Yuji Furutani

Excited-state dynamics of rhodopsin probed by femtosecond fluorescence spectroscopy

Abstract Nonexponential fluorescence dynamics of bovine rhodopsin were measured at various wavelengths from 530 to 780 nm. No dynamic Stokes shift was detected but the fastest part of the fluorescence at both blue and red sides of the spectrum decayed faster than that at the center, which has led to the conclusion that the Franck–Condon state→fluorescence state conversion occurs within time-resolution of apparatus (

In situ spectroscopic, electrochemical, and theoretical studies of the photoinduced host-guest electron transfer that precedes unusual host-mediated alkane photooxidation.

In the unusual photooxidation of alkanes within the cavity of a self-assembled M(6)L(4) cage complex (1), the proposed reaction mechanism involves the generation of the host anion radical and guest cation radical via guest-to-host photoinduced electron transfer (PET). In the present study, the postulated host anion radical was elucidated by in situ IR spectroscopy. The difference IR spectrum before and after the irradiation of the clathrate complex 1a [symbol: see text] (2)(4) (2 = adamantane) features a dominant negative peak at approximately 1500 cm(-1), which is attributed to the decrease in the CN stretching vibration and thus indicative of the structural change in the triazine core of the cage. Electrochemical measurements and theoretical calculations show the remarkably great electron-accepting ability of the triazine core. These results strongly support the proposal of guest-to-host PET.

Salinibacter Sensory Rhodopsin SENSORY RHODOPSIN I-LIKE PROTEIN FROM A EUBACTERIUM

Halobacterium salinarum sensory rhodopsin I (HsSRI), a dual receptor regulating both negative and positive phototaxis in haloarchaea, transmits light signals through changes in protein-protein interactions with its transducer, halobacterial transducer protein I (HtrI). Haloarchaea also have another sensor pigment, sensory rhodopsin II (SRII), which functions as a receptor regulating negative phototaxis. Compared with HsSRI, the signal relay mechanism of SRII is well characterized because SRII from Natronomonus pharaonis (NpSRII) is much more stable than HsSRI and HsSRII, especially in dilute salt solutions and is much more resistant to detergents. Two genes encoding SRI homologs were identified from the genome sequence of the eubacterium Salinibacter ruber. Those sequences are distantly related to HsSRI (∼40% identity) and contain most of the amino acid residues identified as necessary for its function. To determine whether those genes encode functional protein(s), we cloned and expressed them in Escherichia coli. One of them (SrSRI) was expressed well as a recombinant protein having all-trans retinal as a chromophore. UV-Vis, low-temperature UV-Vis, pH-titration, and flash photolysis experiments revealed that the photochemical properties of SrSRI are similar to those of HsSRI. In addition to the expression system, the high stability of SrSRI makes it possible to prepare large amounts of protein and enables studies of mutant proteins that will allow new approaches to investigate the photosignaling process of SRI-HtrI.

Functional Importance of the Interhelical Hydrogen Bond between Thr204 and Tyr174 of Sensory Rhodopsin II and Its Alteration during the Signaling Process

Sensory rhodopsin II (SRII), a receptor for negative phototaxis in haloarchaea, transmits light signals through changes in protein-protein interaction with its transducer HtrII. Light-induced structural changes throughout the SRII-HtrII interface, which spans the periplasmic region, membrane-embedded domains, and cytoplasmic domains near the membrane, have been identified by several studies. Here we demonstrate by site-specific mutagenesis and analysis of phototaxis behavior that two residues in SRII near the membrane-embedded interface (Tyr174 on helix F and Thr204 on helix G) are essential for signaling by the SRII-HtrII complex. These residues, which are the first in SRII shown to be required for phototaxis function, provide biological significance to the previous observation that the hydrogen bond between them is strengthened upon the formation of the earliest SRII photointermediate (SRIIK) only when SRII is complexed with HtrII. Here we report frequency changes of the S-H stretch of a cysteine substituted for SRII Thr204 in the signaling state intermediates of the SRII photocycle, as well as an influence of HtrII on the hydrogen bond strength, supporting a direct role of the hydrogen bond in SRII-HtrII signal relay chemistry. Our results suggest that the light signal is transmitted to HtrII from the energized interhelical hydrogen bond between Thr204 and Tyr174, which is located at both the retinal chromophore pocket and in helices F and G that form the membrane-embedded interaction surface to the signal-bearing second transmembrane helix of HtrII. The results argue for a critical process in signal relay occurring at this membrane interfacial region of the complex.

