PHOTOCHEMICAL REACTION OF 13-CIS-BACTERIORHODOPSIN STUDIED BY LOW TEMPERATURE SPECTROPHOTOMETRY

Abstract— The photoreaction cycle of 13- cis -bacteriorhodopsin (13- cis -bR) was investigated by low temperature spectrophotometry using two different preparations; 13- cis -bR constituted from bacterioopsin and 13- cis -retinal, and dark-adapted bacteriorhodopsin (bR^D), which is an equi-molar mixture of 13- cis -bR and trans -bR.
By irradiation with 500 nm light at — 190°C, 13- cis -bR was converted to its batho-product, batho-13- cis -bR (batho-bR¹³), which is different from batho-product from trans -bR, batho-bR^t. On warming batho-bR¹³ to -5°C in the dark, it completely changed to trans -bR. We estimated the composition of 13- cis -bR and trans -bR in the warmed sample spectrophotometrically and then the absorption spectrum of batho-bR¹³ was calculated. The absorption maximum lies at 608 nm, 1250 cm⁻¹ longer than that of 13- cis -bR; the molar extinction coefficient (ε) is about 74000 M ⁻¹ cm⁻¹, larger than that of 13- cis -bR (52000 M ⁻¹ cm⁻¹).
On the warming the sample containing batho-bR¹³ formed by irradiating 13- cis -bR or bR^D at — 190°C, we could not detect other intermediates such as the lumi- or meta-intermediates seen in trans-bR system.     

THE REGENERATION OF VISUAL PIGMENTS AND THE CHANGE OF ROD HYPERSENSITIVITY AFTER IRRADIATION BY BLEACHING LIGHT IN FROG RETINA

Abstract— The regeneration processes of visual pigments and the dark adaptation processes of rod photoreceptor after irradiation by bleaching light were studied by spectrophotometric, electroretinographic(ERG) methods and the measurement of early receptor potentials (ERPs) in bullfrog retina. After irradiation by bleaching light, rhodopsin in the isolated retina regenerated to an extent depending on the wavelength and intensity of the bleaching light as well as pH. Intense blue light and a weak alkaline environment (pH 7.5–9.5) favoured the regeneration. The regeneration of pigment in the green rods could not be detected in these experiments on the isolated retina. The regeneration of cone pigment was studied by measuring ERPs from both isolated retinas and retinas with pigment epithelium-choroid complex separated from scleras, which are called PEC-retinas. In the PEC-retinas, cone pigment regenerated more rapidly and with better efficiency than in the isolated retinas.
Rod photoreceptors desensitized permanently by bleaching light did not demonstrate hypersensitivity at 0.1 m M [Ca²⁺]_out, which induced hypersensitivity in non-desensitized photoreceptor, but showed the hypersensitivity when the [Ca²⁺]_out, was lowered further by the addition of EGTA.     

QUANTUM EFFICIENCY FOR THE PHOTOCONVERSION OF BACTERIORHODOPSIN AT VERY LOW TEMPERATURES

Abstract— Quantum efficiencies for photoconversion of bacteriorhodopsin (trans-bR) to a bathochromic product (batho-bR^t) and its photoreversion in purple membrane at 77 and 9 K were investigated with low temperature spectrophotometry. The kinetics of the photoconversion and its photoreversion cannot be expressed by a single exponential curve. The photoconversions at 77 and 9 K showed the same slope in the early stage. The kinetics of the photoreversions were identical at the two temperatures. These results indicate that the quantum efficiencies for the conversion of trans-bR to batho-bR^t or for its photoreversion are identical at the two temperatures.
The fact that the photoreversion cannot be expressed by a single exponential curve suggests the existence of several conformational states of batho-bR^t due to the trimer structure of the purple membrane.     

