Cytochrome c oxidase: structure, function and malfunction

To live we need a permanent supply of energy. This is provided to our cells by a cascade of reactions that breaks down the food we eat into a universal fuel: the ATP. This process mainly occurs in organelles called mitochondria and is known as cellular respiration. The main machinery that mitochondria use to produce ATP is the respiratory chain. It is composed of four complexes, embedded in the mitochondrial inner membrane, that work together to build up an electrochemical gradient called the proton motive force and which drives ATP synthesis. Most of this gradient is in the form of protons which are pumped across the inner mitochondrial membrane by the respiratory chain complexes.

An increasing number of human pathologies are associated with defects in components of the respiratory chain. In many instances, this is because the malfunction has a direct impact on their primary role in energy production via the proton gradient that they form, or because it leads to an increased production of damaging free radicals. Cytochrome c oxidase (CcO) is the terminal enzyme of our respiratory chain. It transforms the oxygen we breathe into water and greatly contributes to the generation of the proton gradient. Alterations (or mutations) in its structure have been linked with diverse pathologies such as myopathy, therapy-resistant epilepsy, neurological diseases and prostate cancer.

Although the overall chemistry of mitochondrial CcO is fairly well understood, it has proven much more difficult to determine how this produces the essential proton gradient. Various hypotheses were formulated based on the available structures of the enzyme (of which only one is of mitochondrial origin, in this case bovine), but were challenged by mutagenesis work performed on smaller bacterial homologues. Today it appears that the major drawback in understanding the mechanism of mitochondrial CcO, and the effects of human disease-related mutations in particular, is the lack of a system to generate large amounts of purified protein containing defined point mutations.

Remarkably, the CcO that is present in Baker's yeast mitochondria is almost identical to that in human mitochondria. The nuclear and mitochondrial DNAs which encode CcO are both amenable to mutagenesis so alterations can be made in any part of the CcO structure to investigate its function. We have thus engineered a yeast system to allow large-scale production of mutants and will use it to address fundamental questions relative to human mitochondrial CcOs.

At first, we will identify the route taken by the protons to cross the protein structure by measuring CcO's ability to pump protons after alterations have been made in chosen area. We will then use advanced techniques like infrared spectroscopy to look at the concerted movement of atoms within CcO's structure and bring experimental evidences of the mechanism following which protons are being pumped. This should tell us more about the principles that govern and control the complex activity and will be our starting point to investigate how factors or signals external to the reaction centre can, in vivo, regulate CcO's activity. This will be of particular interest to understand how the human CcO has adapted to different energy requirements depending on tissue type. We will aim to obtain a detailed 3D structure of the yeast CcO to confirm our hypotheses. As we unravel the details of CcO's action, we will introduce identified human disease-related mutations in our yeast system in order to investigate the nature of their malfunction. Finally, we will aim at progressively incorporating the human genes or parts of the human enzyme in our yeast system. This will create as even better model for the study of human diseases and the development and testing of new therapies.

The aim of this project is to elucidate the pumping mechanism of CcO and to investigate the effect of allosteric factors and isoforms on catalysis, using, for the first time, a yeast system that allows production and large scale purification of mitochondrial mutant forms of the enzyme. We will also start developing yeast as a platform for the construction of a chimeric yeast/human CcO.

Initial focus will be put on identifying the internal hydrophilic pathway responsible for proton pumping. This will involve accurate measurements of ADP/Oxygen ratios on preparations of intact mitochondria from mutant strains.

We will then address the coupling mechanism using time-resolved FTIR spectroscopy to detect concerted movements of key amino acids and water molecules on chosen reaction steps with the purified CcO. This will be done on photolysis of the fully reduced CO-bound enzyme, comparing the signals recorded for the wild-type and mutant forms and, subsequently, be extended to redox reactions.

We will investigate how long range factors can regulate catalysis. For instance, we will follow by FTIR spectroscopy the structural changes induced by the binding of ADP or ATP to allosteric sites on yeast supernumerary subunits. This will include the design of a range of mutants to mimic post-translational modifications such as phosphorylation.

We will aim to crystallise the yeast CcO and resolve its 3D X-ray structure.

Finally, we will use our yeast system to investigate the effect of human disease-related mutations in CcO. We will use a range of biochemical methods to determine level of expression/stability, turnover number, affinity for its substrate, ability to pump proton and at which stoichiometry. If required, more advanced biophysical techniques including FTIR spectroscopy and fast kinetics recording will be used. Ultimately, as the project develops, we will look at the effect of such alterations on a chimeric yeast/human enzyme.

Who will benefit from this research?
-researchers in biological electron transfer and energy coupling;
-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 CM1306 (Understanding Movement and Mechanism in Molecular Machines) whose main objective is to improve understanding of the dynamics of protein complexes on catalysis by bridging neighbouring scientific fields and fostering applicative outcomes;
-patients and families of patients affected by CcO-related mitochondrial diseases.

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. Moreover, if the behaviour of yeast mutants is as predicted, a common picture for all forms of CcO will have been established, providing a resolution of current CcO mechanism controversies.
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. Its extension into a system that assembles a chimeric yeast/human enzyme will also be of great benefit for patients as it can be used for the development and testing of new therapies.

Timescales. Within the timeframe of the project, we will bring new experimental evidences of the coupling mechanism, establish the function of the H channel region of mitochondrial forms of CcO and provide 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. We will work closely with our clinical collaborator Dr Rahman, involved in collecting such mutational/clinical data to gain a mechanistic understanding of their malfunction. During this project lifetime, and in collaboration with Dr Meunier, we will extend the technology and expertise specifically to create a chimeric yeast/human system. On a longer term, this should lead to a viable platform for the detailed characterisation of the mechanisms underlying CcO-related human pathologies.

Staff development. The PDRA involved in the project will gain expertise with a wide range of biochemical and advanced biophysical methods. Through our collaborations with other laboratories in London (Peter Rich, respiratory complexes), France (Brigitte Meunier, genetics) and Cambridge (Leo Sazanov, crystallography) they will gain additional skills. Through my participation in EU COST Action, funds will be available for exchange between laboratories and myself and the PDRA will apply for COST-funded 'Short Term Scientific Missions' to learn expertise of our collaborators and in particular with the group of Peter Brzezinski (Sweden). 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 for 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.