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Fourier Transform Infrared Spectroscopy

Flow-flash FTIR
The setup for FTIR flow-flash, i.e. for time-resolved measurements of oxygen reaction on cytochrome c oxidase, consists of an FTIR spectrometer equipped with a silicon ATR-prism1.

Scheme of the home-made ATR-chamber for FTIR flow-flash

Scheme of the home-made ATR-chamber for FTIR flow-flash

The needle of the syringe with oxygen-saturated buffer is directed to the center of the protein film at ~0.5 mm distance to produce a high local concentration of O2 for the time of the reaction. Injection of O2-saturated buffer by a syringe pump is controlled by a timing board. The reaction of oxygen with fully-reduced enzyme inhibited with CO is initiated by a laser pulse that is delivered to the CcO film with a laser guide after O2-buffer injection.

Scheme of setup for FTIR flow-flash

Scheme of setup for FTIR flow-flash

The laser pulse was controlled through the timing board. Control of redox state of CcO and measurement kinetics of absorbance changes in the visible range are achieved by the spectrophotometer HR2000+ equipped with the light-guide which is positioned on a distance of ~2 mm from the enzyme surface.

Setup for FTIR flow-flash in real-life based on IFS 66/s FTIR spectrometer

Setup for FTIR flow-flash in real-life based on IFS 66/s FTIR spectrometer

After measurement of the oxygen reaction, enzyme re-reduction is accelerated by the buffer flow from the vacuumized bottle through the ATR-chamber. For this purpose, inlet and outlet of the ATR chamber are connected to a reservoir filled with the buffer. The air in the reservoir is exchanged with 100% CO by a home-made vacuum/gas line. Pumping of the buffer is accomplished by the flow pump. The internal volume of the ATR-chamber is ~1 mL. Oxygen injection, laser pulse, flow pump and FTIR spectrometer are operated in an automatic mode by the timing board through TTL pulses.

Time-resolved FTIR spectra

Time-resolved FTIR spectra measured with help of FTIR flow-flash setup on Asp124Asn mutant CcO in which fast steps of oxygen reaction proceed till ferryl compound and its relaxation to fully-oxidized compound is slowed down. The mutation allows resolve FTIR spectra of both compounds. The time-resolved FTIR spectrum of appearance of ferryl compound (relative to fully-reduced CO inhibited state) is shown in red and its relaxation to fully-oxidized compound - in black (A). The summed FRCO->O spectrum is in green (B).


The kinetics of oxygen reaction of Asp124Asn mutant cytochrome c oxidase at specific wavenumbers

The kinetics of oxygen reaction of Asp124Asn mutant CcO at specific wavenumbers. The kinetics correspond to different redox centers and aminoacids that take part in the reaction.


References
1. Gorbikova EA, Belevich NP, Wikstrom M, Verkhovsky MI. Time-resolved ATR-FTIR spectroscopy of the oxygen reaction in the D124N mutant of cytochrome c oxidase from Paracoccus denitrificans. // Biochemistry, 2007, 46(45), 13141-8.


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