MRC Transition Support CDA Amandine MARECHAL

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: 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 five 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 have been formulated based on the available structures of the enzyme 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.

We have made significant progress over the past four years. We have identified the route taken by the protons to cross the protein structure and have found an ideally uncoupled mutation with which to study the important pumping mechanism. We have also determined the 3D structure of the yeast CcO at the highest resolution which has confirmed its similarity to human and revealed important details of its interaction with other respiratory proteins. The latter might be responsible for changes in CcO activity in the context of health and disease and we have developed the tools to investigate this further. These achievements will guide our efforts to progressively incorporate the human genes in our yeast system to create an even better model for the study of human diseases and the development and testing of new therapies.

Given more time and funding continuity, we will complete our research programme and make several significant contributions to the field of mitochondria and metabolism. This will allow me to build a solid track record, establish an international reputation and reach my full potential in support of future applications for a senior fellowship or programme grant to grow my laboratory within the competitive scientific environment. I have given my best to reach this stage but have also suffered significant delays while going through some very difficult times and I still have to make the most of my CDA. The Transition support would enable me to achieve exactly this.

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 a yeast system that allows production and large scale purification of mitochondrial mutant forms of the enzyme. We will also progress the construction of a chimeric yeast/human CcO as a platform to investigate human diseases.

Having identified the internal hydrophilic pathway responsible for proton pumping, we will now 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 purified CcOs. 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 from preparation of CcO-containing respiratory supercomplexes, purified from strains in which CcO isoform expression is tightly controlled.
We will complete our structural biology investigations on the two CcO isoforms to reveal the molecular details responsible for the change in catalytic activity observed when the yeast is grown in normoxic and hypoxic conditions.

Finally, we will look at the effect of human disease-related mutations in a chimeric yeast/human enzyme CcO. We will use our range of now established biochemical methods to determine level of expression/stability, turnover number, affinity for substrate and efficiency to pump protons. If required, more advanced biophysical techniques including FTIR spectroscopy, fast kinetics and cryo-electron microscopy will be used.

The impact summary detailed in my original CDA will apply to the Transition fellowship.