Performance of a time-resolved IR facility for assessment of protonation states and polarity changes in carboxyl groups in a large membrane protein,mammalian cytochrome c oxidase,under turnover conditions in a sub-millisecond time resolution

Time-resolved IR analyses for the protonation and polarity changes of carboxyl groups involved in proton pump enzymes under turnover conditions are indispensable for elucidation of their proton-pump mechanisms. We have developed a new time-resolved infrared facility by introducing a flow system for transferring highly concentrated and thus viscous protein solution to a thin (50?μm) flow cell equipped in a highly sensitive IR spectrometer constructed with the femtosecond mid-IR pulse laser with spectral width of 350?cm^?1 as an IR white light source equipped with multi-channel MCT detector. This facility equipped with O₂ supply system enables the sub-millisecond time scale infrared measurements of the O₂ reduction coupled with proton pumping by bovine cytochrome c oxidase (CcO) initiated by CO-flash photolysis in the COOH (1725–1770?cm^?1) region with the accuracy of about 10?μO.D. under the background O.D. of 1. The facility identifies a band intensity change at ~1744?cm^?1 assignable to protonation of a carboxyl group coupled with a single electron transfer to the O₂ reduction center within 1?ms after initiation of the reaction. The results suggest that the facility detects protonation of a single carboxyl group included in large proteins like as CcO (210?kDa). The present facility sensitively identifies also polarity changes in COOH group by detecting shifts of the bands near 1750?cm^?1 and 1760?cm^?1, without significant intensity changes. These findings show the performance of this facility sufficiently high for providing crucial information for understanding the proton transferring mechanisms of protein carboxyl groups.