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

摘要

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?1as an IR white light source equipped with multi-channel MCT detector. This facility equipped with O2supply system enables the sub-millisecond time scale infrared measurements of the O2reduction coupled with proton pumping by bovine cytochromecoxidase (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?1assignable to protonation of a carboxyl group coupled with a single electron transfer to the O2reduction 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?1and 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.