Importance of the broad regional interaction for spectral tuning in Natronobacterium pharaonis phoborhodopsin (sensory rhodopsin II)

Natronobacterium pharaonis phoborhodopsin (ppR; also called N. pharaonis sensory rhodopsin II, NpsRII) is a photophobic sensor in N. pharaonis, and has a shorter absorption maximum (lambdamax, 500 nm) than those of other archaeal retinal proteins (lambdamax, 560-590 nm) such as bacteriorhodopsin (bR). We constructed chimeric proteins between bR and ppR to investigate the long range interactions effecting the color regulation among archaeal retinal proteins. The lambdamax of B-DEFG/P-ABC was 545 nm, similar to that of bR expressed in Escherichia coli (lambdamax, 550 nm). B-DEFG/P-ABC means a chimera composed of helices D, E, F, and G of bR and helices A, B, and C of ppR. This indicates that the major factor(s) determining the difference in lambdamax between bR and ppR exist in helices DEFG. To specify the more minute regions for the color determination between bR and ppR, we constructed 15 chimeric proteins containing helices D, E, F, and G of bR. According to the absorption spectra of the various chimeric proteins, the interaction between helices D and E as well as the effect of the hydroxyl group around protonated Schiff base on helix G (Thr-204 for ppR and Ala-215 for bR) are the main factors for spectral tuning between bR and ppR.     

Ser-130 of Natronobacterium pharaonis halorhodopsin is important for the chloride binding

Pharaonis halorhodopsin (phR) is an inward light-driven chloride ion pump from Natronobacterium pharaonis. In order to clarify the role of Ser-130(phR) residue which corresponds to Ser-115(shR) for salinarum hR on the anion-binding affinity, the wild-type and Ser-130 mutants substituted with Thr, Cys and Ala were expressed in E. coli cells and solubilized with 0.1% n-dodecyl beta-D-maltopyranoside The absorption maximum (lambda(max)) of the S130T mutant indicated a blue shift from that of the wild type in the absence and presence of chloride. For S130A, a large red shift (12 nm) in the absence of chloride was observed. The wild-type and all mutants showed the blue-shift of lambda(max) upon Cl(-) addition, from which the dissociation constants of Cl(-) were determined. The dissociation constants were 5, 89, 153 and 159 mM for the wild-type, S130A, S130T and S130C, respectively, at pH 7.0 and 25 degrees C. Circular dichroic spectra of the wild-type and the Ser-130 mutants exhibited an oligomerization. The present study revealed that the Ser-130 of N. pharaonis halorhodopsin is important for the chloride binding.     

Binding of calcium ions to bacteriorhodopsin.

Adding Ca2+ or other cations to deionized bacteriorhodopsin causes a blue to purple color shift, a result of deprotonation of Asp85. It has been proposed by different groups that the protonation state of Asp85 responds to the binding of Ca2+ either 1) directly at a specific site in the protein or 2) indirectly through the rise of the surface pH. We tested the idea of specific binding of Ca2+ and found that the surface pH, as determined from the ionization state of eosin covalently linked to engineered cysteine residues, rises about equally at both extracellular and cytoplasmic surfaces when only one Ca2+ is added. This precludes binding to a specific site and suggests that rather than decreasing the pKa of Asp85 by direct interaction, Ca2+ increases the surface pH by binding to anionic lipid groups. As Ca2+ is added the surface pH rises, but deprotonation of Asp85 occurs only when the surface pH approaches its pKa. The nonlinear relationship between Ca2+ binding and deprotonation of Asp85 from this effect is different in the wild-type protein and in various mutants and explains the observed complex and varied spectral titration curves.     

Intramolecular charge transfer in the bacteriorhodopsin mutants Asp85-->Asn and Asp212-->Asn: effects of pH and anions.

