Photodamage to Pigment in the Photosystem Ⅱ Reaction Center D1/D2/Cytochrome b559 Complex

Strong light (800 μmol photons/m2 per s)-induced bleaching of the pigment in the isolated photosystem Ⅱ reaction center (PSII RC) under aerobic conditions (in the absence of electron donors or acceptors) was studied using high-pressure liquid chromatography (HPLC), absorption spectra, 77K fluorescence spectra and resonance Raman spectra. Changes in pigment composition of the PSll RC as determined by HPLC after light treatment were as follows: with increasing illumination time chlorophyll (Chi) a and β-carotene (β-car)content decreased. However, decreases in pheophytin (Pheo) could not be observed because of the mixture of the Pheo formed by degraded chlorophyll possibly. On the basis of absorption spectra, it was determined that, with a short time of illumination, the initial bleaching occurred maximally at 680 nm but that with increasing illumination time there was a blue shift to 678 nm. It was suggested that P680 was destroyed initially, followed by the accessory chlorophyll. The activity of P680 was almost lost after 10 min light treatment. Moreover, the bleaching of Pheo and β-car was observed at the beginning of illumination.After illumination, the fluorescence emission intensity changed and the fluorescence maximum blue shifted,showing that energy transfer was disturbed. Resonance Raman spectra of the PSII RC excited at 488.0 and 514.5 nm showed four main bands, peaking at 1 527 cm-1 (υ1), 1 159 cm-1 (υ2), 1 006 cm-1 (υ3), 966 cm-1 (υ4) for 488.0 nm excitation and 1 525 cm-1 (υ1), 1 159 cm-1 (υ2), 1 007 cm-1 (υ3), 968 cm-1 (υ4) for 514.5 nm excitation.It was confirmed that two spectroscopically different β-car molecules exist in the PSII RC. After light treatment for 20 min, band positions and bandwidths were unchanged. This indicates that carotenoid configuration is not the parameter that regulates photoprotection in the PSII RC.     

Changes in Intensity and Spectral Distribution of Fluorescence: Effect of Light Treatment on Normal and DCMU*-Poisoned Anacystis nidulans‡

The intensity of the "steady-state" fluorescence of "aerobic" Anacystis nidulans is variable under prolonged illumination with orange (590 mmu) or blue (440 mmu) light for both normally photosynthesizing and DCMU-poisoned cells. In general, orange light illumination causes an increase of the fluorescence intensity followed by a decrease, while blue light causes an increase until a steady level is reached. Poisoned Anacystis cells show four to eight times larger changes in fluorescence intensity than the normal cells; the detailed time course of fluorescence changes is also different in poisoned and normal cells. When algae are cooled to -196 degrees C in light, the light-induced changes in the "steady-state" fluorescence disappear in both types of cells. Difference fluorescence spectra, constructed by subtracting the fluorescence spectra taken after 5-15 min of illumination from those after 60-90 min of illumination, show a doublet structure of the difference band with a major peak coinciding with the Anacystis emission maximum (685 mmu) and a minor peak located at about 693 mmu.     

Photodamage to Pigment in the Photosystem Ⅱ Reaction Center D1/D2/Cytochrome b559 Complex

