Molecular mechanism of proton pumping by complex I: A single enzyme study

Mitochondria are the powerhouses of the cells in our body. Complex I is an enzyme complex that plays a crucial role in this process. It uses the energy obtained from chemical reactions to pump positively charged protons across a 5nm thin membrane. This process is not unlike the charging of a battery. Complex I does not always function under optimal conditions and is known to be source of radical oxygen species, which is believed to have important roles in aging. Furthermore, malfunction of complex I has important consequences for (human) health.

We like to know how complex I pumps protons across the membrane. Many studies have already been performed in the past, but the pumping mechanism is still unclear and although hypotheses have been formulised, none of these have been experimentally proven. All studies in the past have looked at large ensembles of complex I and form a picture of how these complexes function on average. However, we think that a better understanding of the pumping mechanism can be obtained if we look at these complexes one by one.

We have recently developed new experimental methods that are so sensitive they can detect proton pumping by single complexes. In this proposal we want to apply these experiments to study complex I. The results will give us a better understanding on how these complexes function in our mitochondria and what happens when they do not work optimally. The questions we aim to address are of fundamental, 'blue-sky' nature, but on the long term we hope that deeper understanding of the processes that generate energy in our body will help us to tackle diseases often related with aging, including Alzheimer's disease and cancer.

Complex I (NADH-ubiquinone oxidoreductase) is the first enzyme in the mitochondrial electron-transfer chain and plays a crucial role in bioenergetics, where it couples the oxidation of NADH to proton transfer across the mitochondrial inner membrane, contributing to the proton motive force (pmf) for ATP production. Complex I is a known source of radical oxygen species (ROS) and its malfunctions have important consequences for (human) health and aging. Structural information from both crystallography and cryo-EM has been used to formulate several models on how complex I pumps protons, but none of them have been experimentally verified. In its deactive state, formed under hypoxic conditions, bovine complex I has been shown to exhibit Na+-H+ antiport activity with potential physiological effects during ischaemia-reperfusion. Here, we propose to follow proton transfer by complex I and as a function of the transmembrane pH gradient and the Na+ concentration. Our results will be compared with the predictions of candidate mechanistic models to gain insight into the molecular mechanism of proton pumping.

In addition, we have recently developed a single-enzyme methodology for proton pumping enzymes and reported, for the first time, that two very different classes of proton pumping complex (a heme-copper oxidase and a P-type ATPase) display long-lived leak states. When the complexes adopt these leak states, protons rapidly and freely flow backwards dissipating the pmf energy gradient and we propose that this could help regulate the pmf in bacteria and/or mitochondria. Here, we aim to capitalise on our recently developed methodology to directly observe the abundance, occupancy and activity of the functional states of complex I. If long-lived leak states are detected for complex I, which is unrelated to the above proton pumps, they may be considered a fundamental property of proton pumping enzymes.

SKILLS TRAINING:
There are excellent opportunities for professional development and discipline hopping for the PDRA and PIs engaged in this collaborative, cross-disciplinary research programme where the frequent and effective exchange of materials, methods (protein handling and characterisation, spectroscopy, electrochemistry and lipid handling) and analysed data will be key to success. The University of Leeds (UL) will develop skills in (i) handling and characterisation of membrane- and metallo-proteins, (ii) modification and characterisation of gold and SiO2 surfaces, (iii) electro-chemical characterisation of protein modified electrodes, (iv) fluorescent imaging of single liposomes and (v) data treatment of large datasets to determine statistical relevance of features in single-enzyme data. The Mitochondrial Biology Unit (MBU) will offer skill development in membrane protein purification, in particular complex I, and in the reconstitution of complex I in proteoliposomes. Additional opportunities for the PDRAs to enhance their (transferable) skills in line with the Researcher Development Framework will be provided at UL.

PUBLIC ENGAGEMENT:
Increased public understanding of how science contributes to our understanding of 'How proteins work' and how they function in mitochondria is an important aspect of our programme. Several events, open to the general public and visited by adults and children, will be developed by the PDRAs and PIs to incorporate the motivations for, and results from, the proposed research on "microbial electricity".
Throughout the project the PDRA and PIs will also take the opportunity to present aspects of this research through talks to the general public. They will be alert to opportunities to contribute articles in popular science magazines and position papers from professional societies. The PIs and PDRAs will also ensure that throughout the project their novel results are made available on the respective institutes' and group websites (after protection of IP and publication), and that they are available to give interviews to newspapers and magazines. Advice on disseminating information will be provided by the institutes' press offices.

In the first or second year, we will commission a 'science meets art' outreach activity in collaboration with The Superposition (http://www.thesuperposition.org/, and the daughter activity BioLeeds, https://bioleeds.wordpress.com/), a network and space for artists, scientists and makers in the Leeds area. The PI, Jeuken, has an existing track record of collaboration with bioLeeds. The commission brief will be on "Energising Membranes" to capture the concept of how charge transfer plays a crucial role in bioenergetics. We plan to exhibit the artwork at science festivals in the Yorkshire region, in the first instance, while using connections within the Superposition to display it at other art festivals/exhibitions in the UK. This will help engage a section of the public that would not traditionally attend science outreach events, particularly the over 25s, who are known to be a difficult demographic to reach with science outreach.

COMMERCIALISATION AND IP PROTECTION:
This proposal describes a project of fundamental nature and the proposed outcomes are not of direct commercial value. However, the IP position of the research, in particular the technology development aspect, will be monitored throughout the project and the understanding of the IP position will be continuously evaluated. Future research, beyond the lifetime of this project, might apply knowledge obtained during this project in the pharmaceutical industry and, if identified, commercially valuable IP will thus be actively protected, through filing and licensing patents, prior to presentation of results at international meetings, in peer-reviewed papers or as popular science reports. The PIs will be alert to opportunities for spin-off or joint venture opportunities.