Time-resolved Fourier transform infrared spectroscopy of the polarizable proton continua and the proton pump mechanism of bacteriorhodopsin

Nanosecond-to-microsecond time-resolved Fourier transform infrared (FTIR) s pectroscopy in the 3000-1000-cm(-1) region has been used to examine the pol arizable proton continua observed in bacteriorhodopsin (bR) during its phot ocycle. The difference in the transient FTIR spectra in the time domain bet ween 20 ns and 1 ms shows a broad absorption continuum band in the 2100-180 0-cm(-1) region, a bleach continuum band in the 2500-2150-cm(-1) region, an d a bleach continuum band above 2700 cm(-1). According to Zundel (G. Zundel , 1994, J. Mol. Struct. 322.33-42), these continua appear in systems capabl e of forming polarizable hydrogen bonds. The formation of a bleach continuu m suggests the presence of a polarizable proton in the ground state that ch anges during the photocycle. The appearance of a transient absorption conti nuum suggests a change in the polarizable proton or the appearance of new o nes. It is found that each continuum has a rise time of less than 80 ns and a decay time component of similar to 300 mus. In addition, it is found tha t the absorption continuum in the 2100-1800-cm(-1) region has a slow rise c omponent of 190 ns and a fast decay component of similar to 60 mus. Using t hese results and those of the recent x-ray structural studies of bR(570) an d M-412 (H. Luecke, B. Schobert, H.T. Richter, J.-P. Cartailler, and J. K. Lanyi, 1999, Science 286:255-260), together with the already known spectros copic properties of the different intermediates in the photocycle, the poss ible origins of the polarizable protons giving rise to these continua durin g the bR photocycle are proposed. Models of the proton pump are discussed i n terms of the changes in these polarizable protons and the hydrogen-bonded chains and in terms of previously known results such as the simultaneous d eprotonation of the protonated Schiff base (PSB) and Tyr185 and the disappe arance of water molecules in the proton release channel during the proton p ump process.