Mitochondria are the major energy providers of our cells. They have a chain of proteins - the mitochondrial respiratory chain - embedded in their inner membrane that oxidise products derived from the metabolism of food and reduce oxygen to water. Cytochrome c oxidase (CcO) is the final component of this chain that catalyses the oxygen reduction reaction; in fact, it is this enzyme that is responsible for consumption of most of the oxygen that we breathe and it provides a significant fraction of our energy needs. Energy is released during its reaction with oxygen and CcO is able to trap it by creating a charge difference across the mitochondrial membrane in which it is located - rather equivalent to charging of a capacitor in electronics. This charge gradient is then be used by another enzyme, called ATP synthase, to make ATP. ATP is a stable chemical that can release energy when it is hydrolysed; it diffuses around the cell and supplies a large part of energy needed by the myriad of reactions required for healthy functioning of our cells and tissues.

We already know a great deal about the atomic structure of the enzyme and the way in which it safely reacts with oxygen to release energy. We also have a very good idea of the general way that it uses the energy to make the charge gradient. However, in order to elucidate the specific details of the coupling mechanism we have had to rely heavily on studies of simpler bacterial forms of the enzyme, particularly aided by the ability to change individual parts of these bacterial enzymes by introducing mutations. However, such bacterial enzyme studies cannot address all aspects of how our own human, more complicated, CcO works. The structures of several different types of bacterial CcOs are known but, to date, the only mitochondrial CcO structure that has been resolved is that from cow hearts. Although all forms of CcO share a similar core structure around the region where oxygen in reduced, other structural differences between bacterial and mammalian CcOs have led to suggestions that the energy storage reactions are different from those seen in bacterial CcOs.. An even more striking difference is that mitochondrial CcOs have up to 10 additional subunits, none of which are found in bacterial CcOs and to date we have little understanding of their functions. There is also good evidence that, in contrast to bacterial CcOs, those in more complex organisms can occur in different forms and are regulated differently in the cellular environments of different tissues. It is important to understand these aspects because a range of human genetic diseases arise from malfunctioning of CcO. Unfortunately, studies of these uniquely mitochondrial CcO questions are severely hampered by the lack of a good system to introduce mutations.

Remarkably, the CcO that is present in mitochondria of Baker's yeast cells is structurally extremely similar to human CcO mitochondria in terms of both its core structure and the structures of all of its additional subunits. Because of this, and because we can make alterations in parts of yeast CcO in the same way as has been done successfully with bacterial CcOs, we can now investigate just how these mitochondrial (and therefore human) CcOs function and can be controlled. The understanding of the unique aspects of the more complex mitochondrial enzymes will provide a platform for testing and understanding how the human enzyme functions and malfunctions in health and disease.

Technical Abstract:
The project addresses whether the mitochondrial CcOs have a different coupling mechanism to the better studied bacterial CcOs and whether the mitochondrial CcO core reactions can be influenced allosterically by additional supernumerary subunits.

Resolutions of these issues have been hampered by the lack of a system to produce purified mutant forms of mitochondrial CcO in good yields. We now have a system to produce such mutants in his-tagged form and to purify the mutant CcOs. To date, 14 mutants have been produced and WT and two mutant CcOs have been purified. Simple whole cell tests of all mutant strains have established that the H channel region is functionally important. The mutation system will be expanded to produce more core subunit mutations, switching of isoforms of yeast subunit 5 (implicated in allosteric control and proposed to act by modulation of the core H channel properties), generation of supernumerary subunit 2nd site reversions to highlight key intersubunit interactions and production of other mutations/domain changes of supernumerary subunits.

Initial fast simple screening will be based on cell growth with respiratory substrate and spectroscopic estimation of levels of expressed CcO in whole cells. This will establish which mutations result in impairment of function versus poor expression (to date, almost all mutant CcOs have been found to be expressed at near WT levels).

CcO will be purified by our standardised technique (DDM solubilisation, Ni- and DEAE Sepharose CL-6B column chromatographies) from many of the mutant stains that show normal levels of expression of impaired CcO.

A range of biochemical and biophysical tests will be performed to establish enzyme integrity, classical enzyme catalytic constants, efficiency of coupling of oxygen chemistry to proton translocation, major sites of impairment and influence of mutation, loss or gain of control function by other external factors.

Potential Impact:
1 Who will benefit from this research?
- researchers in biological electron transfer and energy coupling;

- the UCL Consortium for Mitochondrial Research that brings together basic and clinical scientists across the UCL campus. This Consortium recognises the fundamental role of mitochondria in the wellbeing of cells, tissues and the organism and their major role in major diseases. Its broad goal is to generate innovative experimental approaches and applications to illuminate major questions from fundamental mechanisms of mitochondrial biology and bioenergetics to understanding the role of mitochondria in disease;

- UK and worldwide Research Centres (e.g. the Mitochondrial Biology Unit in Cambridge, The Mitochondrial Centre, Newcastle, the Mitochondrial Research & Innovation Group, Rochester, USA) and medical charities and public domain sites (e.g. The United Mitochondrial Disease Foundation) devoted to mitochondrial research and its wider implications;

- members of the EU Cost Action CM0902 (Molecular Machineries for Ion Translocation Across Biomembranes), of which I am UK representative, whose main objective is to improve understanding of proton and metal ion translocation across bio-membranes by bridging neighbouring scientific fields and fostering applicative outcomes.

2 How will they benefit from this research?
Basic researchers in related areas will benefit from the mechanistic understanding and structures important for energy coupling that should find applicability in other biological redox systems. Members of the more diverse organisations above will be particularly interested in our development of this yeast CcO system as a platform for understanding mechanism and control of human mitochondrial CcO function and its malfunction in diseases. This yeast system will provide the only currently viable genetic system to test effects of known mutations of human mitochondrial CcO, using an enzyme that closely resembles its structure in all key aspects.

3 Timescales
Within the timeframe of the project, we will establish the function of the H channel region of mitochondrial forms of CcO and provided insights into how such enzymes are modulated by environmental factors. This yeast system is already usable to test effects of known human CcO mutations that have been linked to disease states. Indeed, we have already been contacted by clinical colleagues involved in collecting such mutational/clinical data (Dr. S. Rahman and colleagues, Inst. Child Health, UCL) who have an interest in our pursuing this route. We have therefore agreed to run exploratory work during this project lifetime with a view, in collaboration with Dr. Meunier and clinical colleagues, to securing EU or other international funding to extend the technology and expertise specifically to define effects of selected CcO mutations been implicated in human mitochondrial diseases.

4 Staff Development
The researchers involved in the project will gain expertise on-site with a wide range of biochemical and advanced biophysical methods. Through our collaborations with other laboratories in France (Meunier, this project), Sweden (Brzezinski, Swedish funding) and Cambridge (Walker, Cambridge, crystallography, PhD project) they will gain additional skills. Through my participation in EU COST Action CM0902, funds are available for exchange between my laboratory and those of collaborators and I will encourage the PDRA (and PhD students) to make COST-funded 'Short Term Scientific Missions' to learn expertise of our collaborators. The research skills that will be gained during this project are applicable to other major enzymes, including several of medical interest. In addition, professional skills that staff will develop will be applicable in other employment sectors foe example ability to multi-task, work in a team and communicate with scientists and non-scientists. Hence, the project will provide skills for onward employment and career development.