Recent bioorganic studies on rhodopsin and visual transduction

Rhodopsin, the pigment responsible for vision in animals, insect and fish is a typical G protein (guanyl-nucleotide binding protein) consisting of seven transmembrane alpha helices and their interconnecting extramembrane loops. In the case of bovine rhodopsin, the best studied of the visual pigments, the chromophore is 11-cis retinal attached to the terminal amino group of Lys296 through a protonated Schiff base linkage. Photoaffinity labeling with a 3-diazo-4-oxo-retinoid shows that C-3 of the ionone ring moiety is close to Trp265 in helix F (VI) in dark inactivated rhodopsin. Irradiation causes a cis to trans isomerization of the 11-cis double bond giving rise to the highly strained intermediate bathorhodopsin. This undergoes a series of thermal relaxation through lumi-, meta-I and meta-II intermediates after which the retinal chromophore is expelled from the opsin binding pocket. Photoaffinity labeling performed with 3-diazo-4-oxoretinal at -196 degrees C for batho-, -80 degrees C for lumi-, -40 degrees C for meta-I, and 0 degrees C for meta-II rhodopsin showed that in bathorhodopsin the ring is still close to Trp265. However, in lumi-, meta-I and meta-II intermediates crosslinking occurs unexpectedly at A169 in helix D (IV). This shows that large movements in the helical arrangements and a flip over of the ring moiety accompanies the transduction (or bleaching) process. These changes in retinal/opsin interactions are necessarily accompanied by movements of the extramembrane loops, which in turn lead to activation of the G protein residing in the cytoplasmic side. Of the numerous G protein coupled receptors, this is the first time that the outline of transduction pathway has been clarified.     

SPECTRAL PROPERTIES OF THE RHODOPSIN-SYSTEM OF THE CRAYFISH Astacus leptodactylus

Abstract— The absorption spectra of the membrane-bound and of the digitonin-solubilized visual pigment of crayfish Astacus leptodactylus were investigated by conventional spectrophotometry. A method was developed to isolate purified rhabdoms almost entirely free from screening pigments from a single retina. The quantity of isolated and purified rhabdoms from a single retina was sufficient to measure the absorption spectra of the visual pigment.
The absorption spectra of the chromoprotein system (R and M) show that both the membrane-bound and the digitonin-solubilized visual pigment isomers are stable at 0°C and pH 7.0. Rhodopsin and metarhodopsin are photoreversible under these conditions without any light-induced denaturation. The difference spectra for the chromoprotein isomers and those of different photostationary states yield maximal values for ΔE at 570 and 485 nm.
At neutral pH, 0°C, Λ_max of rhodopsin is 530 nm. Irradiation with light of Λ= 630 to 640 nm isomerizes rhodopsin nearly quantitatively to metarhodopsin with Λ_max, of 500 nm. The molar extinction coefficient of metarhodopsin is greater than that of rhodopsin by a factor of ˜ 1.41. each measured at its respective Λ_max Metarhodopsin can be isomerized to rhodopsin by irradiating at Λ > 630 nm. As the absorption spectra of the two chromoprotein isomers overlap, only part of the metarhodopsin can be reversed to rhodopsin. The maximal photoreversion can be achieved by irradiating at 460 nm. The stability of the digitonin-solubilized chromoprotein is remarkably dependent on temperature. Warming the digitonin extract of rhabdoms from 0 to 20 or 30°C caused a shift of the rhodopsin spectrum to shorter wavelengths (Λ_max= 485 nm) accompanied by a decrease of E_Λmax by about 30%.     

RESONANCE RAMAN STUDIES OF BACTERIORHODOPSIN ANALOGUES

Abstract— We present the results of resonance Raman measurements on a series of bacteriorhodopsin (bR) analogues formed from synthetic retinals which have replaced the native chromophore in the active site. Specifically, 5,6-dihydro-bR, 13-desmethyl-bR, 10-methyl-bR, 14-methyl-bR, and 10.14-dimethyl-bR have been studied. All five analogues bind and form Schiff base retinal-apoprotein linkages. While the Schiff base linkages of 5,6-dihydro-bR, 13-desmethyl-bR, and 10-methyl-bR are protonated, like the native chromophore, the 14-methyl-bR, and 10,14-dimethyl-bR Schiff bases are unprotonated. These results suggest that the binding site of bacteriorhodopsin near the Schiff base moiety is different from that of rhodopsin. The protonated Schiff base -C=NH- stretching frequency of 5.6-dihydro-bR lies at 1660 cm^-1 which is unusually high for a bacteriorhodopsin based pigment. The downward shift upon deuteration is 16 cm^-1, essentially identical to that measured for bacteriorhodopsin. This and the other analogue results strongly reinforce our previous arguments that the Schiff base stretching frequency is determined in large part by two factors, the C=N force constant and the stretch interaction with C=N-H bend. On the other hand, the deuterium isotope effect is determined primarily by the stretch-bend interaction.     

