Exposure to low irradiances favors the synthesis of 9-cis beta, beta-carotene in Dunaliella salina (Teod.)

We examined the effect of irradiance on the synthesis of beta-carotene and its isomers by Dunaliella salina. Growth irradiance had a marked effect both on growth of the alga (which was suppressed at both low and high irradiances) and on the accumulation of beta-carotene. The accumulation of beta-carotene but not alpha-carotene was closely linked to an increase in irradiance. Growth at low irradiances (20-50 micromol m(-2) s(-1)) promoted a high ratio of 9-cis to all-trans beta-carotene (>2:1), while exposure to high irradiances (200-1,250 micromol m(-2) s(-1)) resulted in a large reduction in this ratio (to <0.45:1). A similar pattern was seen for the geometric isomers of alpha-carotene, with exposure to low irradiance favoring the accumulation of the 9-cis form. The carotenoid biosynthesis inhibitors 4-chloro-5(methylamino)-2-(alpha-alpha-alpha-trifluoro-m-tolyl)-3-(sH )-pyridazinone and 2-(4-chlorophenylthio)triethylamine caused the accumulation of the precursors phytoene and lycopene, respectively, in D. salina. High-performance liquid chromatography and infrared analysis showed that phytoene adopted the 15-cis and all-trans forms (as in higher plants), and that lycopene primarily adopted the all-trans form. This indicates that isomerization of beta-carotene takes place during or after cyclization.     

Identification of carotenoids in Erwinia herbicola and in a transformed Escherichia coli strain

The yellow pigments of Erwinia herbicola Eho 10 and of a transformed Escherichia coli LE392 pPL376 have been identified as carotenoids. HPLC separation, spectra and in some cases mass spectroscopy demonstrated the presence of phytoene (15-cis isomer), beta-carotene (all-trans, 9-cis and 15-cis), beta-cryptoxanthin ( = 3-hydroxy beta-carotene), zeaxanthin (3,3'-dihydroxy beta-carotene) and corresponding carotene glycosides. In addition, lycopene and gamma-carotene accumulated in the presence of the inhibitor 2-(4-chlorophenylthio)-triethylamine.HCl. Carotenoid content in the transformed E. coli was two-fold higher than in E. herbicola. The pattern of the carotenoids was similar in the two organisms. Inactivation of the katF gene in E. coli resulted in an 85% lowering of carotenoid formation, as did the addition of 0.5% glucose to the medium. Suppression of carotenoid formation by inactivation of the katF gene lowered, but did not abolish, the protection offered by carotenoids against inactivation by alpha-terthienyl plus near-ultraviolet light (320-400 nm).     

Determination of 9-cis beta-carotene and zeta-carotene in biological samples

Concentrations of 9-cis beta-carotene (9-cis betaC) and zeta-carotene (zetaC) in biological samples may provide crucial information on the biological activities of these carotenoids. However, in high-performance liquid chromatography (HPLC) these carotenoids are often co-eluted. Therefore, there is an urgent need to develop a method for 9-cis betaC and zetaC quantitation. Both 9-cis betaC and zetaC have peak absorbance at 400 and 450 nm, respectively, whereas only 9-cis betaC has peak absorbance at 475 nm. We developed a HPLC method to quantitate 9-cis betaC and zetaC by using peak absorbance ratios. The 9-cis betaC/zetaC peak area was monitored at 475, 450 and 400 nm. The 9-cis betaC was quantified by using absorbance value at 475 nm; zetaC was then calculated from the 9-cis betaC/zetaC peak at 400 nm by subtracting 9-cis betaC contribution at 400 nm using the 400-nm/475-nm peak absorbance ratio of 9-cis betaC (0.39). This method was applied to determine 9-cis betaC and zetaC concentrations in serum and breast milk samples (n=12) from American lactating women and serum and breast adipose tissue samples (n=16) from Korean women with either benign or malignant breast tumors. 9-cis betaC concentrations in serum and breast milk of American women, and serum and adipose tissue of Korean women were 7.1+/-0.8 and 1.1+/-0.2 nM, and 15.6+/-1.1 nM and 0.2+/-0.1 nmol/g, respectively. zetaC concentrations in the above samples were 54.2+/-7.2 and 8.3+/-1.8 nM, and 49.0+/-3.9 nM and 0.3+/-0.1 nmol/g, respectively.     

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.     

