The voltage-gated proton channel Hv1 (or VSOP) has a voltage-sensor domain (VSD) with dual roles of voltage sensing and proton permeation. Its gating is sensitive to pH and Zn(2+). Here we present a crystal structure of mouse Hv1 in the resting state at 3.45-Å resolution. The structure showed a 'closed umbrella' shape with a long helix consisting of the cytoplasmic coiled coil and the voltage-sensing helix, S4, and featured a wide inner-accessible vestibule. Two out of three arginines in S4 were located below the phenylalanine constituting the gating charge-transfer center. The extracellular region of each protomer coordinated a Zn(2+), thus suggesting that Zn(2+) stabilizes the resting state of Hv1 by competing for acidic residues that otherwise form salt bridges with voltage-sensing positive charges on S4. These findings provide a platform for understanding the general principles of voltage sensing and proton permeation.

Anabaena sensory rhodopsin (ASR) is an archaeal-type rhodopsin found in eubacteria, and is believed to function as a photosensor interacting with a 14 kDa soluble protein. Most of the residues in the retinal binding pocket are similar in ASR except proline 206, where the corresponding amino acid in other archaeal-type rhodopsins is highly conserved aspartate that constitutes the counterion complex of the positively charged protonated Schiff base. The recently determined X-ray crystallographic structure of ASR revealed a water molecule between the Schiff base and Asp75 [Vogeley, L., Sineshchekov, O. A., Trivedi, V. D., Sasaki, J., Spudich, J. L., and Luecke, H. (2004) Science 306, 1390-1393], as well as the case for bacteriorhodopsin (BR), a typical transport rhodopsin working as a proton pump. In this study, we applied low-temperature Fourier transform infrared (FTIR) spectroscopy to the all-trans form of ASR at 77 K, and compared the local structure around the chromophore and their structural changes upon retinal photoisomerization with those of BR. The K intermediate minus ASR difference spectra were essentially similar to those for BR, indicating that photoisomerization yields formation of the distorted 13-cis form. In contrast, little amide I bands were observed for ASR. The presence of the proline-specific vibrational bands suggests that peptide backbone alterations are limited to the Pro206 moiety in the K state of ASR. The N-D stretching of the Schiff base is presumably located at 2163 (-) and 2125 (-) cm(-)(1) in ASR, suggesting that the hydrogen bonding strength of the Schiff base in ASR is similar to that in BR. A remarkable difference between ASR and BR was revealed from water bands. Although ASR possesses a bridged water molecule like BR, the O-D stretching of water molecules was observed only in the >2500 cm(-)(1) region for ASR. We interpreted that the weak hydrogen bond of the bridged water between the Schiff base and Asp75 originates from their geometry. Since ASR does not pump protons, our result supports the working hypothesis that the existence of strongly hydrogen bonded water molecules is essential for proton pumping activity in archaeal rhodopsins.

Protein-controlled photochemical reactions often mediate biological light-signal and light-energy conversions. Microbial rhodopsins possess all-trans or 13-cis retinal as the chromophore in the dark, and in the light-driven proton pump, bacteriorhodopsin (BR), the stable photoproduct at the end of the functional cycle of the all-trans form is 100% all-trans. In contrast, a microbial rhodopsin discovered in Anabaena PCC7120 is believed to function as a photochromic sensor. For Anabaena sensory rhodopsin (ASR), the photoreaction is expected to be not cyclic, but photochromic. The present low-temperature UV-visible spectroscopy of ASR indeed revealed that the stable photoproduct of the all-trans form in ASR is 100% 13-cis, and that of the 13-cis form is 100% all-trans. The complete photocycle for the proton pump in BR and the complete photochromism for the chromatic sensor of ASR are highly advantageous for their functions. Thus, the microbial rhodopsins have acquired unique photoreactions, in spite of their similar structures, during evolution.

Archaeal-type rhodopsins can accommodate either all-trans- or 13-cis,15-syn-retinal in their chromophore binding site in the dark, but only the former isomer is functionally important. In contrast, Anabaena sensory rhodopsin (ASR), an archaeal-type rhodopsin found in eubacteria, exhibits a photochromic interconversion of both forms, suggesting that ASR functions as a photosensor which interacts with its 14 kDa soluble transducer differently in the all-trans and 13-cis,15-syn forms. In this study, we applied low-temperature Fourier transform infrared (FTIR) spectroscopy to the 13-cis,15-syn form of ASR (13C-ASR) at 77 K and compared the local structure around the chromophore and its structural changes upon retinal photoisomerization with those of the all-trans form (AT-ASR) [Furutani, Y., Kawanabe, A., Jung, K. H., and Kandori, H. (2005) Biochemistry 44, 12287-12296]. By use of [zeta-15N]lysine-labeled ASR, we identified the N-D stretching vibrations of the Schiff base (in D2O) at 2165 cm(-1) for 13C-ASR and at 2163 and 2125 cm(-1) for AT-ASR. The frequencies indicate strong hydrogen bonds of the Schiff base with a water molecule for both 13C-ASR and AT-ASR. In contrast, the N-D stretching vibration appears at 2351 cm(-1) and at 2483 cm(-1) for the K states of 13C-ASR (13C-ASR(K)) and AT-ASR (AT-ASR(K)), respectively, indicating that the Schiff base still forms a hydrogen bond in 13C-ASR(K). Rotational motion of the Schiff base upon retinal isomerization is probably smaller for 13C-ASR than for AT-ASR, the latter altering hydrogen bonding of the Schiff base similar to bacteriorhodopsin (BR), a light-driven proton pump. Appearance of several hydrogen-out-of-plane vibrations and amide I vibrations in 13C-ASR(K), but not in AT-ASR(K), suggests that structural changes are distributed widely along the polyene chain for 13C-ASR. On the other hand, retinal photoisomerization in AT-ASR breaks the hydrogen bond of the Schiff base, and localized structural changes in the Schiff base region are induced.