LOW-TEMPERATURE TRAPPING OF EARLY PHOTOINTERMEDIATES OF α-ISORHODOPSIN

Abstract— a-Isorhodopsin, an artificial visual pigment with a 9- cis -4,5-dehydro-5,6-dihydro(a)retinal chromophore, was photolyzed at low temperatures and absorption difference spectra were collected as the sample was warmed. A bathorhodopsin (Batho)-like intermediate absorbing at ca 495 nm was detected below 55 K, a blue-shifted intermediate (BSI)-like intermediate absorbing at ca 453 nm was observed when the temperature was raised to 60 K and a lumirhodopsin (Lumi)-like intermediate absorbing at ca 470 nm was found when the sample was warmed to 115 K. Photointermediates from this pigment were compared to those of native rhodopsin and 5,6-dihydroisorhodopsin. As in native rho-dopsin, Batho is the first intermediate detected in a-isorhodopsin, though unlike native rhodopsin at low temperatures BSI is observed prior to Lumi formation. a-Isorhodopsin behaves similarly to 5,6-dihydroisorhodopsin, with the same early intermediates observed in both artificial visual pigments lacking the C₅-C₆ double bond. The transition temperature for BSI formation is higher in a-isorhodopsin, suggesting an interaction involving the chromophore ring in BSI formation. The transition temperature for Lumi formation is similar for these two pigments as well as for native rhodopsin, suggesting comparable changes in the protein environment in that transition.     

MIGRATION OF EXCITATION ENERGY FROM THIONINE TO METHYLENE BLUE IN MICELLES

Abstract— The main absorption bands of thionine (Th⁺) and methylene blue (MB⁺) in aqueous solution lie at 598 nm and 664 nm, respectively. This position permits excitation energy transfer from Th⁺ to MB⁺, but not vice versa. We describe here studies of such transfer between these molecules adsorbed on micelles of sodium lauryl sulfate (SLS), imitating, at least to some extent, the state of pigments in chloroplasts.
The SLS concentration was varied from 3.0 to 11 × 10^-3 M. In the presence of dye, aggregation to micelles, each containing 70–100 detergent molecules, begins at about 3.0 × 10^-3 M SLS. Practically all dye ions are adsorbed on these micelles as soon as their formation begins.
Energy transfer from adsorbed Th⁺ ions to adsorbed MB⁺ ions can be demonstrated by observing the quenching of the fluorescence of thionine and the sensitization of that of methylene blue.
At [Th⁺] = [MB⁺] = 1 × 10^-5 M , the most efficient energy transfer (82 per cent efficiency, as derived from measurements of the quenching of Th⁺ fluorescence, or 90 per cent, as derived from sensitization of MB⁺ fluorescence) is observed at the lowest SLS-concentration (3.0 × 10^-3 M ), when the only micelles present are those formed by aggregation of dye-carrying low molecular complexes of SLS with dye cations. Each micelle carries, under these conditions, 10–14 molecules of the two dyes, and the distance between two closest dye ions is about 16 A. Transfer becomes less efficient as the SLS-concentration increases, causing pigment molecules to distribute themselves among a greater number of micelles.     