Resonance Raman spectroscopy of carotenoids in Photosystem I particles

Low-temperature resonance Raman (RR) spectroscopy was used for the first time to study the spectral properties, binding sites and composition of major carotenoids in spinach Photosystem I (PSI) particles. Excitation was provided by an argon ion laser at 457.9, 476.5, 488, 496.5, 502 and 514.5 nm. Raman spectra contained the four known groups of bands characteristic for carotenoids (called from nu(1) to nu4). Upon 514.5, 496.5 and 476.5 nm excitations, the nu(1)-nu(3) frequencies coincided with those established for lutein. Spectrum upon 502-nm excitation could be assigned to originate from violaxanthin, at 488 nm to 9-cis neoxanthin, and at 457.9 nm to beta-carotene and 9-cis neoxanthin. The overall configuration and composition of these bound carotenoid molecules in Photosystem I particles were compared with the composition of pigment extracts from the same PSI particles dissolved in pyridine, as well as to configuration in the main chlorophyll a/b light-harvesting protein complex of photosystem II. The absorption transitions for lutein, violaxanthin and 9-cis neoxanthin in spinach photosystem I particles are characterized, and the binding sites of lutein and neoxanthin are discussed. Resonance Raman data suggest that beta-carotene molecules are also present in all-trans and, probably, in 9-cis configurations.     

The effect of temperature and irradiance on the growth and carotenogenic capacity of seven strains of Dunaliella salina (Chlorophyta) cultivated under laboratory conditions

The carotenogenic microalga Dunaliella salina is cultivated as a natural source of beta-carotene. The 9-cis isomer of beta-carotene is found only in natural sources having commercial advantages over the all-trans isomer due to its high liposolubility and antioxidant power. High irradiance appears to stimulate specifically all-trans beta-carotene accumulation in D. salina, whereas low temperature apparently elicits a-carotene and 9-cis betacarotene production. We studied the effect of temperature and irradiance on the growth and the carotenogenesis of three Chilean (CONC-001, CONC-006 and CONC-007) and four non-Chilean (from Mexico, China, Australia and Israel) strains of D. salina cultivated under two photon flux densities (40 and 110 micromol photons x m(-2) x s(-1)) and two temperatures (15 and 26 degrees C). The Chilean strain CONC-001 and all of the non-Chilean strains exhibited the highest growth rates and the maximum cell densities, whereas the Chilean strains CONC-006 and CONC-007 showed the lowest values in both parameters. The Australian strain showed the highest accumulation of total carotenoids per unit volume (40.7 mg x L(-1)), whereas the Chilean strains CONC-006 and CONC-007, the only ones isolated from Andean environments, yielded the highest amounts of carotenoids per cell (61.1 and 92.4 pg x cell(-1), respectively). Temperature was found to be more effective than irradiance in changing the qualitative and quantitative carotenoids composition. The Chilean strains accumulated 3.5-fold more alpha-carotene than the non-Chilean strains when exposed to 15 degrees C and, unlike the non-Chilean strains, also accumulated this pigment at 26 degrees C. The 9-cis/all-trans beta-carotene ratio was > 1.0 in all treatments for all strains, and the values were not greatly influenced by either temperature or photon flux density. Physiological and biotechnological implications of these results are discussed.     

Effect of salinity on the quantity and quality of carotenoids accumulated by Dunaliella salina (strain CONC-007) and Dunaliella bardawil (strain ATCC 30861) Chlorophyta

Dunaliella salina and D. bardawil are well-known microalgae accumulating high levels of beta-carotene under growth-limiting conditions. In both taxa, this pigment is primarily composed of the isomers 9-cis and all-trans. The 9-cis beta-carotene occurs only in natural sources and is the most attractive from a commercial point of view. The conditions that enhance the preferred accumulation of 9-cis beta-carotene in D. salina are controversial and they have not been well established yet. This study examined the effect of salinity on the quantity and quality of total carotenoids and beta-carotene isomers accumulated by D. salina (strain CONC-007) and D. bardawil (strain ATCC 30861) grown in two media with different nutritional compositions (PES and ART) and at salt concentrations of 1M, 2M and 3M NaCl. Total carotenoids were determined by spectrophotometry and beta-carotene isomers, by HPLC. The highest carotenoid contents per cell were obtained at 2M NaCl in both taxa. In both media, an increase of the 9-cis/all-trans beta-carotene ratio was observed in D. bardawil when the salt concentration increased, with a maximum value of 2.6 (in ART medium at 3M NaCl). In D. salina this ratio did not exhibit the same pattern, and the salt concentrations for maximal ratios were different in both media. The highest ratio obtained for this strain was 4.3 (in ART medium at 2M NaCl).     