Proteorhodopsin (PR) genes are widely distributed among marine prokaryotes and functions as light-driven proton pump when expressed heterologously in Escherichia coli, suggesting that light energy passing through PR may be substantial in marine environment. However, there are no data on PR proton pump activities in native marine bacteria. Here, we demonstrate light-driven proton pump activity (c. 124 H(+) PR(-1)  min(-1) ) in recently isolated marine Flavobacteria. Among 75 isolates, 38 possessed the PR gene. Illumination of cell suspensions from all eight tested strains in five genera triggered marked pH drops. The action spectrum of proton pump activity closely matched the spectral distribution of the sea surface green light field. Addition of hydroxylamine to a solubilized membrane fraction shifted the spectrum to a form characteristic of PR photobleached into retinal oxime, indicating that PRs in flavobacterial cell membranes transform the photon dose in incident radiation into energy in the form of membrane potential. Our results revealed that PR-mediated proton transport can create the sufficient membrane potential for the ATP synthesis in native flavobacterial cells.

The cytoplasmic region of voltage-sensing phosphatase (VSP) derives the voltage dependence of its catalytic activity from coupling to a voltage sensor homologous to that of voltage-gated ion channels. To assess the conformational changes in the cytoplasmic region upon activation of the voltage sensor, we genetically incorporated a fluorescent unnatural amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), into the catalytic region of Ciona intestinalis VSP (Ci-VSP). Measurements of Anap fluorescence under voltage clamp in Xenopus oocytes revealed that the catalytic region assumes distinct conformations dependent on the degree of voltage-sensor activation. FRET analysis showed that the catalytic region remains situated beneath the plasma membrane, irrespective of the voltage level. Moreover, Anap fluorescence from a membrane-facing loop in the C2 domain showed a pattern reflecting substrate turnover. These results indicate that the voltage sensor regulates Ci-VSP catalytic activity by causing conformational changes in the entire catalytic region, without changing their distance from the plasma membrane.

ATP is synthesized by an enzyme that utilizes proton motive force, and thus, nature has created various proton pumps. The best-understood proton pump is bacteriorhodopsin (BR), an outward-directed, light-driven proton pump in Halobacterium salinarum. Many archaeal and eubacterial rhodopsins are now known to show similar proton transport activity. We previously converted BR into an inward-directed chloride ion pump, but an inward proton pump has never been created. Proton pumps must have a specific mechanism to exclude transport in the reverse direction in order to maintain a proton gradient, and in the case of BR, a highly hydrophobic cytoplasmic domain may constitute such machinery. Here we report that an inward-directed proton transport can be engineered from a bacterial rhodopsin by a single amino acid replacement. Anabaena sensory rhodopsin (ASR) is a photochromic sensor in freshwater cyanobacteria that possesses little proton pump activity. When we replaced Asp217 in the cytoplasmic domain (a distance of approximately 15 A from the retinal chromophore) by Glu, ASR exhibited an inward proton transport activity driven by absorption of a single photon. FTIR spectra clearly showed an increased proton affinity for Glu217, which presumably controls the unusual directionality opposite to that in normal proton pumps.

Voltage-sensing phosphatase (VSP) contains a voltage sensor domain (VSD) similar to that in voltage-gated ion channels, and a phosphoinositide phosphatase region similar to phosphatase and tensin homolog deleted on chromosome 10 (PTEN). The VSP gene is conserved from unicellular organisms to higher vertebrates. Membrane depolarization induces electrical driven conformational rearrangement in the VSD, which is translated into catalytic enzyme activity. Biophysical and structural characterization has revealed details of the mechanisms underlying the molecular functions of VSP. Coupling between the VSD and the enzyme is tight, such that enzyme activity is tuned in a graded fashion to the membrane voltage. Upon VSP activation, multiple species of phosphoinositides are simultaneously altered, and the profile of enzyme activity depends on the history of the membrane potential. VSPs have been the obvious candidate link between membrane potential and phosphoinositide regulation. However, patterns of voltage change regulating VSP in native cells remain largely unknown. This review addresses the current understanding of the biophysical biochemical properties of VSP and provides new insight into the proposed functions of VSP.