DRUG SENSITIZATION: PHOTOBINDING OF SULFANILAMIDE TO BOVINE SERUM ALBUMIN

Abstract— Photobinding of sulfanilamide to bovine serum albumin (BSA) was investigated by irradiating BSA and buffered BSA/drug solutions with UV light (Λ= 300 nm) under anaerobic conditions. The protein solutions were then denatured and the unbound sulfanilamide removed. Marked differences in the UV and fluorescence spectra of the solutions before and after irradiation were observed, suggesting covalent binding of the drug to BSA. This was confirmed using [¹⁴C]labelled sulfanilamide. The extent of photobinding of sulfanilamide determined using the radiolabeled drug, was concentration dependent. The binding ratio varied from 3 mol drug per mol BSA for a 10^-4 M drug concentration, to 10 mol drug per mol BSA for 10^-2 M drug concentration.
The protein solutions were hydrolysed under acid conditions and the amino acids obtained were analysed by ion exchange chromatography. The hydrolysate of irradiated BSA (10^-4 M ) -sulfanilamide (10^-2 M ) mixture lost about 10 mol of cystine per mol of BSA. This loss was not observed after hydrolysis of irradiated alone or non-irradiated BSA. Irradiation of cystine with [¹⁴C]sulfanilamide in HC1 solutions produced the same compound as was found after hydrolysis of irradiated BSA/sulfanilamide mixtures. This was demonstrated by autoradiography of paper chromatograms. The same compound was also detected in an irradiated [³⁵S]cystine non-labelled sulfanilamide mixture. It was not detected, however, after irradiation of a mixture of all amino acids of BSA excluding cystine. These data suggest that cystine residues are involved in the photobinding of sulfanilamide (or its photoproducts) to BSA.     

QUANTUM EFFICIENCY AND PHOTOSENSITIVITY OF THE RHODOPSIN ⇌ METARHODOPSIN CONVERSION IN CRAYFISH PHOTORECEPTORS

Photosensitivity (K_λ) of a visual pigment is the product of the molecular absorption coefficient (α_λ) and the quantum efficiency for photoconversion (γ). Among the invertebrates, many visual pigments are stable not only in the rhodopsin (R) conformation but also as the photoproduct, metarhodopsin (M), We here employ a method for determining the photosensitivities of the two stable pigments of a rhodopsin-metarhodopsin pair, using kinetic analysis of fluorescence from metarhodopsin combined with measurements of spectral absorption made before and after saturation at the isosbestic wavelength of the pigment pair. A curve fitting technique, in which a theoretical function is scaled for best fit to the measured absorption spectrum of the photosteady-state mixture, yields values for the photosensitivity of rhodopsin at λ._max, the ratio of quantum efficiencies for rhodopsin—metarhodopsin interconversion, and the fractional composition of the steady-state mixture. With knowledge of the molecular extinction coefficient, the absolute values of quantum efficiency can be calculated. For crayfish ( Orconectes, Procambarus ) rhodopsin, measured in isolated rhabdoms, K_max= 1.05 x 10^-16 cm² at 535 nm with >7λR→M0.69. These values are similar to the photosensitivity and quantum efficiency of bleaching of vertebrate rhodopsins in digitonin solution (Dartnall, 1972). For the metarhodopsin, K_max= 1.02 x 10^-16 cm² at 510 nm, and λ_M-R= 0.49.     

PICOSECOND TRANSIENT BEHAVIOR OF REACTION-CENTER PARTICLES OF PHOTOSYSTEM I ISOLATED FROM SPINACH CHLOROPLASTS. ENERGY AND ELECTRON TRANSFER UPON SINGLE AND MULTIPLE PHOTON EXCITATION

Abstract— Both [15-¹³C] and [14-¹³C] all-trans-retinals were synthesized. Bacteriorhodopsin containing [14-¹³C]retinal as a chromophore, when solubilized with octyl-β-D-glucoside, showed characteristic resonances at 125 and 118 ppm from tetramethyl silane. The former was assigned to the signal from free retinal and the latter from protonated Sehiff base. When the bacteriorhodopsin was denatured in sodium dodecyl sulfate, the signal at 118 ppm disappeared, while the signal at 125 ppm rather increased.
In the case of bacteriorhodopsin containing [15-¹³C]retinal, when solubilized with Triton X-100, a characteristic resonance at 169 ppm was distinguishable as a shoulder peak superimposed on the broad signal of carbonyl carbons and it was assigned to the signal from the protonated Sehiff base. The other signal observed at 191 ppm was from free retinal.
These results suggested that the Sehiff base of bacteriorhodopsin is protonated in the dark.     