Characterization of the chromophore of the third rhodopsin-like pigment of Halobacterium halobium and its photoproduct.

Halobacterium halobium contains at least three retinal-containing pigments: bacteriorhodopsin, halorhodopsin, and a third rhodopsin-like pigment (tR) absorbing at approximately 590 nm, tR590. Illumination of tR590 gives rise to a very long-lived blue absorbing photoproduct, tR370. Using high-performance liquid chromatography we show that the chromophore of tR590 is primarily all-trans retinal and its conversion by light to tR370 causes the chromophore to isomerize primarily to the 13-cis conformation. Irradiation of the tR370 gives rise to a transient photoproduct absorbing at approximately 520 nm that decays back to the initial pigment tR590. In addition to all-trans retinal, the apomembrane of tR can also combine with 13-cis retinal but not with the 9- or 11-cis isomers.     

Reconstitution of ancestral green visual pigments of zebrafish and molecular mechanism of their spectral differentiation

We previously reported that zebrafish have four tandemly duplicated green (RH2) opsin genes (RH2-1, RH2-2, RH2-3, and RH2-4). Absorption spectra vary widely among the four photopigments reconstituted with 11-cis retinal, with their peak absorption spectra (lambda(max)) being 467, 476, 488, and 505 nm, respectively. In this study, we inferred the ancestral amino acid (aa) sequences of the zebrafish RH2 opsins by likelihood-based Bayesian statistics and reconstituted the ancestral opsins by site-directed mutagenesis. The ancestral pigment (A1) to the four zebrafish RH2 pigments and that (A3) to RH2-3 and RH2-4 showed lambda(max) at 506 nm, while that (A2) to RH2-1 and RH2-2 showed a lambda(max) at 474 nm, indicating that a spectral shift had occurred toward the shorter wavelength on the evolutionary lineages A1 to A2 by 32 nm, A2 to RH2-1 by 7 nm, and A3 to RH2-3 by 18 nm. Pigment chimeras and site-directed mutagenesis revealed a large contribution (approximately 15 nm) of glutamic acid to glutamine substitution at residue 122 (E122Q) to the A1 to A2 and A3 to RH2-3 spectral shifts. However, the remaining spectral differences appeared to result from complex interactive effects of a number of aa replacements, each of which has only a minor spectral contribution (1-3 nm). The four zebrafish RH2 pigments cover nearly an entire range of lambda(max) distribution among vertebrate RH2 pigments and provide an excellent model to study spectral tuning mechanisms of RH2 in vertebrates.     

Examination of molar-based distribution of A,B and C chains of amylopectin by fluorescent labeling with 2-aminopyridine

A method for determination of a molar-based distribution of A, B and C chains of amylopectin was developed. Labeling with fluorescent 2-aminopyridine was proportional to the number-average degree of polymerization (dp(n)) of the chains in the range of 6-440. Number-average chain lengths (cl(n)) of amylopectins from six different plant sources (rice, maize, wheat, potato, sweet potato and yam) determined by the labeling method were in good agreement with values obtained by determination of non-reducing residues. The molar-based distributions were polymodal (A, B(1) and B(2)+B(3) fractions) and characteristic to botanical sources. Amylopectins from starches with A-crystalline type had higher amount of A+B(1) chains (90-93% by mole) than starches with B-type (68-87%). Molar ratios of (A+B(1))/(B(2)+B(3)) were 8.9-12.9 for the A-type starches and 2.1-6.5 for the B-type starches, suggesting that amylopectins of A-type starches had 1.5-2 times more branches per cluster than B-type. The distributions of C chains, except for amylomaize, showed a broad, asymmetrical profile from dp approximately 10 to approximately 130 with a peak at dp approximately 40 and were very similar among botanical sources, suggesting that the biosynthetic process for C chains is similar in different plant species.     

Light-stable rhodopsin. I. A rhodopsin analog reconstituted with a nonisomerizable 11-cis retinal derivative.

