Kinetic resonance Raman spectra of native and isotopically labelled purple membranes are compared. Using these data and the assignments of the previous paper in this sequence, we have confirmed that the Schiff base is deprotonated at times that are short in comparison to M412 evolution. In addition, by monitoring the kinetic resonance Raman spectra in 2H2O with 488.0 nm excitation we have been able to characterize in more detail the vibrational features associated with this unprotonated intermediate that precedes M412. Furthermore, the kinetic spectra of fully deuterated purple membranes in H2O have allowed us to assign the 1465 cm-1 band in these spectra to the C=C stretching frequency of BR570 and the 1512 cm-1 band to the C=C stretching frequency of M412. These spectra have also provided an indication of a Raman spectral feature associated with O640 and, finally, our kinetic spectra have provided evidence that there is a significant alteration in the rate constants for the evolution of the various intermediates when the non-exchangeable protons on the membrane are replaced by deuterons.
Bibliographical noteFunding Information:
This work was supported by a National Institutes of Health Grant No. EY 01377 and the Division of Basic Energy Sciences of the Department of Energy.
- Chemical modification
- Purple membrane
- Resonance Raman spectroscopy