Low-temperature Spectrophotometry Research Articles

Characteristics of the Photoconversion of Rhodopsin in the Early Stages of Photolysis

Low-temperature spectrophotometry was used to study the primary stages of rhodopsin photolysis. A digitonin extract of rhodopsin was irradiated at -155 degrees C with blue light of wavelength 436 nm. The stage of the bathorhodopsin --> lumirhodopsin conversion was accompanied by the simultaneous formation of several products. Formation of an intermediate product spectrally similar to the known "blue-shifted intermediate" (BSI) was demonstrated. It is suggested that the appearance of more than one intermediate product at each stage of photolysis reflects the existence of several conformational states of the rhodopsin molecule during its photoconversion.

Photolysis of [PtBr6]2- complex in frozen methanol matrix

Photochemistry of the [PtBr6]2– complex in the low-temperature methanol matrix (77 K) was studied by low-temperature spectrophotometry and ESR spectroscopy. The [PtBr4]2– complex is the main photolysis product. The mechanism of two-electron reduction of the platinum(IV) ion is proposed. The primary photochemical process is electron transfer from the solvent molecule to the photoexcited initial complex to form the intermediate radical complex ([PtBr6]3–...·CH2OH). The transfer of the second electron in the radical complex produces the final reaction products.

Photochemistry of the IrCl 2−6 complex in methanol matrices

Low-temperature spectrophotometry and electron spin resonance (ESR) were used to study the IrC 2− 6 photochemistry in methanol matrices frozen at 77 K. Photoreaction gives rise to the absorption band of an IrCl 3− 6 complex with a maximum at 215 nm and to a new band with a maximum at 287 nm that is unobservable during IrCl 2− 6 photolysis in solutions at room temperature. The appearance of this new band is assigned to the formation of a weak complex between the .CH 2OH radical and IrCl 3− 6 ion (i.e. a IrCl 3− 6 … .CH 2OH radical complex). The irradiation of methanol matrix with IrCl 2− 6 gives rise to the .CH 2OH radical signal in the ESR spectrum with additional splitting which can be attributed to the partial delocalization of spin density from radical to the IrCl 3− 6 complex and the appearance of hyperfine splitting (HFS) on the iridium nucleus. When the matrix is heated to 115 K the IrCl 3− 6 … .CH 2OH complex disappears.

Spectral properties and photoactivities of intermediates of photoconversion from the red-light-absorbing to far-red-light-absorbing form of pea phytochrome

The pathway for the phototransformation of the red-light-absorbing (Pr) to the far-red-light-absorbing (Pfr) form of native phytochrome A from pea was investigated by low temperature spectrophotometry. Four intermediates between Pr and Pfr were recognized. The first intermediate (lumi-R) was stable below −100°C. The other intermediates and Pfr appeared during warming of lumi-R to −80 °C (meta-Ra), −40 °C (meta-Rb′), −20 °C (meta-Rc) and 0 °C (Pfr). Each intermediate (lumi-R, meta-Ra and meta-Rc) and Pr was photointerconvertible as a two-component system at the temperature at which the intermediate had initially appeared. Meta-Rb′ is also photoconvertible to Pr. Prolonged red light irradiation of meta-Rb′ at −40 °C resulted in appearance of another intermediate (meta-Rb).

Resolution of the Aerobic Respiratory System of the Thermoacidophilic Archaeon, Sulfolobus sp. Strain 7:: I. THE ARCHAEAL TERMINAL OXIDASE SUPERCOMPLEX IS A FUNCTIONAL FUSION OF RESPIRATORY COMPLEXES III AND IV WITH NO c-TYPE CYTOCHROMES