With the aim of preparing a light-stable rhodopsin-like pigment, an analog, II, of 11-cis retinal was synthesized in which isomerization of the C11-C12 cis-double bond is blocked by a cyclohexene ring built around the C10 to C13-methyl. The analog II formed a rhodopsin-like pigment (rhodopsin-II) with opsin expressed in COS-1 cells and with opsin from rod outer segments. The rate of rhodopsin-II formation from II and opsin was approximately 10 times slower than that of rhodopsin from 11-cis retinal and opsin. After solubilization in dodecyl maltoside and immunoaffinity purification, rhodopsin-II displayed an absorbance ratio (A280nm/A512nm) of 1.6, virtually identical with that of rhodopsin. Acid denaturation of rhodopsin-II formed a chromophore with lambda max, 452 nm, characteristic of protonated retinyl Schiff base. The ground state properties of rhodopsin-II were similar to those of rhodopsin in extinction coefficient (41,200 M-1 cm-1) and opsin-shift (2600 cm-1). Rhodopsin-II was stable to hydroxylamine in the dark, while light-dependent bleaching by hydroxylamine was slowed by approximately 2 orders of magnitude relative to rhodopsin. Illumination of rhodopsin-II for 10 s caused approximately 3 nm blue-shift and 3% loss of visible absorbance. Prolonged illumination caused a maximal blue-shift up to approximately 20 nm and approximately 40% loss of visible absorbance. An apparent photochemical steady state was reached after 12 min of illumination. Subsequent acid denaturation indicated that the retinyl Schiff base linkage was intact. A red-shift (approximately 12 nm) in lambda max and a 45% recovery of visible absorbance was observed after returning the 12-min illuminated pigment to darkness. Rhodopsin-II showed marginal light-dependent transducin activation and phosphorylation by rhodopsin kinase.     

Quantum efficiency distributions of photo-induced side-pathway donor oxidation at cryogenic temperature in photosystem II

We monitored illuminated-minus-dark absorption difference spectra in the range of 450-1100 nm induced by continuous illumination at 8 K of photosystem II (PSII) core complexes from Thermosynechococcus elongatus. The photo-induced oxidation of the side-path donors Cytb(559), beta-carotene and chlorophyll Z, as well as the concomitant stable (t(1/2) > 1 s) reduction of the first plastoquinone electron acceptor, Q(A) (monitored by the well-known 'C550' shift), were quantified as a function of the absorbed photons per PSII. The Q(A) photo-induced reduction data can be described by three distinct quantum efficiency distributions: (i) a very high efficiency of approximately 0.5-1, (ii) a middle efficiency with a very large range of approximately 0.014-0.2, and (iii) a low efficiency of approximately 0.002. Each of the observed side-path donors exhibited similar quantum efficiency distributions, which supports a branched pathway model for side-path oxidation where beta-carotene is the immediate electron donor to the photo-oxidized chlorophyll (P680(+)). The yields of the observed side-path donors account quantitatively for the wide middle efficiency range of photo-induced Q(A) reduction, but not for the PSII fractions that exhibit the highest and lowest efficiencies. The high-efficiency component may be due to Tyr(Z) oxidation. A donor that does not exhibit an identified absorption in the visible-near-IR region is mainly responsible for the lowest efficiency component.     

Favourable mutation test outcomes for individuals at risk for Huntington disease change the perspectives of first-degree relatives

In mutation testing for Huntington disease, an autosomal dominant hereditary late-onset disorder, unfavourable test outcomes in at-risk individuals provide important information about other family members at risk. On the other hand, common counselling practice considers favourable outcomes as non-informative for at-risk relatives, except for the offspring of the tested individual. We shall show, however, that favourable outcomes also change the perspectives for the tested individual's first-degree relatives at risk. In the case of a (prospective) parent originally at 50% risk, and with n equalling the number of children or fetuses identified as non-carriers, the probability of being a non-carrier equals 2 (n)/(2 (n)+1) for the at-risk parent, providing that none of the offspring of this parent has been identified as a carrier. Likewise, the probability of being a non-carrier equals (2 (n+1)+1)/(2 (n+1)+2) for the (future) siblings of the tested individual. These changes in probabilities are important for individuals who are considering prenatal or presymptomatic DNA-testing for autosomal dominant hereditary late-onset disorders, such as Huntington disease and hereditary forms of cancer (BRCA1/2, FAP, HNPCC). Consequences can be far reaching in the case of pregnancies, where the risk of miscarriage after a prenatal test is 1%-2%. Parents initially at 50% risk may consider not having a prenatal test in successive pregnancies, knowing that favourable test results in previous pregnancies have considerably reduced their personal risk.     

