Deciphering the proton-translocation mechanism of complex I

The way our body produces energy has been puzzling whole generations of scientists for decades. Complex I is the first and the largest enzyme of the respiratory chain and plays a central role in this process, coupling transfer of two electrons from NADH to ubiquinone to the translocation of four protons across the membrane. The L-shaped complex includes two large domains, the hydrophilic arm, which protrudes into the mitochondrial matrix or bacterial cytoplasm, and transports electrons to ubiquinone, and a membrane arm, which pumps four protons across the membrane. The medical importance of complex I is prominent. The vast majority of the mitochondrial diseases, mainly neurodegenerative disorders, have been linked to multiple mutations in genes encoding structural subunits of complex I. These mutations disrupt structural integrity of complex I, which impairs the generation of the proton gradient and consequently generation of ATP. These alterations result also in increased production of the reactive oxygen species, further damaging complex I, mitochondrial membrane, mitochondrial DNA and other proteins. Thus, understanding action of this enzyme is of primary importance as it might lead to the formulation of novel and innovative therapeutic strategies. However, despite decades of extensive structural and biochemical studies, one fundamental issue has remained unresolved, that is, how reduction of ubiquinone enables translocation of protons across the membrane. This is primarily due to the lack of high-resolution structural data, which would unravel conformations of complex I at various stages of the proton translocation cycle. This project aims to change this. Through the work on the proposed project, the researchers from the IST Austria, Dr. Karol Kaszuba and Prof. Leonid Sazanov, will decipher all relevant details of the proton translocation mechanism, relying on atomic structures of complex I, which will be derived for different redox states of the enzyme from single-particle cryo-electron microscopy (cryo-EM) maps. The cryo-EM technique is currently revolutionizing structural biology and it constitutes a preferred method for structural studies of large macromolecular complexes such as complex I. Obtained structures will be subsequently analysed by applying a wide spectrum of computer simulations techniques. If successful, this project will finally provide us with the understanding of the enigmatic process of proton transfer, one of the most intriguing mysteries in the research fields of Bioenergetics and Natural Sciences generally.

The way our body produces energy has been puzzling whole generations of scientists for decades. Complex I is the first and the largest enzyme of the respiratory chain and plays a central role in this process, coupling transfer of two electrons from NADH to ubiquinone to the translocation of four protons across the membrane. The L-shaped complex includes two large domains, the hydrophilic arm, which protrudes into the mitochondrial matrix or bacterial cytoplasm, and transports electrons to ubiquinone, and a membrane arm, which pumps four protons across the membrane. The medical importance of complex I is prominent. The vast majority of the mitochondrial diseases, mainly neurodegenerative disorders, have been linked to multiple mutations in genes encoding structural subunits of complex I. These mutations disrupt structural integrity of complex I, which impairs the generation of the proton gradient and consequently generation of ATP. These alterations result also in increased production of the reactive oxygen species, further damaging complex I, mitochondrial membrane, mitochondrial DNA and other proteins. Thus, understanding action of this enzyme is of primary importance as it might lead to the formulation of novel and innovative therapeutic strategies. However, despite decades of extensive structural and biochemical studies, one fundamental issue has remained unresolved, that is, how reduction of ubiquinone enables translocation of protons across the membrane. This is primarily due to the lack of high-resolution structural data, which would unravel conformations of complex I at various stages of the proton translocation cycle. This project has changed this. In this project I built different conformations of bacterial complex I, which were obtained from the single-particle cryo-electron microscopy (cryo-EM) and studied these conformations by computer simulations, which allowed me to establish a correlation between structural changes within binding site of a complex (induced by binding of ubiquinone) and structural changes within its membrane, proton-transferring region. An existence of such correlation, has been long speculated, but never demonstrated. My work also allowed me to propose a mechanism, by which this correlation operates. The results of simulations clearly show that placement of charge on amino acids residues, which interact with ubiquinone, as it has been speculated before, plays a dominant role in introducing structural changes in the region of membrane domain. Presumably, changes, which would enable proton transfer. In summary, this work has substantially strengthened a hypothesis about a central role of ubiquinone in proton transfer, as it showed that the reduction of ubiquionone can lead to changes in membrane domain of a complex and that these changes likely propagate via the charge-driven mechanism. Thus, this project substantially contributed to the understanding of the enigmatic process of proton transfer, one of the most intriguing mysteries in the research fields of Bioenergetics and Natural Sciences generally.