The aerobic respiratory system of the thermoacidophilic archaeon, Sulfolobus sp. strain 7, is unusual in that it consists of only a- and b-type cytochromes but no c-type cytochromes. In previous studies, a novel cytochrome oxidase a583-aa3 subcomplex has been purified, which showed a ferrocytochrome c oxidase but no caldariellaquinol oxidase activity (Wakagi, T., Yamauchi, T., Oshima, T., Müller, M., Azzi, A., and Sone, N. (1989) Biochem. Biophys. Res. Commun. 165, 1110-1114). We show here that the cytochrome subcomplex could be copurified with a non-CO-reactive cytochrome b562 as a novel terminal oxidase "supercomplex," which also contained a Rieske-type FeS cluster at gy = 1.89. It contained one copper and all four heme centers detectable in the archaeal membranes by the low temperature spectrophotometry and the potentiometric titration: cytochromes b562 (+146 mV), a583 (+270 mV), and aa3 (+117 and +325 mV). The presence of one copper atom indicates that it contains the conventional heme a3-CuB binuclear center for reducing molecular oxygen. In conjunction with the presence of a Rieske-type FeS center, inhibitor studies suggest that the terminal oxidase segment of the respiratory chain of Sulfolobus sp. strain 7 is a functional fusion of respiratory complexes III and IV, where cytochrome b562 and the Rieske-type FeS center probably play a central role in the oxidation of caldariellaquinol. This archaeal terminal oxidase supercomplex reconstitutes the in vitro succinate oxidase respiratory chain for the first time together with caldariellaquinone and the purified cognate succinate:caldariellaquinone oxidoreductase complex. The reconstitution system requires caldariellaquinone for the activity, and is highly sensitive to cyanide and 2-heptyl-4-hydroxy-quinoline-N-oxide. These results are also discussed in terms of the evolutionary considerations.

Thermal recovery of iodopsin from its meta I-intermediate

The thermal reaction of meta I-intermediate of iodopsin (metaiodopsin I), a chicken red-sensitive cone pigment, was studied by low-temperature spectrophotometry at −20°C. Irradiation of iodopsin at −20°C produced metaiodopsin I, whose absorption maximum was at about 470 nm. An incubation of metaiodopsin I at −20 °C resulted in a conversion to metaiodopsin II having absorption maximum at about 380 nm, as well as a concurrent formation of a red-shifted product stable at room temperature. Since the absorption spectrum and photo-reactivity of the red-shifted product were identical with those of iodopsin, the red-shifted product should be iodopsin. Thus a part of metaiodopsin I can revert to iodopsin by the thermal reaction unlike metarhodopsin I.

Photocycle of phoborhodopsin from haloalkaliphilic bacterium (Natronobacterium pharaonis) studied by low-temperature spectrophotometry.

Phoborhodopsin (pR) is the fourth retinal pigment of Halobacterium halobium and works as a photoreceptor for the negative phototactic response. A similar pigment was previously found in haloalkaliphilic bacterium (Natronbacterium pharaonis) and also works as the receptor of the negative phototactic response; this pigment is called pharaonis phoborhodopsin (ppR). In this paper, the photocycle of ppR was investigated by means of low-temperature spectrophotometry. The absorption maximum of ppR is located at 498 nm, while that of pR is at 487 nm. The absorption spectra of the two have similar vibrational structures. Irradiation of ppR below -100 degrees C produced a K-like intermediate (ppRK) which was a composite of two components. The original ppR and ppRK were perfectly photoreversible. On warming, ppRK was directly converted to an M-like intermediate without formation of the L-like intermediate. The M-like intermediate was converted to the O-like intermediate at pH 7.2, but the O-like intermediate was not detected at pH 9.0. The O-like intermediate then reverted to the original pigment. On the basis of these findings, the photocycle and the primary photochemical process of ppR are presented.

Photoreaction cycle of phoborhodopsin studied by low-temperature spectrophotometry

The photochemical and subsequent thermal reactions of phoborhodopsin (pR490), which mediates the negative phototaxis (phobic reaction) of Halobacterium halobium, were investigated by low-temperature spectrophotometry. At room temperature, the absorption spectrum of pR490 displayed vibrational structure with a maximum at 490 nm and a shoulder at 460 nm, which were remarkably sharpened by cooling, resulting in the appearance of two well-separated peaks. On irradiation of pR490 at -170 degrees C, a photo-steady-state mixture composed of pR490 and two photoproducts, P520 and P480, was formed. P480 had an absorption maximum at 480 nm and thermally converted to pR490 above -160 degrees C, while P520 had an absorption maximum at 515 nm and thermally converted to P350, the next intermediate, above -60 degrees C. Above -30 degrees C, P350 was converted to P530, and then reverted to pR490. P520, P350, and P530 may correspond to K, M, and O intermediates of bacteriorhodopsin, respectively, on the basis of their absorption spectra, but the intermediates corresponding to L and N intermediates were not observed. On the basis of these results, a new scheme of the photoreaction cycle of pR490 was presented.