Effects of salinity on beta-carotene production by Dunaliella tertiolecta DCCBC26 isolated from the Urmia salt lake, north of Iran

This study examined the effect of different salt concentrations (0.05-3 M of NaCl) on the kinetics of growth, total carotenoids and beta-carotene (all-trans and 9-cis) accumulated in Dunaliella tertiolecta DCCBC26, a microalgae strain isolated from the Urmia hypersaline lake, northwest of Iran. Results indicated that the highest amount of carotenoids detected (11.73 mg/l) was in the salinity of 0.5 M NaCl during the stationary growth phase. The percentage of the all-trans and 9-cis-beta-carotene in the exponential phase were 92% and 32% in salinities of 3 M and 0.5 M, respectively. However, only 23% of the beta-carotene was detected in the stationary growth phase of the microalgae in 0.5 M salinity and was 9-cis isomer.     

Pulse radiolysis studies of beta-carotene in oxygenated DMSO solution. Formation of beta-carotene radical cation

The spectroscopic and kinetic characteristics of beta-carotene radical cation (beta-carotene(.+)) were studied by pulse radiolysis in aerated DMSO solution. The buildup of beta-carotene(.+) with k(1) = (4.8 +/- 0.2) x 10(8) dm(3) mol(-1) s(-1) [lambda(max) = 942 nm, epsilon = (1.6 +/- 0.1) x 10(4) dm(3) mol(-1) cm(-1)] results from an electron transfer from beta-carotene to DMSO(.+). The beta-carotene(.+) species decays exclusively by first-order reaction, k = (2.1 +/- 0.1) x 10(3) s(-1), probably by two processes: (1) at low substrate concentration by hydrolysis and (2) at high concentrations also by formation of dimer radical cation (beta-carotene)(2)(.+). Under the experimental conditions, a small additional beta-carotene triplet-state absorption ((3)beta-carotene) in the range of 525 to 660 nm was observed. This triplet absorption is quenched by oxygen (k = 7 x 10(4) s(-1)), resulting in singlet oxygen ((1)O(2)), whose reactions can also lead to additional formation of beta-carotene(.+).     

Responses of the photosynthetic machinery of Spirulina maxima to induced reactive oxygen species

The photosynthetic machinery of Spirulina maxima was studied when subjected to induced reactive oxygen species (ROS) to examine the organism's responses to stress. Significant decreases in both photosynthetic efficiency and growth rate were observed. Exposure to 0.01 mmol H(2)O(2)/(g cell), which induced the lowest specific intracellular ROS level (siROS) led to a 15% decrease in specific growth rate; an increase in siROS by 70-fold led to a 25% decrease in specific growth rate. Similarly, siROS induced by 0.01 mmol H(2)O(2)/(g cell) led to 15% inhibition in photosynthetic efficiency, while an increase in siROS by 40- or 70-fold led to about 60% inhibition in photosynthetic efficiency. To further understand the effects of induced ROS on photosynthetic machinery, we performed a detailed pigmentation analysis as well as analyzed Phycobilisomes (PBS), Photosystem II (PSII), and Photosystem I (PSI), the three important components of cyanobacterial photosynthetic apparatus. We found carotenoids (beta-carotene and lutein) to be most sensitive to siROS. Also, specific levels of phycocyanin and allophycocyanin, which are important PBS pigments, decreased significantly in response to H(2)O(2). Further, electron transport assays revealed that ROS cause damage primarily to PSII, whereas they do not significantly affect PSI in comparison; siROS induced by 0.01 mmol H(2)O(2)/(g cell) led to a 15% inhibition of PSII, and increase in siROS by 9-, 40-, and 70-fold led to 22%, 36%, and 46% inhibition, respectively.     

Biochemical and biophysical characterization of photosystem I from phytoene desaturase and zeta-carotene desaturase deletion mutants of Synechocystis Sp. PCC 6803: evidence for PsaA- and PsaB-side electron transport in cyanobacteria

In photosystem I, oxidation of reduced acceptor A(1)(-) through iron-sulfur cluster F(X) is biphasic with half-times of approximately 5-30 ns ("fast" phase) and approximately 150-300 ns ("slow" phase). Whether these biphasic kinetics reflect unidirectional electron transfer, involving only the PsaA-side phylloquinone or bi-directional electron transfer, involving both the PsaA- and PsaB-side phylloquinones, has been the source of some controversy. Brettel (Brettel, K. (1988) FEBS Lett. 239, 93-98) and Joliot and Joliot (Joliot, P., and Joliot, A. (1999) Biochemistry 38, 11130-11136) have attributed to nearby carotenoids electrochromic band shifts, accompanying A(1) reduction, centered at approximately 450 and 500-510 nm. As a test of these assignments, we separately deleted in Synechocystis sp. PCC 6803 the genes that encode phytoene desaturase (encoded by crtP (pds)) and zeta-carotene desaturase (encoded by crtQ (zds)). The pds(-) and zds(-) strains synthesize phytoene and zeta-carotene, respectively, both of which absorb to shorter wavelength than beta-carotene. Compared with wild type, the mutant A(1)(-) (FeS) - A(1)(FeS)(-) difference spectra, measured in cells and photosystem I complexes, retain the electrochromic band shift centered at 450 nm but show a complete loss of the electrochromic band shifts centered at 500-510 nm. Thus, the latter clearly arise from beta-carotene. In the wild type, the electrochromic band shift of the slow phase (centered at 500 nm) is shifted by 6 nm to the blue compared with the fast phase (centered at 506 nm). Thus, the carotenoid pigments acting as electrochromic markers during the fast and slow phases of A(1)(-) oxidation are different, indicating the involvement of both the PsaA- and the PsaB-side phylloquinones in photosystem I electron transport.     

