The main producer of energy in the living cells is a respiratory chain located in the inner membrane of the mitochondria. The respiratory chain consists of sequentially connected macromolecular complexes (energy generators), which transform the chemical energy of redox reactions into delocalized transmembrane electro-chemical potential.
Respiratory chain of mitochondria

Terminal oxidase are the final energy generators in the respiratory chain. They catalyze the final step of respiration - reduction of molecular oxygen to water. The energy released in this reaction is conserved in the form of a transmembrane electrochemical gradient of protons across the membrane, which is mostly used by the ATP synthase (complex V) for formation of ATP.

In contrast to eukaryotes, where only one type of terminal oxidases (aa3-type cytochrome c oxidase) is present, the respiratory chains in bacteria can vary extensively and have multiple types of terminal oxidases. The main function of such branching is to provide bacteria with better elasticity in a variety of environmental growth conditions.

Scheme of Respiratory Chains of mitochondria and bacteria

Our group uses biophysical methods to investigate the molecular mechanism of proton translocation by terminal oxidases. To understand the mechanism of proton translocation, it is imperative to identify those partial reactions in the catalytic cycle that are coupled to this process. One of the most promising approaches is to track the events in the enzyme in real time during single enzyme turnover.

For monitoring the sequential appearance of the catalytic intermediates, we use transient optical spectroscopy. To follow proton translocation during catalytic turnover, we have developed an electrometric method, which allows measurements of proton translocation across the membrane in real time. The last development was the building of an ultra-fast freeze-quench system, which enables us to stop enzyme catalysis by freezing in a time scale from tens of microseconds to tens of milliseconds. The frozen sample can then be analyzed by EPR or optical spectroscopy. Application of these methods has let us estimate the intermediates of the catalytic cycle and establish the time of their formation and decay. So far, we have identified all four intermediates of the catalytic cycle, the reduction of which results in single proton translocation.

To verify the most important general molecular mechanisms providing coupling between electron transfer reactions and ion translocation, we have started a comparative study of the proton-translocating cytochrome c oxidase and the sodium-translocating NADH:quinone oxidoreductase (Complex I): two very different enzymes but essentially accomplishing the same function – the redox driven transmembrane transport of ions. The goal of the project is to understand the molecular mechanism of ion translocation by the protein energy generators.

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