(1) We make use of chemical principles to understand fundamentally important reactions in biology. Reduction-oxidation (redox) reactions underpin innumerable chemical reactions and much of the chemistry of life. Many redox reactions proceed via radical intermediates and these are often located in mechanistically key locations (see our recent book chapter). We investigate how oxidation-state changes govern respiration (respiratory complex I) and photosynthesis (photosynthetic complex I) and how nature has fine-tuned the redox properties of its many intricate molecular machines. Membranes play a fundamentally important role for many proteins and we are investigating the role of membranes on protein activity and function through spin labels and protein-intrinsic paramagnetic centres.  

(2) We are developing film-electrochemical EPR (FE-EPR) as a new method to study the evolution of radicals during redox and catalytic reactions in real time. This enables detailed and previously impossible mechanistic understanding of both chemical and biochemical reactions. The catalytic reactions we investigate range from well-known long-established chemical transformations such as nitroxide-catalysed alcohol oxidation, to complex metal-containing enzymes that may prove to be novel targets for the development of new antimicrobials. Our FE-EPR methodology is even finding applications in battery research. 

(3) Radicals often hold key mechanistic information - but trapping them in sufficient numbers for detailed EPR investigations can be a major challenge.  We specialise in working with minute sample concentrations and volumes, and are seeking to make the impossible possible with our current collaborative grant SpinSUPER. 

Protein Film Electrochemistry-EPR

PFE-EPR

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The combination of electron paramagnetic resonance (EPR) spectroscopy with protein film electrochemistry (PFE) provides a novel platform for the investigation of redox-active species including metalloproteins. The development of PFE-EPR enables the detection of paramagnetic species with direct and accurate potential control, providing new mechanistic insight to redox-based processes in biomolecules, including catalysis. Current EPR spectroelectrochemical methods to study proteins are based on generating radical species in solution and therefore limited by diffusion (precluding accurante potnetial control) and do not enable catalytic investivations. We exploit the advantages of PFE, i.e. 'wiring' electrons directly to the working electrode surface, to eliminate diffusion limitations. 

Project members and collaborators

• Adam Sills (PhD student, ICL)
• Yunfei Dang (PhD student, ICL)
• Davide Facchetti (PhD student, ICL)
• Dr. Sam Cobb (PDRA, Cambridge)
• Prof. Erwin Reisner (PI, Cambridge)

Project alumni

• Dr. Maryam Seif Eddine (PDRA, ICL)
• Dr. Kaltum Abdiaziz (PDRA, QMUL)

Respiratory Complex I (NADH:ubiquinone oxidoreductase)

Complex I – the entry point of electrons into the respiratory chain of all higher organisms – is the last of the respiratory enzymes whose mechanism remains unknown. In mitochondria, complex I oxidises NADH from sugars and fats, reduces ubiquinone and transports protons across the inner mitochondrial membrane, thereby contributing to the proton motive force that enables ATP-synthase to make ATP.

We are working on elucidating some of the key elements of the mechanism of complex I using advanced EPR methods, in particular we are fascinated how the redox energy built up through electron transfer is used to drive proton translocation. Given the large size of the enzyme (the size of the mitochondrial enzyme is ca. 1 MDa), many biophysical methods routinely applied to study protein cannot be used (e.g. NMR). We are exploiting the fact that EPR spectroscopy is blind to all the many paired electrons in the complex, and that unpaired electrons are situated in mechanistically key locations. For instance, many of the iron-sulfer clusters ([2Fe-2S] and [4Fe-4S] clusters) become EPR-visible when reduced. We have recently divised a potentiometric method that enables us to adjust very small amounts of protein to very precise reduction potentials - an important prerequisite for studying these redox-active proteins spectroscopically. This has enabled us, in conjunction with pulse EPR spectroscopy and site-directly mutagenesis, to investigate the role of Fe-S cluster N2. 

Project members and collaborators

• Adam Sills (PhD student, ICL)
• Fang Fang (PhD student, ICL)
• Eleanor Clifford (PhD student, ICL)
• Dr John Wright (PDRA, Medical Research Council, Mitochondrial Biology Unit, Cambridge)
• Dr Judy Hirst (PI, Medical Research Council, Mitochondrial Biology Unit, Cambridge)

Project alumni

• Dr. Maryam Seif Eddine (PDRA, ICL)
• Dr. Kaltum Abdiaziz (PDRA, QMUL)

Photosynthetic Complex I (the NAD(P)H dehydrogenase 'like' complex (NDH) )

We have teamed up with Dr Guy Hanke to investigate the fascinating molecular machine NDH, in many ways related to the mitochondrial enzyme (complex I, above), present in plants and cyanobacteria. Our goal is to unravel the mechanism of NDH, and ultimately to exploit this knowledge in order to increase stress tolerance in plants (in collaboration with Prof. Nestor Carrillo, Rosario, Argentina). Increasing crop yields is an especially important challenge facing the developing world, but may affect us all one day given the ever rising world population. Research on NDH has thus far focused on genetic investigations and very little is known about the function of NDH, and no biophysical studies are reported. Using our experience with the mitochondrial enzyme, we are using EPR spectroscopy and protein film electrochemistry to investigate the co-factors in and the electron donors to NDH. Moreover, the knowledge gained through NDH is also likely relevant for understanding the mechanism of complex I. 

See Dr Hanke's webpage for more details on the biological context. 

Project members and collaborators

• Eleanor Clifford (PhD student, ICL)
• Angeliki Chatziathanasiou (PhD student, ICL)
• Dr Guy Hanke (PI, School of Biological and chemical Sciences, QMUL)

Project Alumni

• Gemma McGuire (PhD student, QMUL & ICL)
• Katherina Richardson (PhD student, LIDo program, QMUL & ICL)