Photocycles of bacteriorhodopsin in light- and dark-adapted purple membrane studied by time-resolved absorption spectroscopy.

Nanosecond time-resolved absorption spectra have been measured throughout the photocycle of bacteriorhodopsin in both light-adapted and dark-adapted purple membrane (PM). The data from dark-adapted samples are interpretable as the superposition of two photocycles arising independently from the all-trans and 13-cis retinal isomers that coexist in the dark-adapted state. The presence of a photocycle in dark-adapted PM which is indistinguishable from that observed for light-adapted PM under the same experimental conditions is demonstrated by the observation of the same five relaxation rates associated with essentially identical changes in the photoproduct spectra. This cycle is attributed to the all-trans component. The cycle of the 13-cis component is revealed by scaling the data measured for the light-adapted sample and subtracting it from the data on the dark-adapted mixture. At times less than 1 ms, the resulting difference spectra are nearly time-independent. The peak of the difference spectrum is near 600 nm, although there appears to be a slight (approximately 2 nm) blue-shift in the first few microseconds. Subsequently the amplitude of this spectrum decays and the peak of the difference spectrum shifts in two relaxations. Most of the amplitude of the photoproduct difference spectrum (approximately 80%) decays in a single relaxation having a time constant of approximately 35 ms. The difference spectrum remaining after this relaxation peaks at approximately 590 nm and is indistinguishable from the classical light-dark difference spectrum, which we find, in experiments performed on a much longer time scale, to peak at 588 nm. The decay of this remaining photo-product is not resolvable in the nanosecond kinetic experiments, but dark adaptation of a completely light-adapted sample is found to occur exponentially with a relaxation time of approximately 2,000 s under the conditions of our experiments.     

On the photocycle and light adaptation of dark-adapted bacteriorhodopsin.

Pulsed Nd laser (25 ns, 530 nm) photolysis experiments were carried out at room temperature in aqueous suspensions of dark- and light-adapted fragments of the purple membrane of Halobacterium halobium. It is shown that the (50%) 13-cis isomeric component (BR13-cis) of dark-adapted bacteriorhodopsin (BRDA) undergoes a photocycle involving a characteristic transient absorbing in the neighborhood of 610 nm. At relatively high excitation intensities BR13-cis is converted to the same 410 nm (M) transient that characterized the photocycle of the all-trans isomer (BRtrans) of light-adapted bacteriorhodopsin (BRLA). This process, which competes with the generation of the "610" species, is attributed to the photo-induced conversion, during the pulse, of BR13-cis (or of its primary photoproduct "X") to a species in the BRtrans photocyte. The relationship between these observations and the mechanism of BRDA hv leads to BRLA adaptation at low excitation intensities (for which a quantum yield limit, 0 less than or equal to (3.5 +/- 0.7) X 10(-2) , is established) is discussed.     

Reaction center of photosystem II with no peripheral pigments in D2 allows secondary electron transfer in D1

A pigment-deficient reaction center of photosystem II (PSII)-with all the core pigments (two molecules of chlorophyll a and one of pheophytin a in each D protein) but with only one molecule each of peripheral chlorophyll a (Chlz) and beta-carotene (Car)-has been investigated by pump-probe spectroscopy. The data imply that Car and Chlz are both bound to D1. The absence of Car and Chlz in D2 allows the unprecedented observation of secondary electron transfer in D1 of PSII reaction centers at room temperature. The absorption band of the Car cation in D1 (Car(D1)(+*)) peaks around 910 nm (as against 990 nm for Car(D2)(+*)), and its positive hole is shared by ChlzD1, whereas Car(D2)(+*) can disappear by capturing an electron from ChlzD2.