THE FORMATION OF TWO FORMS OF BATHORHODOPSIN AND THEIR OPTICAL PROPERTIES

Abstract— Using two kinds of rhodopsin preparations (digitonin extract and rod outer segments suspension), we measured changes in absorption spectra during the conversion of rhodopsin or isorhodopsin to a photosteady state mixture composed of rhodopsin, isorhodopsin and bathorhodopsin by irradiation with blue light (437 nm) at 77 K and during the reversion of bathorhodopsin to a mixture of rhodopsin and isorhodopsin by irradiation with red light (> 650 nm) at 77 K. The reaction kinetics could be expressed with only one exponential in the former case and with two exponentials in the latter case. These data suggest that both rhodopsin and isorhodopsin are composed of a single molecular species, while bathorhodopsin is composed of two molecular species, designated as bathorhodopsin₁ and bathorhodopsin₂. The absorption spectra of these bathorhodopsin were calculated by two different methods (kinetic method and warming-cooling method). The former was based on the kinetics of the conversion of two forms of bathorhodopsin by irradiation with the red light. The spectra obtained by this method were consistent with those obtained by the warming-cooling method. Bathorhodopsin₁ and bathorhodopsin₂ have Λ_max at 555 and 538 nm, respectively. The two forms of bathorhodopsin are interconvertible in the light, but not in the dark. Thus, we suggest that a rhodopsin molecule in the excited state relaxes to either bathorhodopsin₁ or bathorhodopsin₂ through one of the two parallel pathways.     

THE EFFECT OF THE ANTIPSORIATIC DRUG METABOLITE ETRETIN (Ro 10-1670) ON UVB IRRADIATION INDUCED CHANGES IN THE METABOLISM OF ARACHIDONIC ACID IN HUMAN KERATINOCYTES IN CULTURE

[¹⁴C]Arachidonic acid was avidly incorporated into human keratinocytes in culture and following exposure to UVB irradiation of 9 mJ/cm² (erythemally effective, EE) substantial amounts of ¹⁴C-radiolabel were released from the cells. The release of radiolabel was accompanied by a decrease in the labelling of phosphatidylethanolamine whereas the labelling of triacylglycerols and cholesteryl esters was increased. Keratinocytes produced significant amounts of prostaglandin E₂ (PGE₂) and following UVB irradiation of 9 mJ/cm² (EE) the formation of prostaglandin E₂ was increased.
Etretin (Ro 10-1670), the active metabolite of the antipsoriatic drug etretinate (Ro 10-9359), affected significantly neither the total release of radiolabel induced by UVB nor the formation of prostaglandin E₂. However, in the presence of etretin the UVB irradiation induced transfer of [^l4C]arachidonic acid into triacylglycerols and cholesteryl esters was not increased as much as in the corresponding experiments without etretin. On the basis of the present study it appears that etretin does not interfere with the release of arachidonic acid in amounts which could be related to the therapeutic effects of the combination of retinoids with UVB irradiation (Re-UVB) in the treatment of psoriasis.     

