprises forty 50-minute lectures and eight laboratory sessions for each student. Students are assessed by the quality of their lab reports (25%), regular on-line quizzes (10%), and their performance in an end-of-year exam (65%).

I also contribute a few lectures and labs to BS1090 and BS2510.