QUANTITATIVE-DETERMINATION OF THE PROTEIN CATALYTIC EFFICIENCY FOR THE RETINAL EXCITED-STATE DECAY IN BACTERIORHODOPSIN

It was previously found that by removing the negative charge of Asp85 in bacteriorhodopsin (bR), either by protonating it (as in deionized b R) or by mutation to Asn, the decay time of the retinal excited state increases from 0.5 ps to either 1.5 or 10 ps. The two decay components result from the presence of all-trans and 13-cis,15-syn (13-cis) reti nal isomers in the modified retinal protein. To quantitatively determi ne the protein catalysis for the primary process in native bR, we need to determine which decay component results from the excited state of the all-trans isomer (present in the native bR). It is known that the all-trans isomer absorbs at longer wavelength than the 13-cis isomer i n blue bR. In this communication, we report the results of pump-probe experiments using 100 fs laser pulses. Probing is carried out at 490 n m, where the excited state in both isomers absorbs. It is found that t he ratio of the amplitudes of the two decay components in blue bR chan ges with variation of the excitation wavelength. The shorter-lived com ponent is found to increase in amplitude as the excitation wavelength increases, i.e., as we excite more of the all-trans isomer. This leads to the conclusion that the short-lived component (1.5 ps) is for the decay of the all-trans excited state while the long-lived component (1 0 ps) is for the 13-cis retinal excited-state decay. Thus, the presenc e of the negative charge of Asp85 in native bR catalyzes the rate of t he excited-state decay of the all-trans retinal by 300% and that of th e 13-cis isomer by >2000%.