The photovoltage kinetics of the bacteriorhodopsin mutants Asp212-->Asn and Asp85-->Asn after excitation at 580 nm have been investigated in the pH range from 0 to 11. With the mutant Asp85-->Asn (D85N) at pH 7 no net charge translocation is observed and the signal is the same, both in the presence of Cl- (150 mM) and in its absence (75 mM SO4(2-)). Under both conditions the color of the pigment is blue (lambda max = 615 nm). The time course of the photovoltage kinetics is similar to that of the acid-blue form of wild-type, except that an additional transient charge motion occurs with time constants of 60 microseconds and 1.3 ms, indicating the transient deprotonation and reprotonation of an unknown group to and from the extracellular side of the membrane. It is suggested that this is the group XH, which is responsible for proton release in wild-type. At pH 1, the photovoltage signal of D85N changes upon the addition of Cl- from that characteristic for the acid-blue state of wild-type to that characteristic for the acid-purple state. Therefore, the protonation of the group at position at 85 is necessary, but not sufficient for the chloride-binding. At pH 11, well above the pKa of the Schiff base, there is a mixture of "M-like" and "N-like" states. Net proton transport in the same direction as in wild-type is restored in D85N from this N-like state. With the mutant Asp212-->Asn (D212N), time-resolved photovoltage measurements show that in the absence of halide ions the signal is similar to that of the acid-blue form of wild-type and that no net charge translocation occurs in the entire pH range from 0 to 11. Upon addition of Cl- in the pH range from 3.8 to 7.2 the color of the pigment returns to purple and the photovoltage experiments indicate that net proton pumping is restored. However, this Cl(-)-induced activation of net charge-transport in D212N is only partial. Outside this pH range, no net charge transport is observed even in the presence of chloride, and the photovoltage shows the same chloride-dependent features as those accompanying the acid-blue to acid-purple transition of the wild-type.     

Effects of chloride on the absorption spectrum and photoreactions of halorhodopsin

The absorption maximum of halorhodopsin in a membrane fraction prepared from the cells of Halobacterium halobium under low-salt conditions shifted to longer wavelenghts upon addition of NaCl (Ogurusu, T., Maeda, A., Sasaki, N. and Yoshizawa, T. (1981) J. Biochem. (Tokyo) 90, 1267–1273). This bathochromic shift was due to chloride, not sodium. Bromide and iodide were also effective. The bathochromic shift of the absorption maximum was not accompanied by any change in the isomer composition of retinal in halorhodopsin. The same ionic species were essential for the formation of the hypsochromic photoproduct at −75°C. These effects of NaCl on halorhodopsin are discussed in terms of the presence of the two forms of halorhodopsin, a form binding chloride and a chloride-free form.     

Influence of the size and protonation state of acidic residue 85 on the absorption spectrum and photoreaction of the bacteriorhodopsin chromophore.

The consequences of replacing Asp-85 with glutamate in bacteriorhodopsin, as expressed in Halobacterium sp. GRB, were investigated. Similarly to the in vitro mutated and in Escherichia coli expressed protein, the chromophore was found to exist as a mixture of blue (absorption maximum 615 nm) and red (532 nm) forms, depending on the pH. However, we found two widely separated pKa values (about 5.4 and 10.4 without added salt), arguing for two blue and two red forms in separate equilibria. Both blue and red forms of the protein are in the two-dimensional crystalline state. A single pKa, such as in the E. coli expressed protein, was observed only after solubilization with detergent. The photocycle of the blue forms was determined at pH 4.0 with 610 nm photoexcitation, and that of the red forms at pH 10.5 and with 520 nm photoexcitation, in the time-range of 100 ns to 1 s. The blue forms produced no M, but a K- and an L-like intermediate, whose spectra and kinetics resembled those of blue wild-type bacteriorhodopsin below pH 3. The red forms produced a K-like intermediate, as well as M and N. Only the red forms transported protons. Specific perturbation of the neighborhood of the Schiff base by the replacement of Asp-85 with glutamate was suggested by (1) the shift and splitting of the pKa for what is presumably the protonation of residue 85, (2) a 36 nm blue-shift in the absorption of the all-trans red chromophore and a 25 nm red-shift of the 13-cis N chromophore, as compared to wild-type bacteriorhodopsin and its N intermediate, and (3) significant acceleration of the deprotonation of the Schiff base at pH 7, but not of its reprotonation and the following steps in the photocycle.     