FLUORESCENCE KINETICS OF SPINACH CHLOROPLASTS MEASURED WITH A PICOSECOND OPTICAL KERR GATE

Abstract. An overview of the reported chlorophyll a fluorescence lifetimes from green plant photosystems is presented and the problems encountered in the measurement of fluorescence lifetime using two currently available picosecond techniques are discussed.
The fluorescence intensity of spinach chloroplasts exposed to 10 ps flashes was measured as a function of time after the flash and wavelength of observation by the ultrafast Kerr shutter technique. Using a train of 100 pulses separated by 6ns and with an average photon flux per pulse of ˜2 times 10¹⁴ photons/cm², the fluorescence intensity at 685 nm (room temperature) was found to decay with two components, a fast one with a 56 ps lifetime, and a slow one with a 220 ps lifetime. The 730 nm fluorescence intensity at room temperature decays as a single exponential with a 100 ps lifetime. The 730 nm fluorescence lifetime was found to increase by a factor of 6 when the temperature was lowered from room temperature to 90 K while the lifetime of 685 and 695 nm fluorescence were unchanged. At room temperature, the fast and slow components at 685 nm are attributed to the emission from pigment system I (PS I) and PS II, respectively. It is likely that the absolute values of lifetimes, reported here, may increase if single ps low intensity flashes are used for these measurements.     

EVIDENCE FOR A MAGNESIUM DEPENDENT ATPase IN BOVINE ROD OUTER SEGMENT DISK MEMBRANES*†

Abstract—In the presence of Mg²⁺ and adenosine triphosphate (ATP), a rapid. light-induced, light-scattering transient is observed from bovine rod outer segments (ROS). This light-scattering transient we have labelled 'A'. Ca²⁺ cannot replace Mg²⁺. nor can guanosine triphosphate (GTP) replace ATP. 'A' is observed at ATP concentrations as low as a few μM.
The half-time of 'A', 60 ms at 20° and 20 ms at 37°, is consistent with a process possibly involved in visual transduction.
'A' has the action spectrum of rhodopsin bleaching and its amplitude is strictly proportional to the fraction of rhodopsin bleached per flash. 'A' can be regenerated by 11- cis retinal.
Inhibition studics with ATP analogues, which cannot be hydrolysed and fail to evoke an 'A' response, reveal that an ATP hydrolysis process has to precede illumination in order for 'A' to occur.
On the basis of the above findings. it is proposed that there is a Mg²⁺ dependent ATPase in ROS that allows the disk membrane to assume a new membrane state which, upon illumination, is altered. giving rise to the structural phenomenon monitored as light-scattering transient 'A'.     

BACTERIORHODOPSIN AND THE SENSORY PIGMENT OF THE PHOTOSYSTEM 565 IN HALOBACTERIUM HALOBIUM

Abstract Blocking in vivo synthesis of retinal by addition of nicotine to the culture medium leads to the loss of photobehavior in Halobacterium halobium. Addition of rrans -retinol or frans-retinol₂ (3,4-dehy-droretinol) restores the responses to light decreases in the green-yellow spectral range. Action spectra of the reconstituted retinal- and retinal₂-photosystem show maximal sensitivity at 565 and 580 nm, respectively. Addition of retinol or retinol₂ also restores the formation of bacteriorhodopsin (BR) or bacteriorhodopsin₂ (BR₂= 3,4-dehydroretinal-bacterio-opsin complex). The absorption spectra of BR and BR₂, measured in isolated membranes, as well as in living bacteria, show maxima at 568 nm (BR) and at about 600 nm (BR₂), respectively. Comparison of the action spectrum of the retinal2-containing sensory photosystem with the absorption spectrum of BR₂ suggests that a retinal pigment different from BR is responsible for the photosensory behavior to green-yellow light.     

PHOTOPRODUCTION OF HYDROGEN FROM LINKED CHROMOPHORES

Abstract On irradiation of solutions of anthryl-substituted cobalt(III) cage complexes, [(l-(anthryl-9-methylamino)-8-methyl-3,6,10,13,16,19-hexaazabicyclo [6.6.6] eicosane) cobalt(III)]³⁺ or [(l-(4-an-thryl-9)-3-aza-butyl-l-amino)-8-methyl-3,6,10,13,16,19-hexaazabicyclo [6.6.6] eicosane)cobalt (III)]³* in the presence of ethylenediaminetetraacetic acid and platinum catalysts hydrogen was produced. These complexes act as coupled photosensitizers (anthracene moiety) and electron relays (cobalt cage) to produce H₂ via energy trapping and intramolecular electron transfer initially. The intensity of fluorescence and the photochemical reactivity favour the latter complex and the excited singlet state of the anthracene chromophore is invoked as the intermediate state leading to the reduction of Co(III) to Co(II).     