Strong light （800μmol photons/m^2 per s）-induced bleaching of the pigment in the isolated photosystem Ⅱ reaction center （PSII RC） under aerobic conditions （in the absence of electron donors or acceptors） was studied using high-pressure liquid chromatography （HPLC）, absorption spectra, 77K fluorescence spectra and resonance Raman spectra. Changes in pigment composition of the PSII RC as determined by HPLC after light treatment were as follows： with Increasing illumination time chlorophyll （Chl） a and β-carotene （β-car） content decreased. However, decreases in pheophytin （Pheo） could not be observed because of the mixture of the Pheo formed by degraded chlorophyll possibly. On the basis of absorption spectra, it was determined that, with a short time of illuminatlon, the initial bleaching occurred maximally at 680 nm but that with Increasing Illumination time there was a blue shift to 678 nm. It was suggested that P680 was destroyed Initially, followed by the accessory chlorophyll. The activity of P680 was almost lost after 10 mln light treatment. Moreover, the bleaching of Pheo and β-car was observed at the beginning of illumination. After Illumination, the fluorescence emission Intensity changed and the fluorescence maximum blue shifted, showing that energy transfer was disturbed. Resonance Raman spectra of the PSII RC excited at 488.0 and 514.5 nm showed four main bands, peaking at 1 527 cm^-1 （υ101）, 1 159 cm^-1 （υ2）, 1 006 cm^-1 （υ3）, 966 cm^-1 （υ4） for 488.0 nm excitation and 1 525 cm^-1 （υ1）, 1 159 cm^-1 （υ2）, 1 007 cm^-1 （υ3）, 968 cm^-1 （υ4） for 514.5 nm excitation. It was confirmed that two spectroscopically different β-car molecules exist In the PSII RC. After light treatment for 20 mln, band positions and bandwidths were unchanged. This indicates that carotenoid configuration Is not the parameter that regulates photoprotectlon in the PSII RC.     

尼罗红荧光法快速检测培养过程中湛江等鞭金藻的中性脂

研究一种快速准确测定微藻中中性脂的方法。湛江等鞭金藻是一种中性脂含量高且具有开发潜力的能源微藻。以湛江等鞭金藻为实验对象,首先优化尼罗红染色的条件。当二甲基亚砜体积分数为2.0%、尼罗红质量浓度为1.00μg/m L、细胞密度为1.0×106个/m L、激发波长为480 nm、检测波长为580 nm时,优化的染色时间为10min。其次测定了背景荧光对检测的影响。结果表明,在不同细胞状态下,背景荧光强度大约是微藻内荧光强度的20%左右,可以忽略。最后比较了尼罗红荧光法和重量法。结果表明,荧光强度与中性脂含量的相关系数R2=0.946 8,虽然两者相关性并不十分高,但作为一种快速测定微藻中中性脂的方法,尼罗红荧光法依然是研究微藻培养过程中中性脂含量变化的有效方法。     

Quenching of a room temperature fluorescence band at 693 nm during photoactivation of the water-splitting system of Photosystem II in flashed barley leaves

The modifications of the room temperature fluorescence spectrum during the photoactivation of the water-splitting system by continuous illumination were investigated in flashed barley leaves. A blue shift of the chlorophyll fluorescence band was detected during the first 2 min of illumination. During this shift, a decrease of the fluorescence intensity around 693 nm could be demonstrated in difference spectra and in second derivative spectra. This decrease is interpreted as a quenching of PS II fluorescence during the photoactivation. A relative fluorescence increase around 672 nm also occurred during the same period and is thought to reflect rapid light-induced chlorophyll formation. The flashed leaves contained small amounts of photoactive photochlorophyllide which could be removed by a short flash of intense white light given before continuous illumination. The fact that such flash had only weak effect on the 693 nm fluorescence decrease, whereas it strongly reduced the amplitude of the 672 nm fluorescence increase, favours the above interpretations.Abbreviations chl chlorophyll - PS II Photosystem II - PS I Photosystem I     

Characteristics of the relative maximum in the decay of delayed light emission from Pothos leaf following far-red excitation

Following illumination with wavelengths longer than 700 nm, the intensity of light emission from Pothos aurea leaf falls for 1 min and then increases to a maximum after 2 min in the dark. The spectrum of this minute-range liminescence matches that of prompt fluorescence excited at the same wavelength, but differs from that of prompt or minute-range delayed emission excited by wavelengths shorter than 700 nm. This emission is less sensitive to heat damage than millisecond delayed emission, and may originate from photosystem I.     

