Functional Proton-Electron Transfer Elements in Biological Energy Conversion

Project Information

bioPCET

Grant agreement ID: 715311

DOI

10.3030/715311

Project closed

EC signature date 1 December 2016

Start date 1 February 2017

End date 31 January 2023

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 494 368,00

EU contribution

€ 1 494 368,00

Coordinated by

STOCKHOLMS UNIVERSITET
Sweden

Periodic Reporting for period 4 - bioPCET (Functional Proton-Electron Transfer Elements in Biological Energy Conversion)

Reporting period: 2021-02-01 to 2023-01-31

Summary of the context and overall objectives of the project

The ERC project, bioPCET, focuses on understanding molecular principles of how biological systems capture and covert energy. These processes are catalyzed by coupled transfer of protons and electrons (PCET), i.e. two elementary particles, but their fundamental mechanistic principles are still not well understood. The objective of our project is to obtain a molecular-level understanding of how functional elements in proteins power biological energy conversion. This we aim to achieve by studying the catalytic machinery of one of the largest and most intricate enzymes in mitochondria and bacteria, the respiratory complex I. As a main target, we aim to control the functional modules of complex I that we probe by molecular simulation techniques, de novo-protein design, and biophysical studies. The project aims to establish a fundamental understanding of enzyme function that is important for elucidating the molecular basis of life. The information gained can guide the design of novel man-made enzymes for sustainable energy technology, whereas our research on cell respiration is significant for understanding development of disease related to energy metabolism in the cell.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

This project is divided in five workpackages and a methodological subproject that focuses on elucidating how different functional modules in complex I establish a 300 Å charge transport process. The project resulted in 30 peer-reviewed publications, and several manuscripts that are in review or preparation during the finalizing of the project. Many of the publications are published in high-impact journal (JACS, PNAS, Science Adv, Nature Comms) supporting that our project team has secured a high level of research. The work has been presented in multiple leading conferences in the field and communicated in review articles to reach both experts and non-expert audiences. A brief summary of each workpackage (WP) is given below.

In WP1, we have studied functional elements that are used for conversion of chemical energy to redox energy. We have studied how electrons enter the complex I with the redox cofactor NADH, and how this module further bifurcates the electrons into a chain of FeS redox-cofactors. We identified protein residues that are responsible for the catalytic activity and how certain point mutations may enhance the production of reactive oxygen species (ROS), linked to the development of mitochondrial disease. In WP2 and WP3, we have studied functional elements that mediate long-range electron transfer and establish the catalytic core of complex I. We have identified electronic and structural changes linked to the electron transfer process as well as its energetics along the FeS chain. Moreover, we have established the function of key elements that enables complex I to reduce the quinone substrates, and conformational changes linked to this process. In WP4, we have studied elements mediating redox energy for proton pumping, and identified a novel substrate binding-site that may activate the proton pumping activity of the enzyme. In WP5, we have studied how different protein modules establishing the long-range proton-pumping machinery, and how the biological membrane surroundings modulate the biological activity. During the project, we have developed computational simulations methods, linked with protein design and biophysical experiments, that has enabled us to probe the structure and function of the catalytic machinery in complex I on a broad range of time-scales and spatial resolutions.

During the project, we identified functional elements of the Complex I machinery that are responsible for the long-range proton transport process (PNAS 118:e2019498118; PNAS 114:E6314; JACS 142:13718; JACS 142:21758). Based on these, we suggested key principles by which Complex I enables the long-range energy transduction process by coupled conformational and hydration changes, triggering protonation reactions that propagate across the complete (>200 Å) membrane domain (PNAS 114:E6314; JACS 142:13718). We could also resolve how the active site of Complex I reduces the quinone substrates (PNAS 112:11571; PNAS 114:12737; JACS 139:16282), how the enzyme oxidizes biological electron carriers such as NADH (JACS 141:5710), we identified novel substrate binding sites (PNAS 115:E8413; Nat Commun 11:5261), and determined how the lipid environment affects its functional dynamics (Science Adv 5:eaav1850). Moreover, our work unraveled how modular adaptations of the Complex I architecture lead to new functionality that support photosynthetic energy conversion, including the concentration of CO2 in cyanobacteria (Nat Commun 11:494), production of hydrogen gas (H2) in archaea (JACS 143:20873), as well as the electron bifurcation in acetogenic bacteria living in extreme conditions (JACS 145:5696). Despite significant structural differences to other energy transducing enzymes, our studies also allude to conserved mechanistic principles that could be generally employed in biological energy conversion (Acc Chem Res 54:4462). The integrated computational-experimental approaches that we developed to address these mechanistic questions, allowed us to further design artificial proteins as frameworks for testing biological hypotheses (Nat Commun 12:1895), without the "evolutionary baggage" of natural proteins. These methods will be used in future work to address challenging mechanistic questions in biology. The ERC project resulted in several articles published in top journals (Nature Comm, PNAS, Sci. Adv., JACS). The work was disseminated in public talks in conferences, invited talks at various universities, as well as to the general public.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

To probe the connection between protein structure, function, and dynamics, we have developed and applied computational multi-scale methodology that ranges from quantum chemical techniques to large-scale classical approaches that has enabled us to probe the structure, energetics, and dynamics of complex biochemical processes on fs-ms (10-15-10-3 s) timescales for systems comprising up to millions of atoms. Such broad range of methods are normally not applicable for large enzyme complexes. The computational methods have been further combined with protein design and biophysical experiments that have allowed us to validate some of the predicted mechanistic models. During the second half of the project, we aim to further enhance the complementarity between computational-, design-, and biophysical experimental parts.

Several breakthroughs in our project go beyond state of the art in the field. Before the start of the project, there was very limited understanding of the mechanistic principles of the long-range charge transfer reactions, including also limited structural information of the studied system. Our work resulted in several mechanistic proposals of the molecular principles. This work has provided a basis for the current mechanistic discussion in the field, and guiding principles for ongoing experimental work. The project also led to methodological breakthroughs, enabling multi-scale simulations on highly complex biological systems that had not been described before, as well as the development of new integrative computational-experimental approaches that are likely to have a general impact on the field.

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