Differences in the photobleaching process between 7-cis- and 11-cis-rhodopsins: a unique interaction change between the chromophore and the protein during the lumi-meta I transition

The photochemical and subsequent thermal reactions of 7-cis-rhodopsin prepared from cattle opsin and 7-cis-retinal were investigated by low-temperature spectrophotometry and laser photolysis, and compared with those of 11-cis-rhodopsin prepared from cattle opsin and 11-cis-retinal. Low-temperature experiments revealed that the absorption maxima of batho and lumi intermediates from 7-cis-rhodopsin were at slightly shorter wavelengths than those of 11-cis-rhodopsin while the meta I intermediates of both rhodopsin isomers showed the same absorption maxima. Kinetic experiments of the photobleaching process of 7-cis-rhodopsin using picosecond and nanosecond laser pulses revealed the formation of intermediates corresponding to the batho, lumi, meta I, and meta II intermediates from 11-cis-rhodopsin. An intermediate of 7-cis-rhodopsin corresponding to photorhodopsin (a precursor of bathorhodopsin), however, was not detected. Batho and lumi intermediates from 7-cis-rhodopsin had shorter lifetimes (approximately 40 ns and 300 microseconds) than those of 11-cis-rhodopsin (250 ns and 800 microseconds), but the lifetime of the meta I intermediate from 7-cis-rhodopsin was identical with that from 11-cis-rhodopsin (12 ms). These results indicate that the difference in configuration of the original chromophore between 7-cis- and 11-cis-rhodopsins is a cause of different chromophore-opsin interactions in the batho and lumi stages, while in the meta I stage the difference has disappeared by the relaxation of the protein near the chromophores. A possible interaction change between the 9-methyl group of the chromophore and its neighboring protein during the lumi-meta I transition will be discussed.

Spectroscopic study of the batho-to-lumi transition during the photobleaching of rhodopsin using ring-modified retinal analogs

Photochemical and subsequent thermal reactions of rhodopsin containing 9-cis-retinal [Rh(9)] or one of four analogues with 9-cis geometries formed from ring-modified retinals, alpha-retinal [alpha Rh(9)], acyclic retinal [AcRh(9)], acyclic alpha-retinal [Ac alpha Rh(9)], and 5-isopropyl-alpha-retinal [P alpha Rh(9)] were investigated by low-temperature spectrophotometry and nanosecond laser photolysis. Irradiation of each pigment at -180 degrees C produced a photosteady-state mixture containing the original 9-cis pigment, its 11-cis pigment, and a photoproduct, indicating that the primary process of each pigment is a photoisomerization of its chromophore. The photoproduct produced by the irradiation of AcRh(9) had an absorption spectrum red shifted from the original AcRh(9) and was identified as the batho intermediate of AcRh(9). It was converted to the lumi intermediate through a metastable species, the BL intermediate, which has never been detected in Rh(9) at low temperature and whose absorption maximum was at shorter wavelengths than that of the batho intermediate. In contrast, the absorption maxima of the photoproducts produced from the other analogue pigments were at shorter wavelengths than those of the original pigments. They were identified as BL intermediates on the basis of their absorption maxima and thermal stabilities. The formation time constant of the lumi intermediate at room temperature was found to be dependent on the extent of modification of the ring portion of the chromophore, decreasing with the complete truncation of the cyclohexenyl ring [Ac alpha Rh(9)] and increasing with the attachment of the isopropyl group to the ring [P alpha Rh(9)].(ABSTRACT TRUNCATED AT 250 WORDS)

Bathoiodopsin, a primary intermediate of iodopsin at physiological temperature.

Measurement of the primary photochemical reaction of iodopsin, a chicken red-sensitive cone visual pigment, was carried out at room temperature by using picosecond (ps) laser photolysis. Excitation of iodopsin with a ps green pulse (pulse width, 21 ps) caused the instantaneous formation of a bathochromic product, which was stable on a ps time scale. This product may correspond to "bathoiodopsin," which was detected by low-temperature spectrophotometry. Although bathoiodopsin produced at the temperature of liquid nitrogen or helium reverted to the original pigment (iodopsin) on warming (above -170 degrees C), the bathoiodopsin produced at physiological temperature decayed to all-trans-retinal and R-photopsin (the protein moiety of iodopsin) presumably through several intermediates. The absorption maximum of bathoiodopsin at room temperature was at 625 nm, a wave-length slightly shorter than that measured at low temperature (lambda max, 640 nm). The extinction coefficient of bathoiodopsin at room temperature was lower than that at low temperature and close to that of the original iodopsin at room temperature.