Fluorescence fading in quantitative fluorescence microscopy: a cytofluorometer for the automatic recording of fluorescence peaks of very short duration

Synopsis Rates of photodecomposition were studied in some fluorophores during short (milliseconds) and longer (minutes) illumination periods. A xenon burner served as light source, and care was taken to obtain optimum conditions for activation. The fluorophores studied included (i) the formaldehyde-induced fluorescent product from 5-hydroxytryptamine in mast cells, (ii) Berberine sulphate bound to mast cell polyanions, (iii) Feulgen-Pararosaniline-stained DNA, and (iv) Fluorescein isothiocyanate-conjugated IgG in an antinuclear factor test. All fluorophores showed a significant fading during 3 min illumination. The Fluorescein isothiocyanate-conjugate faded the most rapidly; its fluorescence intensity was reduced to 50% of the initial value after 2 sec continuous illumination. No fluorophore faded significantly during the initial few milliseconds of illumination. On the basis of these findings, an inexpensive measuring device was constructed. It contained a peak-reader and memory circuit triggered by the flash synchronization tap of a camera shutter positioned in the activation beam. The peak-reader has a response time of about 2 msec. Repeated measurements on the various fluorophores indicate that this peak-reading device may be used to measure fluorescence intensity without fading.     

Energization and ultrastructural pattern of thylakoids formed under periodic illumination followed by continuous light

Bean leaves grown under periodic illumination (56 cycles of 2 min light and 98 min darkness) were subsequently exposed to continuous illumination, and in connection with granum formation and accumulation of the light-harvesting pigment-protein complex thermoluminescence and light-induced shrinkage of thylakoid membranes were studied. Juvenile chloroplasts with large double sheets of thylakoids obtained under periodic light exhibited low temperature spectra of polarized fluorescence yielding fluorescence polarization (FP) values < 1 at 695 nm, characteristic for pheophytin emission. In the course of maturation under continuous light when normal grana appeared and the chlorophyll a/b light-harvesting photosystem II complex was incorporated into the membrane, at 695 nm the relative intensity of fluorescence dropped and FP changed to a value of > 1, suggesting an overlap between the emission of pheophytin and that of the chlorophyll a/b light-harvesting photosystem II complex. Thermoluminescence glow curves recorded with juvenile thylakoids displayed a relatively high proportion of emission at low temperatures (around -10°C) while with mature chloroplasts, more thermoluminescence originated from energetically deeper traps (discharged around 28°C). This means that during thylakoid development the capacity of the membrane to stabilize the separated charges increases, which might be favourable for the ultimate conservation of energy. The more extensive energization of mature thylakoids was also indicated by a light-induced decrease in the thickness of the membranes upon illumination; a change which could not be detected in juvenile thylakoids.Abbreviations EDTA ethylenediamine tetraacetic acid - Hepes 4-(2-hydroxy ethyl)-1-piperazine ethane sulfonic acid Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

Light-induced Damage of Photosystem Ⅱ Primary Electron Donor P680: a High Performance Liquid Chromatographic Analysis of Pigment Content in D1/D2/Cytochrome b559 Complex Under Photoinhibitory Conditions

By HPLC analytical method, the change of PS Ⅱ RC' s pigment content in the process of photodamage under strong illumination from spinach ( Spinacia oleracea Mill. ) was comparatively studied. The experimental results show that: (1) In authors' analytical conditions, (of which, [Chl] = 150 µg/mL, and the illumination strength was put at 2.3 ×10 6 mJ·m－2·s－1 ), 45 rain of illumination could cause almost the whole loss of A680 in the fourth derivative absorption spectra, while A670 decreased to about one half of its original intensity; the absorption maximum in red, concurrently, was shifted from 676 nm to 671 nm, representing the loss of more than 90% of the photochemical activities of the PS Il RC. (2) During the period of continuous illumination, the Chl concentration decreased in a 3-period style, which meant that the first [Chl] decreased to the 2/3 of its original amount from 20 min to 40 rain after illumination had started, then became stabilized up to about 60 min of illumination, there after a second decrease of [ Chl ] in another about 20 min until it reached about 30 % of the original level and remained unchanged from about 80 min on. The original pigment components of D1/D2/Cyt b559 was approximately as 6 Chl a:2 Pheo:2β-Car which are in support of authors' previous proposal about the minimum Chl/Pheo ratio of 4: 2 in PS Ⅱ RC’s pigment contents. (3) After about 40 min of illumination, a newly appeared elution peak was found between the Pheo andβ-Car peaks in HPLC profile, at the retention time of 7.2 min, a little later than that (6.9 rain) of Pheo molecules, the newly appeared elution peak was supposed to be a kind of accumulated and stable product of the PS II RC's photodamage process and very much possible the Pheo-like molecules.     

