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Methods of the research

Methodological Approaches
The typical rate of oxygen reduction by cytochrome c oxidase (CcO) under steady-state conditions is extremely fast (1 dioxygen per 5-10 ms), which makes it impossible to study CcO by means of conventional steady-state techniques. In order to overcome this limitation we apply the transient kinetics approach to study the enzymes of the respiratory chain. This approach is based these main factors:
  • fast time resolution of the measuring system from nano- to milliseconds
  • synchronous start of the reaction for all molecules in the system
  • faster, than the reaction under investigation, supply of the substrates

Basic transient kinetics techniques
  • the flow-flash method - is a combination of the conventional stopped-flow technique with laser-induced initiation of the reaction. In this method, instead of initiating the reaction by the actual mixing of the fully-reduced CcO with dioxygen, the enzyme is first allowed to react with CO, which traps the enzyme in the fully-reduced state. After that the CO-bound oxidase is mixed in a stopped-flow apparatus with an oxygen-containing buffer. Under these conditions, the reaction of dioxygen with the CO-bound oxidase is limited by the CO-dissociation rate (ca. 0.02 s-1). At the same time CO can be photolyzed away by a laser flash to initiate reaction of the oxidase with oxygen.
  • Electron injecton - is the initiation of the reaction by the injection of a single electron into the enzyme. The electron for injection is supplied by photoactivated dye, such as (2,2-bipyridyl) ruthenium (RubiPy). A laser flash promotes the molecule of RubiPy to an excited state with an Em of about -1.5 V, which can then donate an electron to CuA in less than 0.5 ms.
  • Electron backflow - CO binding to the oxidase is used to maintain the apparent midpoint potentials of heme a3 and CuB at high level. The bound molecule of CO can then be dissociated by a laser flash causing the midpoint potentials of heme a3 and CuB to drop, and allowing electron redistribution among all redox centers according to the new redox equilibrium.
Each of the aforementioned reactions can be monitored by a number of different time-resolved detection techniques such as optical absorption spectroscopy, potentiometric electrometry, or even by time-resolved FTIR.





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