Bacteriorhodopsins with chromophores modified at the β‐ionone site

Abstract

The binding to bacterioopsin of the all-trans isomers of retinal analogues lacking the six-membered ring and differing in length of the conjugated chain, as well as the light-driven action of the proton pump of the resulting bacteriorhodopsin analogues, were studied. The 'opsin shifts' in these modified bacteriorhodopsins are all around 2700 cm-' and do not depend on the number of double bonds in the chromophore. These experimental results suggest that the 4800 cm-' 'opsin shift' in unmodified bacteriorhodopsin consists of a contribution of about 2700 cm-' due to the interaction of the protonated Schiff-base with the counterion. The extra 2100cm-shift in bacteriorhodopsin is due to the specific interaction of the cyclohexene ring and the protein. Only the bacteriorhodopsin analogue with the same number of conjugated double bonds in the chromophore as bacteriorhodopsin itself shows light-driven proton pump action.Bacteriorhodopsin (hereafter bR), the only protein in the purple membrane of the halophilic microorganism Halobacterium halobium, occurs in a dark-adapted ( A, , , = 558 nm) and a light-adapted form ( A, , , = 568 nm), which are interconvertible [l]. The chromophoric part of native bR is derived from all-trans retinal and 13-cis retical in dark-adapted bR, and from all-trans retinal in light-adapted bR [2]. The chromophore is protonated Schiff-base bound to the lysine-216 residue [3,4]. Light-adapted bR functions as a light-driven proton pump, pumping protons from the inside to the outside of the bacterial cell thereby creating a proton gradient across the bacterial membrane to generate the energy for ATP synthesis [5 -81. The release of the chromophore from the bR binding site can be achieved without denaturation by irradiation of bR with visible light in the presence of hydroxylamine, leaving a chromophore-free apoprotein (called bacterioopsin) and all-trans retinal oxime [9]. Addition of all-trans retinal to bacterioopsin results in immediate formation of the so called 430 -460-nm intermediate complex which rapidly converts into all-trans bR [lo] (A, , , = 568 nm). This regenerated bR, after incorporation into phospholipid vesicles pumps protons upon illumination as efficiently as native bR [6]. The red shift in the absorption maximum of dark-adapted bR (in cm-') relative to the protonated all-trans retinal Schiff-base model compound (in cm-', A,,,=440 nm), which is called 'opsin shift' [ll], is believed to be caused by special proteinchromophore interactions. Contributing factors for this 'opsin shift' are an interaction of an anion located near the cyclohexene part of the chromophore in addition to the interaction of the counterion with the protonated Schiff-base part [I I].In order to study the importance of the six-membered ring and of the 5,6 and 7,8 double bond in the chromophore for the purple colour and proton pump activity of bR, our straAbbreviations. bR, bacteriorhodopsin; bR(1) -bR(5), bacteriorhodopsin containing the retinal analogues 1 -5. tegy was to investigate the binding to bacterioo...

Controlling the pKa of the bacteriorhodopsin Schiff base by use of artificial retinal analogues.

“…The smaller (2400-2500 cm-') opsin shifts observed in the case of bR480(III) and bR527(IV) are comparable to those reported for §Difference between the pKa of bR570 and that of the corresponding pigment. analogues characterized by short polyene chains (12)(13)(14). This is consistent with the lack of the ring-chain effects existing in the natural system, also indicating negligible interactions with the proposed opsin dipole.…”

“…By use of resonance Raman spectroscopy, it was shown that bR460 is characterized by a deprotonated Schiff base moiety. The apparent pKa value of 13.3 ± 0.3 observed for the above titration [pKa(I)] was assigned to a simple equilibrium directly involving the Schiff base:…”

“…At present the opsin shift in bR is accounted for in terms of a combination offactors: (a) different interactions between the positively charged Schiff base nitrogen and the counterion in the pigment relative to retinal protonated Schiff base in methanol (11)(12)(13)(14)(15)(16); (b) a planar s-trans ring-chain conformation (14,(17)(18)(19)(20); (c) an ion-pair (rather than a single opsin charge) in the vicinity of the ,B-ionone ring (14,17,18). The smaller (2400-2500 cm-') opsin shifts observed in the case of bR480(III) and bR527(IV) are comparable to those reported for §Difference between the pKa of bR570 and that of the corresponding pigment.…”

Synthetic retinals as probes for the binding site and photoreactions in rhodopsins

“…This applies even in the case of the artificial pigment derived from chromophore 10 bearing only one double bond. Opsin shifts of a similar magnitude were also found (Muradin-Szweykowska et al, 1984) in artificial bR pigments based on a series of short linear polyenes (11-13). More recently, the importance of Schiff base-protein interactions was further demonstrated by breaking the polyene chain using artificial pigments derived from a series of (5,6; 7, 8; 9,10) dihydroetinals Spudich et al, 1986).…”

“…It should be pointed out that this approach assumes that, in the absence of serious steric perturbations, the artificial chromophore adopts a geometry which is close to that of the native chromophore. Although no direct proof justifying this approximation is available, its feasibility is supported by studies with the series of retinal analogs with a perturbed ring region discussed above (Sheves et al, 1985;Muradin-Szweykowska et al, 1984). Independently of the way in which such perturbation is carried out, a similar decrease (of the order of 2000-2500 cm -1) in OSbR is observed.…”