Chloride binding regulates the Schiff base pK in gecko P521 cone-type visual pigment

The binding of chloride is known to shift the absorption spectrum of most long-wavelength-absorbing cone-type visual pigments roughly 30 nm to the red. We determined that the chloride binding constant for this color shift in the gecko P521 visual pigment is 0.4 mM at pH 6.0. We found an additional effect of chloride on the P521 pigment: the apparent pKa of the Schiff base in P521 is greatly increased as the chloride concentration is increased. The apparent Schiff base pKa shifts from 8.4 for the chloride-free form to >10.4 for the chloride-bound form. We show that this shift is due to chloride binding to the pigment, not to the screening of the membrane surface charges by chloride ions. We also found that at high pH, the absorption maximum of the chloride-free pigment shifts from 495 to 475 nm. We suggest that the chloride-dependent shift of the apparent Schiff base pKa is due to the deprotonation of a residue in the chloride binding site with a pKa of ca. 8.5, roughly that of the Schiff base in the absence of chloride. The deprotonation of this site results in the formation of the 475 nm pigment and a 100-fold decrease in the pigment's ability to bind chloride. Increasing the concentration of chloride results in the stabilization of the protonated state of this residue in the chloride binding site and thus increased chloride binding with an accompanying increase in the Schiff base pK.     

Role of charged residues of pharaonis phoborhodopsin (sensory rhodopsin II) in its interaction with the transducer protein

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, NpSRII) is a receptor for negative phototaxis in Natronomonas (Natronobacterium) pharaonis. In membranes, it forms a 2:2 complex with its transducer protein, pHtrII, which transmits light signals into the cytoplasmic space through protein-protein interactions. We previously found that a specific deprotonated carboxyl of ppR or pHtrII strengthens their binding [Sudo, Y., et al. (2002) Biophys. J. 83, 427-432]. In this study we aim to identify this carboxyl group. Since the D75N mutant has only one photointermediate (ppR(O)(-)(like)) whose existence spans the millisecond time range, the analysis of its decay rate is simple. We prepared various D75N mutants such as D75N/D214N, D75N/K157Q/R162Q/R164Q (D75N/3Gln), D75N/D193N, and D75N/D193E, among which only D75N/D193N did not show pH dependence with regard to the ppR(O)(-)(like) decay rate and K(D) value for binding, implying that the carboxyl group in question is from Asp-193. The pK(a) of this group decreased to below 2 when a complex was formed. Therefore, we conclude that Asp-193(p)()(pR) is connected to the distant transducer-ppR binding surface via hydrogen bonds, thereby modulating its pK(a). In addition, we discuss the importance of Arg-162(p)()(pR) with respect to the binding activity.     

Tyr-199 and charged residues of pharaonis Phoborhodopsin are important for the interaction with its transducer

pharaonis Phoborhodopsin (ppR; also pharaonis sensory rhodopsin II, psRII) is a retinal protein in Natronobacterium pharaonis and is a receptor of negative phototaxis. It forms a complex with its transducer, pHtrII, in membranes and transmits light signals by protein-protein interaction. Tyr-199 is conserved completely in phoborhodopsins among a variety of archaea, but it is replaced by Val (for bacteriorhodopsin) and Phe (for sensory rhodopsin I). Previously, we (Sudo, Y., M. Iwamoto, K. Shimono, and N. Kamo, submitted for publication) showed that analysis of flash-photolysis data of a complex between D75N and the truncated pHtrII (t-Htr) give a good estimate of the dissociation constant K(D) in the dark. To investigate the importance of Tyr-199, K(D) of double mutants of D75N/Y199F or D75N/Y199V with t-Htr was estimated by flash-photolysis and was approximately 10-fold larger than that of D75N, showing the significant contribution of Tyr-199 to binding. The K(D) of the D75N/t-Htr complex increased with decreasing pH, and the data fitted well with the Henderson-Hasselbach equation with a single pK(a) of 3.86 +/- 0.02. This suggests that certain deprotonated carboxyls at the surface of the transducer (possibly Asp-102, Asp-104, and Asp-106) are needed for the binding.     

