The photocycle of the photophobic receptor sensory rhodopsin II from N. pharaonis was analyzed by varying measuring wavelengths, temperature, and pH, and by exchanging H2O with D2O. The data can be satisfactorily modeled by eight exponents over the whole range of modified parameters. The kinetic data support a model similar to that of bacteriorhodopsin (BR) if a scheme of irreversible first-order reactions is assumed. Eight kinetically distinct protein states can then be identified. These states are formed from five spectrally distinct species. The chromophore states Si correspond in their spectral properties to those of the BR photocycle, namely pSRII510 (K), pSRII495 (L), pSRII400 (M), pSRII485 (N), and pSRII535 (O). In comparison to BR, pSRII400 is formed approximately 10 times faster than the M state; however, the back-reaction is almost 100 times slower. Comparison of the temperature dependence of the rate constants with those from the BR photocycle suggests that the differences are caused by changes of DeltaS. The rate constants of the pSRII photocycle are almost insensitive to the pH variation from 9.0 to 5.5, and show only a small H2O/D2O effect. This analysis supports the idea that the conformational dynamics of pSRII controls the kinetics of the photocycle of pSRII.

In the present work the light-activated proton transfer reactions of sensory rhodopsin II from Natronobacterium pharaonis (pSRII) and those of the channel-mutants D75N-pSRII and F86D-pSRII are investigated using flash photolysis and black lipid membrane (BLM) techniques. Whereas the photocycle of the F86D-pSRII mutant is quite similar to that of the wild-type protein, the photocycle of D75N-pSRII consists of only two intermediates. The addition of external proton donors such as azide, or in the case of F86D-pSRII, imidazole, accelerates the reprotonation of the Schiff base, but not the turnover. The electrical measurements prove that pSRII and F86D-pSRII can function as outwardly directed proton pumps, whereas the mutation in the extracellular channel (D75N-pSRII) leads to an inwardly directed transient current. The almost negligible size of the photostationary current is explained by the long-lasting photocycle of about a second. Although the M decay, but not the photocycle turnover, of pSRII and F86D-pSRII is accelerated by the addition of azide, the photostationary current is considerably increased. It is discussed that in a two-photon process a late intermediate (N- and/or O-like species) is photoconverted back to the original resting state; thereby the long photocycle is cut short, giving rise to the large increase of the photostationary current. The results presented in this work indicate that the function to generate ion gradients across membranes is a general property of archaeal rhodopsins.

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The halobacterial phototaxis receptors sensory rhodopsin I and II (SRI, SRII) enable the bacteria to seek optimal light conditions for ion pumping by bacteriorhodopsin and͞or halorhodopsin. The incoming signal is transferred across the plasma membrane by means of receptor-specific transducer proteins that bind tightly to their corresponding photoreceptors. To investigate the receptor͞ transducer interaction, advantage is taken of the observation that both SRI and SRII can function as proton pumps. SRI from Halobacterium salinarum, which triggers the positive phototaxis, the photophobic receptor SRII from Natronobacterium pharaonis (pSRII), as well as the mutant pSRII-F86D were expressed in Xenopus oocytes. Voltage-clamp studies confirm that SRI and pSRII function as light-driven, outwardly directed proton pumps with a much stronger voltage dependence than the ion pumps bacteriorhodopsin and halorhodopsin. Coexpression of SRI and pSRII-F86D with their corresponding transducers suppresses the proton transport, revealing a tight binding and specific interaction of the two proteins. T he electrical properties of eukaryotic membrane proteins can easily be analyzed by employing the oocyte expression system from Xenopus laevis. In previous work, it has been demonstrated that bacteriorhodopsin (BR) from Halobacterium salinarum could also be functionally expressed into the plasma membrane of oocytes, which allowed the elucidation of the electrogenic characteristics of this proton pump under well-defined voltageclamp conditions (1, 2). This work indicated that the other members of the bacterial rhodopsin family, the chloride pump halorhodopsin, and the sensory rhodopsins can also be analyzed by this approach. The sensory rhodopsins (SRI and SRII) are, as their names already point to, phototaxis receptors. Although SRII solely transmits the photophobic reaction of the bacteria, SRI displays a dual function. In a one-photon process, a photoattractant response is triggered; however, another blue photon absorbed by an intermediate of the cyclic photoreaction elicits a photophobic response. The excitation of both SRI and SRII leads to an activation of receptor-specific transducers HtrI and HtrII, respectively, which subsequently trigger the bacterial signal transduction chain (reviewed in refs. 3 and 4).Because the sequence homology between the four retinylidene proteins is relatively high, it appeared likely that the sensory rhodopsins could also function as light-driven proton pumps. Indeed, the electrogenic properties of SRI have been extensively studied, and recently data have also been obtained for SRII. However, the picture emerging from these investigations is still controversial.Olson & Spudich (5) and Bogomolni et al. (6) investigated pH changes by using envelope vesicles and reported that SRI is an outwardly directed proton pump that is driven by orange light in a one photon process. Haupts et al. (7,8) performed similar experiments with intact cells and, additionally, analyzed photocurrents of SRI containing membra...

The halobacterial phototaxis receptors sensory rhodopsin I and II (SRI, SRII) enable the bacteria to seek optimal light conditions for ion pumping by bacteriorhodopsin and/or halorhodopsin. The incoming signal is transferred across the plasma membrane by means of receptor-specific transducer proteins that bind tightly to their corresponding photoreceptors. To investigate the receptor/transducer interaction, advantage is taken of the observation that both SRI and SRII can function as proton pumps. SRI from Halobacterium salinarum , which triggers the positive phototaxis, the photophobic receptor SRII from Natronobacterium pharaonis (pSRII), as well as the mutant pSRII-F86D were expressed in Xenopus oocytes. Voltage-clamp studies confirm that SRI and pSRII function as light-driven, outwardly directed proton pumps with a much stronger voltage dependence than the ion pumps bacteriorhodopsin and halorhodopsin. Coexpression of SRI and pSRII-F86D with their corresponding transducers suppresses the proton transport, revealing a tight binding and specific interaction of the two proteins. These latter results may be exploited to further analyze the binding interaction of the photoreceptors with their downstream effectors.