Studies on the Energy Transfer from Phycobilisome to Thylakoid Membrane of Cyanobacteria

Ultrasonic treatment has been introduced to obtain photosynthetic vesicles with hound phycobilisome (PBS). the PBS-thylakoid fraction, from Myxosarcina concinna Printz. The maximal oxygen evolving rate of PBS-thylakoid membrane was around 60 μmol O2/ (mg Chl.h) and the optimum temperature was about 34 ℃. Absorption peaks of the eomplex were located at 680, 628, 490, 438 and 420 nm. When the phyeobiliprotein of the complex was excited by light at 580 nm, the fluorescence emission spectrum at room temperature exhibited a peak at 660 nm and a shoulder at 680 nm, while at liquid nitrogen temperature the peaks were resolved to 664, 695 and 718 nm, The PBS bound with thylakoid membrane depolymerized and the energy transfer from PBS to thylakoid membrane decreased when the complex was put into a solution of low ion intensity. As the PBS was mixed with the PBS desociated thylakoid membrane, by contact with the thylakoid membrane the PBS could again transfer the energy trapped in itself to thylakoid membrane.     

Electron transfer from excited tryptophan to cytochrome c: mechanism of phosphorescence quenching?

Parvalbumin, aldolase and liver alcohol dehydrogenase (ADH), proteins exhibiting long-lived phosphorescence lifetimes at room temperature, were examined for their reactivity with ferricytochrome c (cytochrome c Fe3+) as an external electron acceptor. Illumination of a reaction mixture containing protein and cytochrome c in the absence of oxygen brought about reduction of cytochrome c in relation to the duration of light. The largest portion of reduced cytochrome c was found with a sample containing ADH, where a 50% reduction of cytochrome c was reached after 5 min of illumination with a xenon lamp. Parvalbumin and aldolase were about half as effective under the same conditions. Several lines of evidence support the idea that the reaction of cytochrome c occurred by a long-range electron transfer from the excited triplet state of tryptophan. First, cytochrome c quenches the tryptophan phosphorescence and with parvalbumin, its bimolecular quenching rate constant, kq, was 2.9 x 10(6) M-1 s-1. Second, when the illuminated reaction mixture was supplied with 0.2 mM to 1 mM nitrite, a concentration range of nitrite which quenches the tryptophan phosphorescence but not the fluorescence, the amount of reduced cytochrome c on illumination markedly decreased. Finally, for all illuminated protein samples, the extent of cytochrome c reduction occurred parallel to a decrease in tryptophan content as judged from a decrease in fluorescence intensity and/or a decrease in tryptophan absorption at 280 nm.     

量子点的近场激发荧光特性研究

近场光学显微镜具有nm量级的空间分辨率,量子点(quantum dots,QDs)荧光探针具有激发谱宽、发射谱线窄、荧光强度高、抗光漂白和稳定性高等优点,两者结合用于生物大分子的成像探测和识别具有广泛的应用前景。用近场光学显微镜对链霉亲和素偶联的QDs进行近场荧光激发,并对其荧光发射特性和光稳定性进行研究,结果表明:近场光学显微镜nm量级的空间分辨率,可以同时观察到了QDs的单体、二聚体和三聚体;QDs的荧光发射强度高,近场荧光像对比度好,单量子点的荧光半高宽达到25nm;对一定入射波长的单色激发光,QDs的近场荧光强度随着激发功率密度的增加线性增加,并很快趋于稳定。与传统的荧光染料如异硫氰酸荧光素相比,QDs的稳定性非常好,在激发功率密度为300W/cm2的近场辐射下,量子点的荧光强度超过6h基本保持不变,其抗光漂白能力远远高于普通荧光染料。     

