THE ROLE OF BACK-REACTIONS AND PROTON UPTAKE DURING THE N-]O TRANSITION IN BACTERIORHODOPSINS PHOTOCYCLE - A KINETIC RESONANCE RAMAN-STUDY

The kinetics of bacteriorhodopsin’s photocycle have been analyzed at pH 5, 6, 7, 8, and 8.6 by using time-resolved resonance Raman spectroscopy. The concentrations of the various intermediates as a function of time were determined by following their resonance Raman intensities using 502-nm (L550, N550, BR568), 458-nm (M412), and 752-nm (O640) excitation. The spectral contributions to the pump + probe data from each intermediate were quantitatively separated by least-squares decomposition. These relative concentrations were then converted to absolute concentrations by using a conservation of molecules constraint. This enabled the unambiguous refinement of a variety of kinetic models to find the simplest one that accurately describes the data. The kinetic data, including the biphasic decay of L550 and M412, are best reproduced by a sequential scheme including back-reactions (BR → L ↔ M ↔ N → O → BR). In addition, the kinetics of the L ↔M and N → O steps are found to be pH-dependent. Both the forward and reverse rate constants connecting L550 and M412 increase with pH, confirming earlier proposals of catalyzed Schiff base deprotonation at alkaline pH. Below pH 7, the N550 → O640 rate constant is independent of pH, but it decreases linearly with pH above 7. This indicates that the protein must pick up a proton during the N550 → O640 transition and that this process becomes rate determining above pH 7. There must, therefore, be an intermediate between N550 and which we denote as N+550. A molecular graphics model is presented which incorporates these observations into a mechanism for proton pumping. © 1990, American Chemical Society. All rights reserved.