How does respiratory complex I pumps protons? Finding the missing link using EPR spectroscopy.

The rising age of the UK population presents one of the major challenges to our society and economy and it is vital that we maximise the contribution of the most experienced individuals by increasing the number of years we live in best possible health. The mitochondrial respiratory chain, which is essential for the production of ATP and a prime source of reactive oxygen species, is thought to have a major influence on the rate at which we age. Moreover, defects in the respiratory chain are responsible for mitochondrial diseases - these are rare, but their outcome is often very severe, if not fatal. Their diagnosis is fraught with difficulties and there is no cure.

Complex I is a crucial but poorly understood element in the mitochondrial respiratory chain and we propose to elucidate a particularly important aspect of the mechanism of this essential enzyme by employing a multidisciplinary approach. We will study the bovine enzyme, a close relative of human complex I, employing in particular electron paramagnetic resonance (EPR) spectroscopy, a powerful method for investigating chemical centres having unpaired electrons. In complex I such unpaired electrons are naturally present or can be generated in mechanistically highly informative locations and we will harness the information they provide through advanced pulse EPR and biochemical methods.

Our work will constitute a major step forward in obtaining a complete picture of the molecular mechanism of one of the most important, largest and most enigmatic enzymes in our bodies, with long-term implications for increasing our healthy lifespan and for the recognition and treatment of mitochondrial diseases resulting from complex I dysfunction. Our proposed research program will have immediate impact on UK science, with academic beneficiaries in a diverse range of research disciplines. We will provide top-quality interdisciplinary training for the EPSRC PDRA (and at least two Queen Mary University undergraduate research project students), to provide expertise at the interface of chemistry and biology, with a quantitative approach to fundamental biochemical problems.

We propose to elucidate the mechanism of a fundamentally important respiratory-chain enzyme, complex I, using a highly interdisciplinary approach involving both advanced spectroscopic techniques and biochemical methods. Our research is at the interface of chemistry and biology, and has the potential to generate a number of different types of impact, a summary of which is given below. Please see 'Pathways to Impact' for further details.

1) Societal Impact

As a major contributor of reactive oxygen species, complex I dysfunction has been implicated in ageing. Moreover, complex I has been shown to be the most common site for mitochondrial anomalies, accounting for as much as one third of respiratory chain deficiencies. Leber's hereditary optic neuropathy, MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) and Leigh's Syndrome are some of the mitochondrial disorders specifically associated with complex I. They form part of progressive neuro-degenerative disorders, often present at birth or with onset in early childhood, for which there is no cure. Treatments are largely based on trial and error and at best alleviate symptoms. Recently, aberration of complex I activity has also been shown to enhance the progressiveness of human breast cancer cells and to result in severe hypertrophic cardiomyopathy (a disease in which the heart muscle becomes thickened). Our work in understanding how complex I functions at the molecular level will ultimately lead to rational treatment design and development and thus contribute to a better quality of life and healthy ageing.

The immediate societal impact of the research proposed will be a highly skilled researcher (the PDRA) capable of carrying out top-level research at the biology-chemistry interface. To maximise the impact of our work, we will also engage in outreach activities aimed at inspiring both the general public and the next generation of scientists for research in this field.

2) Economic Impact

As stated in the 2014 governmental budget report "funding pensions and healthcare for an ageing population will create significant cost pressures for the UK as in other countries". Increasing our 'healthspan' (the length of the healthy and active period in our lives) is thus an issue of fundamental importance to our economy and society to which our research will contribute at the fundamental level.

Another burden on our health system is the diagnosis of mitochondrial diseases, a difficult task because symptoms are often similar to a number of better-known diseases. Besides imaging, muscle biopsies have been the most successful in diagnosing mitochondrial disorders, but this invasive method is expensive and still not absolute. Treatment is often ineffective and patients usually have to be closely monitored. Development of effective treatments based on an understanding of the origins of respiratory chain deficiencies will inevitably lead to better and more cost-effective public health services and more tailored advice from charities supporting patients with mitochondrial diseases. In the absence of a clear understanding how the enzyme functions just under 'normal' conditions - the question we seek to answer - effective diagnosis and treatment is like searching for a needle in a haystack.

3) Scientific Impact

In addition to our contributions to understanding the mechanism of complex I, our work will indirectly lead to the development of applications based on proton-coupled electron-transfer reactions. Examples include the development of new catalysts capable of activating oxygen (e.g. for efficient O2 reduction to water in fuel cells) and of splitting water (one avenue to sustainable energy production). Moreover, our work will further the development of electron paramagnetic resonance spectroscopy as an extremely versatile analytical tool in applications ranging from quantum computing to understanding and exploiting processes in biology.