Eradication of Propionibacterium acnes by its endogenic porphyrins after illumination with high intensity blue light

Propionibacterium acnes is a Gram-positive, microaerophilic bacterium that causes skin wounds. It is known to naturally produce high amounts of intracellular porphyrins. The results of the present study confirm that the investigated strain of P. acnes is capable of producing endogenic porphyrins with no need for any trigger molecules. Extracts from growing cultures have demonstrated emission peaks around 612 nm when excited at 405 nm, which are characteristic for porphyrins. Endogenic porphyrins were determined and quantified after their extraction from the bacterial cells by fluorescence intensity and by elution retention time on high-performance liquid chromatography (HPLC). The porphyrins produced by P. acnes are mostly coproporphyrin, as shown by the HPLC elution patterns. Addition of delta-aminolevulinic acid (ALA) enhanced intracellular porphyrin synthesis and higher amounts of coproporphyrin have been found. Eradication of P. acnes by its endogenic porphyrins was examined after illumination with intense blue light at 407-420 nm. The viability of 24 h cultures grown anaerobically in liquid medium was reduced by less than two orders of magnitude when illuminated once with a light dose of 75 J cm(-2). Better photodynamic effects were obtained when cultures were illuminated twice or three times consecutively with a light dose of 75 J cm(-2) and an interval of 24 h between illuminations. The viability of the culture under these conditions decreased by four orders of magnitude after two illuminations and by five orders of magnitude after three illuminations. When ALA-triggered cultures were illuminated with intense blue light at a light dose of 75 J cm(-2) the viability of the treated cultures decreased by seven orders of magnitude. This decrease in viability can occur even after a single exposure of illumination for the indicated light intensity. X-ray microanalysis and transmission electron microscopy revealed structural damages to membranes in the illuminated P. acnes. Illumination of the endogenous coproporphyrin with blue light (407-420 nm) apparently plays a major role in P. acnes photoinactivation. A treatment protocol with a series of several illuminations or illumination after application of ALA may be suitable for curing acne. Treatment by both pathways may overcome the resistance of P. acnes to antibiotic treatment.     

The fluorescence from the chromophore of the purple membrane protein.

The fluorescence from the purple membrane protein (PM) of Halobacterium halobium and its relation to the primary photochemical events have been studied. The emission spectrum at 77 degrees K has structure, with peaks at 680, 710-715, and 730-735 nm. The excitation spectrum shows a single peak centered at 580 nm. This and a comparison of the fluorescence intensity at 77 degrees K under a variety of conditions with the amounts of the bathoproduct (or K, the only photoproduct seen at this temperature) formed suggest that the source of the fluorescence is the purple membrane itself, not the photoproduct. From the difference in several of their properties, we suggest that the fluorescing state of the pigment is different from the excited state which leads to photoconversion.     

Characteristics of Fluorescence and Delayed Light Emission from Green Photosynthetic Bacteria and Algae

Green photosynthetic bacteria exhibit variations in the intensity of their fluorescence during illumination. The initial intensity of fluorescence, measured at the onset of illumination, has a spectrum in which the major pigment Chlorobium chlorophyll predominates. The minor pigment bacteriochlorophyll predominates in the spectrum of the time-varying part of the fluorescence. The spectrum of delayed light emission is identical to that of the time-varying fluorescence. The variations in fluorescence also resemble the delayed light in their kinetics and in their dependence on exciting light intensity. Similar results are obtained for the kinetics of prompt and delayed light emission in the algae Chlorella and Anacystis. These findings raise the possibility that the variations in fluorescence actually represent a fast component of delayed light emission, of intensity comparable to the intensity of fluorescence. In Anacystis there is an outburst of light emission that develops after the exciting light has been turned off, reaching a maximum intensity after 1 to 3 seconds. This emitted light has the spectrum of chlorophyll fluorescence. It appears to be a novel example of bioluminescence with singlet excited chlorophyll as the emitter.     

