Chromophore structure in bacteriorhodopsin's N intermediate: implications for the proton-pumping mechanism

Abstract

By elevating the pH to 9.5 in 3 M KCl, the concentration of the N intermediate in the bacteriorhodopsin photocycle has been enhanced, and time-resolved resonance Raman spectra of this intermediate have been obtained. Kinetic Raman measurements show that N appears with a half-time of 4 +/- 2 ms, which agrees satisfactorily with our measured decay time of the M412 intermediate (2 +/- 1 ms). This argues that M412 decays directly to N in the light-adapted photocycle. The configuration of the chromophore about the C13 = C14 bond was examined by regenerating the protein with [12,14-2H]retinal. The coupled C12-2H + C14-2H rock at 946 cm-1 demonstrates that the chromophore in N is 13-cis. The shift of the 1642-cm-1 Schiff base stretching mode to 1618 cm-1 in D2O indicates that the Schiff base linkage to the protein is protonated. The insensitivity of the 1168-cm-1 C14-C15 stretching mode to N-deuteriation establishes a C = N anti (trans) Schiff base configuration. The high frequency of the C14-C15 stretching mode as well as the frequency of the 966-cm-1 C14-2H-C15-2H rocking mode shows that the chromophore is 14-s-trans. Thus, N contains a 13-cis, 14-s-trans, 15-anti protonated retinal Schiff base.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of a proton pump analyzed with computer simulations

“…The significant energy barrier for retinal isomerisation from 15-trans to 15-syn [32,46] makes it unlikely that a 13-cis, 15-syn geometry [8] could be reached during the K-to-L transition [33]. The conclusion from computations that sampling of 15-syn retinals prior to retinal deprotonation is unlikely [33] is consistent with spectroscopy indicating that in L retinal is 13-cis, all-trans [73,74]. In Ref.…”

Trapping the M sub 1 and M sub 2 substrates of bacteriorhodopsin for electron diffraction studies

“…The importance of conformational changes with respect to the mechanism of active transport is best understood within the framework of the "C-T ModeI" of bR functionality (Fodor et al, 1988). The logic of this model becomes clear when one realizes that the retinal doesn't change configuration between L, M and N, thus eliminating models which rely on retinal isomerization alone to explain transport.…”

“…In view of the results presented in this dissertation, with guidance from published results using molecular vibrational i, spectroscopy, I propose an extension of the C -T Model (Fodor et al, " 1988) to explain how changes in bR's retinal induce changes in bR's protein conformation and vice versa. In proposing to extend the C -T Model, I am guided by the principle of mutual induced fit (Koshland, 1973).…”

“…The transition from the T protein conformation to the C protein conformation is postulated to occur during either the L -M reaction (Fodor et al, 1988) or during the M1 -M 2 reaction Ormos, 1991). However, it can be argued that the T -C transition occurs earlier in the photocycle, as early as the K -L reaction.…”

Proton Translocation In Proteins

“…The coupling between redox activity and protonation in this extreme would be indirect and mediated by conformational transition of the protein. The simplicity of the direct coupling mechanism is quite appealing, but we must point out that increasing evidence favors the indirect mechanism for two other proton pumps, ATP synthase (3) and bactcriorhodopsin (54). As far cytochrome c oxidase is concerned, no compelling evidence favours either coupling mechanism at this juncture (but see the next section on redoxlinked conformational transitions).…”