Active Internal Waters in the Bacteriorhodopsin Photocycle. A Comparative Study of the L and M Intermediates at Room and Cryogenic Temperatures by Infrared Spectroscopy

We present time-resolved room-temperature infrared difference spectra for the bacteriorhodopsin (bR) photocycle at 8 cm (-1) spectral and 5 micros temporal resolution, from 4000 to 800 cm (-1). An in situ hydration method allowed for a controlled and stable sample hydration (92% relative humidity), largely improving the quality of the data without affecting the functionality of bR. Experiments in both H 2 (16)O and H 2 (18)O were conducted to assign bands to internal water molecules. Room-temperature difference spectra of the L and M intermediates minus the bR ground state (L-BR and M-BR, respectively) were comprehensively compared with their low-temperature counterparts. The room-temperature M-BR spectrum was almost identical to that obtained at 230 K, except for a continuum band. The continuum band contains water vibrations from this spectral comparison between H 2 (16)O and H 2 (18)O, and no continuum band at 230 K suggests that the protein/solvent dynamics are insufficient for deprotonation of the water cluster. On the other hand, an intense positive broadband in the low-temperature L-BR spectrum (170 K) assigned to the formation of a water cavity in the cytoplasmic domain is absent at room temperature. This water cavity, proposed to be an essential feature for the formation of L, seems now to be a low-temperature artifact caused by restricted protein dynamics at 170 K. The observed differences between low- and room-temperature FTIR spectra are further discussed in light of previously reported dynamic transitions in bR. Finally, we show that the kinetics of the transient heat relaxation of bR after photoexcitation proceeds as a thermal diffusion process, uncorrelated with the photocycle itself.

Engineering an Inward Proton Transport from a Bacterial Sensor Rhodopsin

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.

Effects of Chloride Ion Binding on the Photochemical Properties of Salinibacter Sensory Rhodopsin I

Microbial organisms utilize light not only as energy sources but also as signals by which rhodopsins (containing retinal as a chromophore) work as photoreceptors. Sensory rhodopsin I (SRI) is a dual photoreceptor that regulates both negative and positive phototaxis in microbial organisms, such as the archaeon Halobacterium salinarum and the eubacterium Salinibacter ruber. These organisms live in highly halophilic environments, suggesting the possibility of the effects of salts on the function of SRI. However, such effects remain unclear because SRI proteins from H. salinarum (HsSRI) are unstable in dilute salt solutions. Recently, we characterized a new SRI protein (SrSRI) that is stable even in the absence of salts, thus allowing us to investigate the effects of salts on the photochemical properties of SRI. In this study, we report that the absorption maximum of SrSRI is shifted from 542 to 556 nm in a Cl(-)-dependent manner with a K(m) of 307+/-56 mM, showing that Cl(-)-binding sites exist in SRI. The bathochromic shift was caused not only by NaCl but also by other salts (NaI, NaBr, and NaNO(3)), implying that I(-), Br(-), and NO(3)(-) can also bind to SrSRI. In addition, the photochemical properties during the photocycle are also affected by chloride ion binding. Mutagenesis studies strongly suggested that a conserved residue, His131, is involved in the Cl(-)-binding site. In light of these results, we discuss the effects of the Cl(-) binding to SRI and the roles of Cl(-) binding in its function.