Photo-induced oxidation of poly- N-vinylcarbazole in the solid phase in presence of methane halides

ESR and low temperature spectrophotometry have been used to investigate the mechanism of the photochemical oxidation of poly- N-vinylcarbazole in solid films containing CHI 3 and CBr 4 additives. The photochemical reaction proceeds in two stages. First, as a result of photodssociation of the complexes with charge transfer chemically active intermediates form—carbazyl radical, iodine or bromine. In the case of the CHI 3 additive iodine forms with the quantum yield 10 −1 and the carbazyl radical with the quantum yield 3 × 10 −4. The carbazyl radicals interacting with iodine initiate the reaction proceeding by the chain mechanism with charge transfer. The reaction product—aminocationic salt of PNVC 2 2+(I 3 −) 2—forms with the quantum yield 10 −2. The scheme of the photochemical reactions is discussed.

Effect of chloride ion on the thermal decay process of the batho intermediate of iodopsin at low temperature.

The photochemical and the subsequent thermal behaviors of iodopsin (Cl(-)-bound form) and N-iodopsin (iodopsin whose Cl- was replaced by NO3-) in CHAPS-phosphatidylcholine (PC) were studied by low-temperature spectrophotometry. Irradiation of the iodopsin preparation at -185 degrees C produced a photo-steady-state mixture composed of iodopsin, bathoiodopsin, and isoiodopsin. Bathoiodopsin was thermally reverted to the original iodopsin. These results were almost the same as those reported previously [Yoshizawa, T., & Wald, G. (1967) Nature 214, 566-571] in which iodopsin was extracted with 2% digitonin. Therefore, photochemical and subsequent thermal behaviors of iodopsin were independent of the detergent to solubilize iodopsin. Irradiation of N-iodopsin at -185 degrees C produced the similar photo-steady-state mixture. However, N-bathoiodopsin was thermally converted to the next intermediate, presumably N-lumiiodopsin. These results suggest that the batho-lumi transition of iodopsin at low temperature is likely to be inhibited by the Cl- bound to the protein moiety of iodopsin, while at room temperature the Cl- bound to iodopsin could be released on the conversion process of batho- to lumiiodopsin.

Low-temperature spectrophotometry of phoborhodopsin

Photoreactions of the fourth rhodopsin-like pigment, phoborhodopsin (pR480), of Halobacterium halobium were studied by low-temperature spectrophotometry. Upon irradiation of pR480 at - 80 as well as - 170°C with 436 nm light, a new intermediate having a difference absorption maximum at about 520 nm was produced. We designate this intermediate 'P520'. Prolonged irradiation caused the formation of a photosteady-state mixture composed of P520 and pR480, indicating that P520 was photoreversible to pR480 at - 80°C. P520 was thermally converted back to pR480 through P350 and P530, both of which were detected previously [(1986) Biochem. Biophys. Res. Commun. 139, 389–395]. Based on these results, a new photochemical reaction cycle of pR is presented.

Low temperature spectrophotometry on the photoreaction cycle of sensory rhodopsin

The photoreaction of sensory rhodopsin (sR) was studied by using low temperature spectrophotometry. Upon irradiation of sR 590 at temperatures ranging from −30 to −60°C, sR 373, a photointermediate was produced without any preceding photoproducts. The formation of sR 373 was temperature-dependent, and an Arrhenius plot gave an activation energy of 15.7 kcal/mol, suggesting that there might be a potential barrier in the excited state of sR 590.

Electrostatic interaction between retinylidene chromophore and opsin in rhodopsin studied by fluorinated rhodopsin analogues.

Photochemical reactions of fluorinated rhodopsin analogues (F-rhodopsins) prepared from 10- or 12-fluorinated retinals (10- or 12-F-retinals) and cattle opsin were investigated by means of low-temperature spectrophotometry. On irradiation with blue light at liquid nitrogen temperature (-191 degrees C), the F-rhodopsins were converted to their respective batho intermediates. On warming, they decomposed to their respective fluororetinals and cattle opsin through lumi and meta intermediates. There was a difference in photochemical behavior between batho-12-F-rhodopsin and batho-10-F-rhodopsin. Upon irradiation with red light at -191 degrees C, batho-12-F-rhodopsin was converted to a mixture of 12-F-rhodopsin and 9-cis-12-F-rhodopsin like that of the natural bathorhodopsin, whereas batho-10-F-rhodopsin was not converted to 9-cis-10-F-rhodopsin but only to 10-F-rhodopsin. This fact suggests that the fluorine substituent at the C10 position (i.e., 10-fluoro) of the retinylidene chromophore may interact with the protein moiety during the process of isomerization of the chromophore or in the state of the batho intermediate. On irradiation with blue light at -191 degrees C, 9-cis-10-F-rhodopsin was converted to another bathochromic intermediate that was different in absorption spectrum from batho-10-F-rhodopsin. 9-cis-10-F-rhodopsin was practically "photoinsensitive" at liquid helium temperature (-265 degrees C), whereas 10-F-rhodopsin was converted to a photo-steady-state mixture of 10-F-rhodopsin and batho-10-F-rhodopsin. The specific interaction between the fluorine atom at the C10 position of the retinylidene chromophore and the opsin was discussed in terms of electrostatic interactions.

