Our bodies derive most of their useable energy from the controlled burning (oxidation) of fats, proteins and carbohydrates by the gas, oxygen. Oxygen delivery to organs (heart, brain etc.) is therefore vital for health, growth and development. Problems with oxygen delivery or consumption are responsible for the bad effects of many disease processes e.g. brain damage after a stroke, heart attacks etc. Nearly all (>95%) of the oxygen in the body is consumed by a small organ (organelle) found inside all cells. This is called the mitochondrion. Inside the mitochondrion one protein catalyst (enzyme) called cytochrome c oxidase (CCO) consumes all the oxygen; the oxygen is converted into water, generating a voltage across the mitochondrion that eventually is used to make a molecule called adenosine triphosphate (ATP); ATP is the universal energy currency of all cells and drives muscle movement, brain signalling, growth, development, tissue repair etc. CCO contains a number of coloured iron and copper centres that, uniquely, make it possible to detect it in intact humans e.g. in the brain during 'thinking' or in the muscle during exercise. The purpose of this grant is to study how CCO is controlled in the body, in particular in order to improve our understanding of these signals that we can measure non-invasively. This is a complex problem that we will address by using a combination of in vitro (test tube) experiments and mathematical modelling. It utilises the expertises of biology, physics, mathematics, biochemistry and cell biology. We will first optimise the measurement of the different coloured signals in the test tube. We will then look at how these signals are controlled in vitro. From this our ultimate aim is to develop a dynamic mathematical model that will demonstrate how it might be controlled in the whole body (in vivo). We will focus in this project on one organ (the brain) for two reasons; the brain is critically dependent on oxygen for survival and there is a lot of in vivo data about its oxygen consumption and delivery to the brain. A key part of our understanding of this complex system is to understand how it works at a range of levels. We will therefore develop models of how oxygen is consumed in CCO on its own, CCO within the mitochondrion and CCO in the whole brain. We will use appropriate experiments at each level of organisation to define and test how the model works. In particular we will ask colleagues from around the world to analyse our model critically, both with respect to their data and their own theories. The ultimate aim will be to understand how this complex biological system works at both a 'reductionist' molecular and more 'holistic' organ level. As well as being of interest for its own sake, an improved description of this system is likely to have significance for healthcare and industry (in particular for people manufacturing machines that measure parameters relating to oxygen and energetics in the body.

Technical Abstract:
Mitochondrial oxygen consumption is the dominant energy transduction pathway in mammalian systems. The control of this system is complex and integrates with other pathways at both biochemical and physiological levels. There is lot of knowledge about the mitochondrial electron transfer system at the level of mechanistic enzymology, but the detailed regulation of electron flow within the enzymes is still a matter of live debate. The purpose of this grant is to model the regulation of this complex energy transduction pathway at multiple levels / the individual enzyme (cytochrome c oxidase, CCO), the organelle (mitochondrion) and the organ (brain). The collection of new in vitro steady state date will be integrated with model building with experiment and model feeding off each other. A particular interest will be how to make optimal use of the data generated via non-invasive monitoring of haemoglobin and CCO in the brain by near infrared (NIR) techniques. First we will optimise the deconvolution of the in vitro spectra of the relevant CCO chromophores that are detectable by visible and NIR spectroscopy. This is required for the measurements of intermediate concentrations in the in vitro and in vivo steady state. We will then perform a detailed study of the factors regulating CCO intermediates in the purified enzyme, artificial proteoliposomes, rat brain mitochondria and cells, enabling us to develop a complete steady state kinetic model of CCO . This will then be incorporated into a kinetic model of mitochondrial energy metabolism. Finally the revised mitochondrial model will be incorporated into an ongoing model of brain blood flow, autoregulation and metabolism. This will allow current in vivo measurements of CCO redox state to be accessible to this model, assisting with both the in vivo testing of the model and its relevance to accessible real time measurements in animal studies and human volunteers and patients.