Early Photocycle Structural Changes in a Bacteriorhodopsin Mutant Engineered to Transmit Photosensory Signals

Bacteriorhodopsin (BR) and sensory rhodopsin II (SRII) function as a light-driven proton pump and a receptor for negative phototaxis in haloarchaeal membranes, respectively. SRII transmits light signals through changes in protein-protein interaction with its transducer HtrII. Recently, we converted BR by three mutations into a form capable of transmitting photosignals to HtrII to mediate phototaxis responses. The BR triple mutant (BR-T) provides an opportunity to identify structural changes necessary to activate HtrII by comparing light-induced infrared spectral changes of BR, BR-T, and SRII. The hydrogen out-of-plane (HOOP) vibrations of the BR-T were very similar to those of SRII, indicating that they are distributed more extensively along the retinal chromophore than in BR, as in SRII. On the other hand, the bands of the protein moiety in BR-T are similar to those of BR, indicating that they are not specific to photosensing. The alteration of the O–H stretching vibration of Thr-204 in SRII, which we had previously shown to be essential for signal relay to HtrII, occurs also in BR-T. In addition, 1670(+)/1664(-) cm-1 bands attributable to a distorted α-helix were observed in BR-T in a HtrII-dependent manner, as is seen in SRII. Thus, we identified similarities and dissimilarities of BR-T to BR and SRII. The results suggest signaling function of the structural changes of the HOOP vibrations, the O–H stretching vibration of the Thr-215 residue, and a distorted α-helix for the signal generation. We also succeeded in measurements of L minus initial state spectra of BR-T, which are the first FTIR spectra of L intermediates among sensory rhodopsins.

Molecular insight into intrinsic heme distortion in ligand binding in hemoprotein.

A pair of myoglobins containing inherently distorted alpha-ethyl-2,4-dimethyldeuteroheme or undistorted 2,4-dimethyldeuteroheme were prepared, and the functional consequence of intrinsic heme deformation was investigated. The visible absorption peaks of the myoglobin bearing the distorted heme exhibited a bathochromic shift, indicating that the heme was deformed in the protein pocket. Ligand affinities for the ferric myoglobin with the distorted heme were found to be higher than those of the myoglobin bearing the undistorted heme. The observation suggested that the iron atom was more displaced toward the proximal histidine to weaken the coordination of the water molecule. In the paramagnetic proton NMR spectrum of ferrous deoxy protein, the deformed heme caused a 3.2 ppm lower-field shift of the proximal histidine signal, supporting an enhanced iron-histidine interaction. The deformed heme in ferrous myoglobin lowered the oxygen and carbon monoxide affinities by 25- and 480-fold, respectively, and caused the cleavage of the iron-histidine bond in a fractional population of the nitric oxide derivative. These results demonstrate a distinctive controlling mechanism for ligand binding by the deformed heme. Upon the heme distortion, the iron atom is more attracted by the proximal histidine to reduce the affinity of exogenous ligands for the ferrous heme.

Interaction between Na+ ion and carboxylates of the PomA-PomB stator unit studied by ATR-FTIR spectroscopy

Bacterial flagellar motors are molecular machines powered by the electrochemical potential gradient of specific ions across the membrane. The PomA-PomB stator complex of Vibrio alginolyticus couples Na(+) influx to torque generation in this supramolecular motor, but little is known about how Na(+) associates with the PomA-PomB complex in the energy conversion process. Here, by means of attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy, we directly observed binding of Na(+) to carboxylates in the PomA-PomB complex, including the functionally essential residue Asp24. The Na(+) affinity of Asp24 is estimated to be approximately 85 mM, close to the apparent K(m) value from the swimming motility of the cells (78 mM). At least two other carboxylates are shown to be capable of interacting with Na(+), but with somewhat lower affinities. We conclude that Asp24 and at least two other carboxylates constitute Na(+) interaction sites in the PomA-PomB complex. This work reveals features of the Na(+) pathway in the PomA-PomB Na(+) channel by using vibrational spectroscopy.