Photochemistry in dried polymer films incorporating the deionized blue membrane form of bacteriorhodopsin.

The preparation and photochemical properties of dried deionized blue membrane (dIbR600; lambdamax approximately 600 nm, epsilon approximately 54, 760 cm-1 M-1, f approximately 1.1) in polyvinyl alcohol films are studied. Reversible photoconversion from dIbR600 to the pink membrane (dIbR485; lambdamax approximately 485 nm) is shown to occur in these films under conditions of strong 647-nm laser irradiation. The pink membrane analog, dIbR485, has a molar extinction coefficient of approximately 39,000 cm-1 M-1 (f approximately 1.2). The ratio of pink --> blue and blue --> pink quantum efficiencies is 33 +/- 5. We observe an additional blue-shifted species (dIbR455, lambdamax approximately 455 nm) with a very low oscillator strength (f approximately 0.6, epsilon approximately 26,000 cm-1 M-1). This species is the product of fast thermal decay of dIbR485. Molecular modeling indicates that charge/charge and charge/dipole interactions introduced by the protonation of ASP85 are responsible for lowering the excited-state all-trans --> 9-cis barrier to approximately 6 kcal mol-1 while increasing the corresponding all-trans --> 13-cis barrier to approximately 4 kcal mol-1. Photochemical formation of both 9-cis and 13-cis photoproducts are now competitive, as is observed experimentally. We suggest that dIbR455 may be a 9-cis, 10-s-distorted species that partially divides the chromophore into two localized conjugated segments with a concomitant blue shift and decreased oscillator strength of the lambdamax absorption band.     

Properties of Asp212----Asn bacteriorhodopsin suggest that Asp212 and Asp85 both participate in a counterion and proton acceptor complex near the Schiff base.

The gene coding for bacteriorhodopsin was modified in vitro to replace Asp212 with asparagine and expressed in Halobacterium halobium. X-ray diffraction measurements showed that the major lattice dimension of purple membrane containing the mutated bacteriorhodopsin was the same as wild type. At pH greater than 7, the Asp212----Asn chromophore was blue (absorption maximum at 585 nm) and exhibited a photocycle containing only the intermediates K and L, i.e. a reaction sequence very similar to that of wild-type bacteriorhodopsin at pH less than 3 and the blue form of the Asp85----Glu protein at pH less than 9. Since in the latter cases these effects are attributed to protonation of residue 85, it now appears that removal of the carboxylate of Asp212 has similar consequences as removing the carboxylate of Asp85. However, an important difference is that only Asp85 affects the pKa of the Schiff base. At pH less than 7, the Asp212----Asn protein was purple (absorption maximum at 569 nm) but photoexcitation produced only 15% of the normal amount of M and the transport activity was partial. The reactions of the blue and purple forms after photoexcitation are both quantitatively accounted for by a proposed scheme, K in equilibrium with L1 in equilibrium with L2----BR, but with the addition of an L1 in equilibrium with M reaction with unfavorable pKa for Schiff base deprotonation in the purple form. The latter hinders the transient accumulation of M, and the consequent branching at L1 allows only partial proton transport activity. The results are consistent with the existence of a complex counterion for the Schiff base proposed earlier (De Groot, H. J. M., Harbison, G. S., Herzfeld, J., and Griffin, R. G. (1989) Biochemistry 28, 3346-3353) and suggest that Asp85, Asp212, and at least one other protonable residue participate in it.     

Catalysis of the retinal subpicosecond photoisomerization process in acid purple bacteriorhodopsin and some bacteriorhodopsin mutants by chloride ions.

