wavelength (nm)

Figure 5. Absorption spectra of BR (solid), HR (dash), NpSRII (dot), and SRI (dash, dot, dot). The spectrum of SRI was measured at pH 6, displaying only maxima at 400 and 587 nm. The spectra are corrected for the contribution of light scattering [216].

of the retinal ring with respect to the polyene chain could account for the opsin shift as well as for the fine structure of the absorption maximum only observed in the known HsSRIIs [120]. The structural basis for these observations has been sought in those amino acid residues of the retinal binding site that differ from those of the other archaeal rhodopsins. However, mutational studies were unable to narrow the cause to particular amino acids [121-123]. Even the simultaneous replacement of 10 amino acids from the retinal binding pocket by corresponding residues from BR did not result in a substantial batho-chromic shift (N. Kamo, personal communication). These failures to explain the absorption properties of HsSRII or NpSRII indicate that our knowledge of colour regulation in retinal proteins is not adequate, thus further experiments are mandatory. However, a recent crystal structure of NpSRII at 2.4 À [124] provides new insight into the colour regulation in NpSRII. It appears that the main contribution to the blue-shift is the longer distance of the guanidinium group of Arg72 from the Schiff base as compared to that of Arg82 in BR.

The absorption maximum of NpSRII is slightly dependant on external conditions. Lowering the pH to 3.5 shifts the maximum to 325 nm (pK = 5.6; detergent solubilised NpSRII has a pK of < 4) [125,126], These observations can be explained by the protonation of the Schiff base counter-ion Asp75 which, under physiological conditions, is deprotonated [127]. Removal of this anion from the protonated Schiff base - which can be accomplished either by its protonation or by its mutation into a neutral amino acid - exerts a bathochromic shift of the same order of ~900 cm-1 for BR, pHR, NpSRII, and HsSRII [125,128-130], The addition of highly concentrated solutions of chloride to the acid form of NpSRII reverses the bathochromic shift [125,126],

A similar dependency of the absorption maximum on the external conditions is found for SRI. At neutral pH three maxima, at 587 nm (SRI587), 5 50 nm (SRI550), and 400 nm (SRI400), are detected [32,33,107,131-133] (Figure 5).

The action spectrum of the photo-attractant response of H. salinarum corresponds to the maximum at 587 nm [22]. Fourier-transform infrared (FTIR) data have revealed that the counterion (Asp76) of the Schiff base is protonated [134], as it was also proposed for Asp 85 in the acidified (or deionised) purple membrane [135,136]. Congruent with this observation is the fact that the SRI-mutants D76N and D76A are fully functional as phototaxis receptors [134], The pK of Asp76 is about 7.2, leading to the species absorbing at 550 nm [32,137-139], which turned out to be a light-driven proton pump (see below). There is so far no obvious explanation for the maximum at about 400 nm, although a deprotonated Schiff base might be responsible. It is interesting to note that the binding of the transducer increases the pK of Asp76 to 8.7 [131]. The characteristic pH of the natural habitat of H. salinarum is at about 7.5, implying complete occupancy of the 587 nm state.

1.3.3 Photocycle of sensory rhodopsins

The chromophore in SRI and NpSRII is all-trans retinal [140,141] bound via a Schiff base linkage to a Lys residue on helix G. In NpSRII a trans-\3-cis isomerisation of the retinal chromophore (this so-called light/dark adaptation was first observed in BR) cannot occur because the retinal binding site only accepts all-trans retinal but not 13-cis retinal [142], In HsSRII from H. salinarum the fraction of all-trans retinal has been determined to be 80% [98]. For SRI the light-induced a\\-trans to 13-cis isomerisation is a prerequisite for its functioning [143,144]. In the initial state SRI contains almost only all-trans retinal (95%) which is shifted to 93% 13-cis retinal in the M-state [145], The resonance Raman spectra are neither altered by the mutation D76N nor by the complexation with the transducer Htrl [145].

Steric constraints in the retinal binding pocket have been deduced from experiments using retinal analogues [146-148]. The photoactive site of SRI and BR was probed by a set of 24 retinal analogues [149], This investigation revealed differences in the protein environment near the retinal 13-methyl group and near the (3-ionone ring. It was proposed that the 13-methyl group-protein interaction functions as a trigger for SRI activation. A similar proposal has been made for the retinal 9-methyl group in mammalian rhodopsin [150].

After light excitation the sensory rhodopsins thermally relax back to the original state through several intermediates. Generally, these photocycles are quite similar to that of BR, consisting of the canonical intermediates K,L,M,N and O states ([151] and literature therein); schemes of the photocycles are depicted in Figure 6. The K-intermediate of NpSRII is formed within 5 ps [133]. For SRI (at pH 6; protonated Asp76) a slow biexponential absorbance change indicates a long-lived excited state. Photoacoustic measurements for both pigments revealed volume changes at the stage of the K-intermediate that are considerably larger than those observed for BR [132,152,153], The NpSRII mutant D75N does not influence the production of

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