MITOCHONDRIAL DNA SYNTHESIS IN NON-GROWTH CONDITION (LIQUID HOLDING) IN Saccharomyces cerevisiae. RELATION WITH GROWTH STAGES and MITOCHONDRIAL DNA REPAIR AFTER UV-IRRADIATION

Abstract— The synthesis of DNA as measured by incorporation of [¹⁴C]adenine in Saccharomyces cerevisiae liquid held in non-growth conditions was followed in controls and UV-irradiated cells. The incorporation into mitochondrial DNA relative to total DNA was higher in these conditions than that observed in growth medium, especially in liquid held stationary phase cells. The absolute amount of mitochondrial DNA synthesized during liquid holding was larger in exponential than in stationary phase cells and increased after UV-irradiation.
The data reported here are discussed in relation to the effects of liquid holding on the UV-induction of rho ⁻ mutants, such effects depending upon the growth stage of the cells at the time of irradiation. A correlation has been found between the initial ability of the cells to synthesize mitochondrial DNA in liquid holding conditions and their capacity to repair UV-induced lesions in this DNA. We propose the hypothesis that the opposite effects of liquid holding on the UV-induction of rho ⁻ mutants observed in exponential versus stationary phase cells are not due to the action of different repair pathways, but result essentially from quantitative differences in mtDNA synthesis.     

THE ELECTRON SPIN POLARIZATION OF THE DONOR-TRIPLET STATE IN NATIVE and MODIFIED PHOTOSYNTHETIC REACTION CENTERS FROM

The electron spin polarization (ESP) pattern of the donor-triplet state (P^R) of reaction centers (RC's) of the purple bacterium Rhodobacter (formerly Rhodopseudomonas) sphaeroides R-26 was investigated. δm =±1 triplet EPR spectra were recorded of unmodified RC's as well as of RC's from which Fe²⁺ or ubiquinone was removed, or ubiquinone was substituted by menaquinone.
The relative amplitude of the Y peaks in the triplet EPR powder spectrum of P^R decreases when the temperature is increased from 8 K to 100 K in RC's with an intact quinone-iron complex. This decrease is more pronounced when the primary ubiquinone is substituted by menaquinone. These observations provide further support for the hypothesis that the observed lineshape of the P^R triplet state EPR spectrum reflects the presence of a third electron spin, magnetically coupled to I^- in the P⁺I^- radical pair, as suggested by Van Wijk et al. (1986) (Photobiochem. Photobiophys . 11, 95–100). Our observations suggest that this phenomenon may be general in purple bacteria.     

Effects of Substitutions D73E, D73N, D103N and V106M on Signaling and pH Titration of Sensory Rhodopsin II

Abstract— Several mutations in the repellent phototaxis receptor sensory rhodopsin II (SRII), in residues homologous to residues important in the related proton pump bacteriorhodopsin, were expressed in Pho81Wr^–, a Halobacterium salinarum strain deficient in production of SRII and its transducer protein HtrII. The lack of production of SRII and HtrII is shown to be due to insertion of an ISH2 transposon into the promoter region upstream of the htrII - sopH gene pair. Near wild-type phototaxis responses are rescued in Pho81Wr^– by expression of HtrII with D73E, D103N or V106M receptors. Partial responses are restored by the HtrII-D73N pair. From absorption spectroscopy of his-tag-purified receptor protein from mutants D73N and D73E we conclude that Asp73 is the primary counterion to the protonated Schiff base in SRII, like the corresponding Asp85 in bacteriorhodopsin. The absorption maximum of SRII (487 nm) is shifted to 514 nm in mutant D73N, a 1080 cm⁻¹ shift identical to that caused by D85N in bacteriorhodopsin. Acid titration of SRII also induces the red shift with a pK of 3.0 in wild type. The absorption shift and the pK are nearly the same in V106M and D103N, but the pK is raised to 5.1 in D73E, confirming that Asp73 is the residue responsible for this spectral transition.