The dynamics and the spectra of the excited state of the retinal in bacteriorhodopsin (bR) and its K-intermediate at pH 0 was compared with that of bR and halorhodopsin at pH 6.5. The quantum yield of photoisomerization in acid purple bR was estimated to be at least 0.5. The change of pH from 6.5 to 2 causes a shift of the absorption maximum from 568 to 600 nm (acid blue bR) and decreases the rate of photoisomerization. A further decrease in pH from 2 to 0 shifts the absorption maximum back to 575 nm when HCl is used (acid purple bR). We found that the rate of photoisomerization increases when the pH decreases from 2 to 0. The effect of chloride anions on the dynamics of the retinal photoisomerization of acid bR (pH 2 and 0) and some mutants (D85N, D212N, and R82Q) was also studied. The addition of 1 M HCl (to make acid purple bR, pH 0) or 1 M NaCl to acid blue bR (pH 2) was found to catalyze the rate of the retinal photoisomerization process. Similarly, the addition of 1 M NaCl to the solution of some bR mutants that have a reduced rate of retinal photoisomerization (D85N, D212N, and R82Q) was found to catalyze the rate of their retinal photoisomerization process up to the value observed in wild-type bR. These results are explained by proposing that the bound Cl- compensates for the loss of the negative charges of the COO- groups of Asp85 and/or Asp212 either by neutralization at low pH or by residue replacement in D85N and D212N mutants.     

A halorhodopsin-overproducing mutant isolated from an extremely haloalkaliphilic archaeon Natronomonas pharaonis

The halorhodopsin (hR)-overproducing mutant strain KM-1 was isolated from the extremely haloalkaliphilic archaeon Natronomonas pharaonis type strain DSM2160(T). hR-enriched membranes were easily obtained by washing the cells with distilled water. The membranes were claret colored owing to two pigments: hR and bacterioruberin. The hR component in the absorption spectra changed from blue to purple upon the addition of Cl(-) and had a K(m) value of 1.7mM. Overexpression of hR in strain KM-1 might be caused by the point mutation Asp324-->Asn in the bacteriorhodopsin activator homologues of N. pharaonis. The mutation changed the hR-expression pattern from inducible to constitutive in the late exponential phase.     

Stopped-flow analysis on anion binding to blue-form halorhodopsin from Natronobacterium pharaonis: comparison with the anion-uptake process during the photocycle

Pharaonis halorhodopsin (phR), the light-driven chloride ion pump from Natronobacterium pharaonis with C-terminal histidine tag, was expressed in Escherichia coli cells. The protein was solubilized with 0.1% n-dodecyl beta-D-maltopyranoside and purified with a nickel column. Removal of Cl- from the medium yields blue phR (phR(blue)) that has lost Cl- near the chromophore. Addition of Cl- converts phR(blue) to a red-shifted Cl--bound form (phR(Cl)). Circular dichroic spectra of phR(blue) and phR(Cl) exhibited a bilobe in the visual region, indicating specific oligomerization of the phR monomers. The order of anion concentration which induced a shift from phR(blue) to phR(X) was Br- < Cl- < NO3- < N3-, which was the same as in the case of phR purified from N. pharaonis membranes. Chloride binding kinetics was measured by time-resolved absorption changes with stopped-flow rapid mixing. Rates of Cl- binding consisted of fast and slow components, and the amplitude of the fast component was about 90% of the total changes. The rate constant of the fast component at 100 mM NaCl at 25 degrees C was 260 s(-1) with an apparent activation energy of 35 kJ/mol. These values are in good agreement with the process of Cl- uptake in the photocycle (O --> hR' reaction) reported previously [Váró et al. (1995) Biochemistry 34, 14500-14507]. In addition, the Cl- concentration dependence on both rates was similar to each other. These observations suggest that the O-intermediate is similar to phR(blue) and that Cl- uptake during the photocycle may be ruled by a passive process.     

Guanidinium restores the chromophore but not rapid proton release in bacteriorhodopsin mutant R82Q.

Replacement of the Arg residue at position 82 in bacteriorhodopsin by Gln or Ala was previously shown to slow the rate of proton release and raise the pK of Asp 85, indicating that R82 is involved both in the proton release reaction and in stabilizing the purple form of the chromophore. We now find that guanidinium chloride lowers the pK of D85, as monitored by the shift of the 587-nm absorbance maximum to 570 nm (blue to purple transition) and increased yield of photointermediate M. The absorbance shift follows a simple binding curve, with an apparent dissociation constant of 20 mM. When membrane surface charge is taken into account, an intrinsic dissociation constant of 0.3 M fits the data over a range of 0.2-1.0 M cation concentration (Na+ plus guanidinium) and pH 5.4-6.7. A chloride counterion is not involved in the observed spectral changes, as chloride up to 0.2 M has little effect on the R82Q chromophore at pH 6, whereas guanidinium sulfate has a similar effect to guanidinium chloride. Furthermore, guanidinium does not affect the chromophore of the double mutant R82Q/D85N. Taken together, these observations suggest that guanidinium binds to a specific site near D85 and restores the purple chromophore. Surprisingly, guanidinium does not restore rapid proton release in the photocycle of R82Q. This result suggests either that guanidinium dissociates during the pump cycle or that it binds with a different hydrogen-bonding geometry than the Arg side chain of the wild type.     