Low temperature spectrophotometry of the phototransformation of Pfr to Pr in pelletable pea phytochrome

Manabe, K. 1987. Low temperature spectrophotometry of the phototransformation of Pfr to Pr, in pelletable pea phytochrome.Low temperature spectrophotometry was used to study the phototransformation of Pfr to Pr in 1000–7000 g pelletable fractions extracted from dark grown pea (Pisum sativum L. cv. Alaska) epicotyls which had been irradiated with red and then far‐red light. At ‐170°C, far‐red irradiation of the pelletable phytochrome which had been pre‐irradiated with saturating fluence of red light before freezing caused formation of an intermediate (named I660), the difference spectrum of which showed a marked ab‐sorbance decrease at 740 nm and a concomitant small increase at about 660 nm. The inermediate I660 was converted to another intermediate (I660) when it was warmed above ‐80°C. The difference spectrum of this intermediate showed a positive peak at 670 nm. This intermediate was photoconverted to Pfr by red irradiation and also underwent dark reversion to Pfr at ‐60°C. I660 formed Pr if the temperature was above ‐10°C. The basic features of the phytochrome intermediates resemble those obtained in vivo and in degraded purified phytochrome.

Flash photolysis of chloride complexes of Cu(II) in organic solvents

By means of flash photolysis and low-temperature spectrophotometry, the formation of a complex between a Cu(I) ion and a peroxy radical of the solvent has been detected in ethanol, isopropanol, and dimethylformamide. The peroxy radical is generated in a reaction of a solvent radical with a molecule of dissolved oxygen. The solvent radical appears as a result of photoreduction of chloride complexes of Cu(II). The radical complex has a band in the optical absorption spectrum with a maximum at 415–420 nm in ethanol and isopropanol. The rate of formation of this complex is determined mainly by the reaction of the radical of the matrix (R.) with complexes of bivalent copper. The rate constant of this process in isopropanol at room temperature is (2–3)·108 liters/ mole·sec. Disappearance of the radical complex Cu(I)...RO2. takes place in a reaction with complexes Cu2+solv and CuCl+ with a rate constant of 2.3·107 liters/mole·sec at room temperature.

Photochemical reactions of bacteriorhodopsin in Triton X-100 solution studied by low temperature spectrophotometry

The photochemical reaction of purple membrane solubilized with Triton X-100 (T-BR) was investigated by low temperature spectrophotometry. The batho- and meta-intermediates of T-BR were observed to resemble bacteriorhodopsin in native purple membrane. Two photoproducts characteristic of the T-BR system were found, which were named the "490-nm complex" and the "380-nm complex". The 490-nm complex was in thermal equilibrium with T-BR in the dark. Cooling T-BR to low temperature favoured the 490-nm complex, which was photoinsensitive. On the other hand, the 380-nm complex was produced by warming the batho-intermediate and reverted to the original T-BR. The meta-intermediate of T-BR may possibly be in thermal equilibrium with the 380-nm complex. On the basis of the above results, the possible role of the membrane structure was discussed

Primary intermediates of rhodopsin studied by low temperature spectrophotometry and laser photolysis: Bathorhodopsin, hypsorhodopsin and photorhodopsin

The primary photochemical processes of rhodopsin studied by low temperature spectrophotometry and picosecond laser spectroscopy in our group was summarized. Low temperature spectroscopic experiments demonstrated that the retinylidene chromophores of hypso- and bathorhodopsins are in a twisted all- trans forms. Excitation of rhodopsin with 532 nm laser pulse (width: 25 psec) yielded a new bathochromic photoproduct “photorhodopsin”; its spectrum was located at longer wavelengths than that of bathorhodopsin. Photorhodopsin decays to bathorhodopsin with time constants of about 200 psec in squid and 40 psec in cattle. Squid and octopus hypsorhodopsins were produced within 25 psec by high energy pulse, but not by low energy pulse. Thus hypsorhodopsin is produced by two photon reactions (sequential two photochemical reactions) and decayed to bathorhodopsin with time constant of 125 psec.