Satoru Nakashima

Infrared and Raman spectroscopic investigation of the reaction mechanism of cytochrome c oxidase

Recent progress in studies on the proton-pumping and O₂reduction mechanisms of cytochrome c oxidase (CcO) elucidated by infrared (IR) and resonance Raman (rR) spectroscopy, is reviewed. CcO is the terminal enzyme of the respiratory chain and its O₂reduction reaction is coupled with H⁺ pumping activity across the inner mitochondrial membrane. The former is catalyzed by heme a3 and its mechanism has been determined using a rR technique, while the latter used the protein moiety and has been investigated with an IR technique. The number of H⁺ relative to e⁻ transferred in the reaction is 1:1, and their coupling is presumably performed by heme a and nearby residues. To perform this function, different parts of the protein need to cooperate with each other spontaneously and sequentially. It is the purpose of this article to describe the structural details on the coupling on the basis of the vibrational spectra of certain specified residues and chromophores involved in the reaction. Recent developments in time-resolved IR and Raman technology concomitant with protein manipulation methods have yielded profound insights into such structural changes. In particular, the new IR techniques that yielded the breakthrough are reviewed and assessed in detail. This article is part of a Special Issue entitled: Vibrational spectroscopies and bioenergetic systems.

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 (50u202fμm) flow cell equipped in a highly sensitive IR spectrometer constructed with the femtosecond mid-IR pulse laser with spectral width of 350u202fcm-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 (1725-1770u202fcm-1) region with the accuracy of about 10u202fμO.D. under the background O.D. of 1. The facility identifies a band intensity change at ~1744u202fcm-1 assignable to protonation of a carboxyl group coupled with a single electron transfer to the O2 reduction center within 1u202fms 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 (210u202fkDa). The present facility sensitively identifies also polarity changes in COOH group by detecting shifts of the bands near 1750u202fcm-1 and 1760u202fcm-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.