Chloride regulation of enzyme turnover: application to the role of chloride in photosystem II

Chloride-dependent α-amylases, angiotensin-converting enzyme (ACE), and photosystem II (PSII) are activated by bound chloride. Chloride-binding sites in these enzymes contain a positively charged Arg or Lys residue crucial for chloride binding. In α-amylases and ACE, removal of chloride from the binding site triggers formation of a salt bridge between the positively charged Arg or Lys residue involved in chloride binding and a nearby carboxylate residue. The mechanism for chloride activation in ACE and chloride-dependent α-amylases is 2-fold: (i) correctly positioning catalytic residues or other residues involved in stabilizing the enzyme-substrate complex and (ii) fine-tuning of the pKa of a catalytic residue. By using examples of how chloride activates α-amylases and ACE, we can gain insight into the potential mechanisms by which chloride functions in PSII. Recent structural evidence from cyanobacterial PSII indicates that there is at least one chloride-binding site in the vicinity of the oxygen-evolving complex (OEC). Here we propose that, in the absence of chloride, a salt bridge between D2:K317 and D1:D61 (and/or D1:E333) is formed. This can cause a conformational shift of D1:D61 and lower the pKa of this residue, making it an inefficient proton acceptor during the S-state cycle. Movement of the D1:E333 ligand and the adjacent D1:H332 ligand due to chloride removal could also explain the observed change in the magnetic properties of the manganese cluster in the OEC upon chloride depletion.     

Control of bacteriorhodopsin color by chloride at low pH. Significance for the proton pump mechanism

The chromophore of bacteriorhodopsin undergoes a transition from purple (570 nm absorbance maximum) to blue (605 nm absorbance maximum) at low pH or when the membrane is deionized. The blue form was stable down to pH 0 in sulfuric acid, while 1 M NaCl at pH 0 completely converted the pigment to a purple form absorbing maximally at 565 Other acids were not as effective as sulfuric in maintaining the blue form, and chloride was the best anion for converting blue membrane to purple membrane at low pH. The apparent dissociation constant for Cl- was 35 mM at pH 0, 0.7 M at pH 1 and 1.5 M at pH 2. The pH dependence of apparent Cl- binding could be modeled by assuming two different types of chromophore-linked Cl- binding site, one pH-dependent. Chemical modification of bacteriorhodopsin carboxyl groups (probably Asp-96, -102 and/or -104) by 1-ethyl-3-dimethlyaminopropyl carbodiimide, Lys-41 by dansyl chloride, or surface arginines by cyclohexanedione had no effect on the conversion of blue to purple membrane at pH 1. Fourier transform infrared difference spectroscopy of chloride purple membrane minus acid blue membrane showed the protonation of a carboxyl group (trough at 1392 cm -1 and peak at 1731 cm -1). The latter peak shifted to 1723 cm -1 in D2O. Ultraviolet difference spectroscopy of chloride purple membrane minus acid blue membrane showed ionization of a phenolic group (peak at 243 nm and evidence for a 295 nm peak superimposed on a tryptophan perturbation trough). This suggests the possibility of chloride-induced proton transfer from a tyrosine phenolic group to a carboxylate side-chain. We propose a mechanism for the purple to acid blue to chloride purple transition based on these results and the proton pump model of Braiman et al. (Biochemistry 27 (1988) 8516-8520).     

Constitutive activity in chimeras and deletions localize sensory rhodopsin II/HtrII signal relay to the membrane-inserted domain

Halobacterium salinarum sensory rhodopsin II (HsSRII) is a phototaxis receptor for blue-light avoidance that relays signals to its tightly bound transducer HsHtrII (H. salinarum haloarchaeal transducer for SRII). We found that disruption of the salt bridge between the protonated Schiff base of the receptor's retinylidene chromophore and its counterion Asp73 by residue substitutions D73A, N or Q constitutively activates HsSRII, whereas the corresponding Asp75 counterion substitutions do not constitutively activate Natronomonas pharaonis SRII (NpSRII) when complexed with N. pharaonis haloarchaeal transducer for SRII (NpHtrII). However, NpSRII(D75Q) in complex with HsHtrII is fully constitutively active, showing that transducer sensitivity to the receptor signal contributes to the phenotype. The swimming behaviour of cells expressing chimeras exchanging portions of the two homologous transducers localizes their differing sensitivities to the HtrII transmembrane domains. Furthermore, deletion constructs show that the known contact region in the cytoplasmic domain of the NpSRII-NpHtrII complex is not required for phototaxis, excluding the domain as a site for signal transmission. These results distinguish between the prevailing models for SRII-HtrII signal relay, strongly supporting the 'steric trigger-transmembrane relay model', which proposes that retinal isomerization directly signals HtrII through the mid-membrane SRII-HtrII interface, and refuting alternative models that propose signal relay in the cytoplasmic membrane-proximal domain.     

Vibrational modes of the protonated Schiff base in pharaonis phoborhodopsin

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. Recent X-ray crystallographic structures showed that ppR and bacteriorhodopsin (BR), a light-driven proton pump, possess similar molecular environments of the retinal Schiff base. Nevertheless, absorption spectra are different by 70 nm between ppR and BR, suggesting the different chromophore-protein interactions involving the Schiff base region. In this article, we identify frequencies of the Schiff base vibrations in the ppR(K) minus ppR difference spectra by means of low-temperature FTIR spectroscopy of [zeta-(15)N]lysine-labeled ppR. The N-D stretch in D(2)O was found at 2140 and 2091 cm(-1) for ppR, which are shifted to a lower frequency by 32-33 cm(-1) compared to those for BR. This observation indicates the stronger hydrogen bond of the Schiff base in ppR than in BR. The N-D stretch of the Schiff base and O-D stretch of water molecules are located at the different frequencies in ppR, while they appear in the same frequency region in BR [Kandori, H., Belenky, M., and Herzfeld, J. (2002) Biochemistry 41, 6026-6031]. These differences could be correlated with the distorted pentagonal cluster structure in ppR. In contrast, the N-D stretch of ppR(K) was found at 2474 cm(-1), which is close in frequency to that of BR(K). The O-D stretch of Thr79 was also assigned at 2512 and 2474 cm(-1) for ppR and ppR(K), respectively. These frequencies are close to those of BR, suggesting the interaction of Thr79 and Asp75 in ppR is similar to that of Thr89 and Asp85 in BR.     

Insights into the reaction mechanism of Escherichia coli agmatinase by site-directed mutagenesis and molecular modelling.

Upon mutation of Asp153 by asparagine, the catalytic activity of agmatinase (agmatine ureohydrolase, EC 3.5.3.11) from Escherichia coli was reduced to about 5% of wild-type activity. Tryptophan emission fluorescence (lambdamax = 340 nm), and CD spectra were nearly identical for wild-type and D153N agmatinases. The Km value for agmatine (1.6 +/- 0.1 mm), as well as the Ki for putrescine inhibition (12 +/- 2 mm) and the interaction of the enzyme with the required metal ion, were also not altered by mutation. Three-dimensional models, generated by homology modelling techniques, indicated that the side chains of Asp153 and Asn153 can perfectly fit in essentially the same position in the active site of E. coli agmatinase. Asp153 is suggested to be involved, by hydrogen bond formation, in the stabilization and orientation of a metal-bound hydroxide for optimal attack on the guanidinium carbon of agmatine. Thus, the disruption of this hydrogen bond is the likely cause of the greately decreased catalytic efficiency of the D153N variant.