Glucose concentration determination by means of fluorescence emission spectra of soluble and insoluble glucose oxidase: some useful indications for optical fibre-based sensors

By using soluble and insoluble glucose oxidase, the changes in intrinsic emission fluorescence in the visible spectral region were studied as a function of glucose concentration. Insoluble glucose oxidase (GOD) was obtained by entrapment in a gelatine membrane or by covalent attachment on an agarose membrane grafted with hexamethylendiamine. The intensity of the fluorescence emission peak at 520 nm or the value of the integral fluorescence area from 480 to 580 nm were taken as physical parameters representative of the glucose concentration during the enzyme reaction. By using these parameters, linear calibration curves for glucose concentration were obtained. The extension of the calibration curve and the sensitivity of the adopted systems were found to be dependent on the enzyme state (free or immobilized) and on the immobilization method. In particular, it was found that the extent of the linear range of the calibration curves is increased of one order of magnitude when the glucose oxidase is immobilized, while the sensitivity of the measure is decreased of one order of magnitude by the immobilization process. Measures carried out by using the integral fluorescence area resulted more sensitive than those obtained with the peak size. Useful indications for the construction of optical fibre-based sensors were drawn from the reported results.     

Fluorescence spectral properties of troponin C mutant F22W with one-, two-, and three-photon excitation.

We report the first measurements of protein fluorescence with three-photon excitation, using a mutant of troponin C (TnC) that contains a single tryptophan residue F22W. From the emission intensity dependence on laser power we determine that TnC F22W displays one-, two-, and three-photon excitation at 285, 570, and 855 nm, respectively. The emission spectra and intensity decays are identical for one-, two-, or three-photon excitation. The steady-state and time 0 anisotropies are distinct for each mode of excitation, but the correlation times were the same, suggesting that three-photon excitation of proteins can be accomplished without significant effects of the locally intense illumination. The excitation anisotropy spectrum from 830 to 900 nm displays only negative values, suggesting dominant excitation via the 1Lb state of tryptophan from 830 to 900 nm.     

Microscopical and spectroscopic studies on the fluorescence of a daunomycin-aluminum complex.

In this study, the spectroscopic features and microscopical applications of the fluorescent daunomycin-Al3+ complex have been analyzed. In the presence of Al3+, the absorption spectrum of daunomycin showed a deep bathochromic shift and new peaks at 529 and 566 nm, whereas the fluorescence emission was considerably modified. The emission of daunomycin alone (peak at 560 nm under optimal excitation at 470 nm) decreased continuously from 0.5 to 24h after addition of Al3+ ions, and a new emission peak appeared at 580 nm (optimal excitation at 530 nm). Under the fluorescence microscope using green exciting light, nuclei from chicken blood smears and paraffin sections of rat embryos stained with daunomycin showed a weak emission, which greatly increased after treatment with Al3+ ions. The bright and stable fluorescence of chromatin DNA induced by daunomycin-Al3+ could be a valuable labelling method in fluorescence microscopy and DNA cytochemistry.     

Reversible inactivation of serpins at acidic pH

The inhibitory activity of the serpins alpha(1)-proteinase inhibitor, alpha(1)-antichymotrypsin, alpha(2)-antiplasmin, antithrombin and C(1)-esterase inactivator is rapidly lost at pH 3 but slowly recovers at pH 7.4 with variable first-order rates (t(1/2)=1.4-19.2 min). All except alpha(1)-antichymotrypsin undergo a variation in intrinsic fluorescence intensity upon acidification (midpoint ca. 4.5) with a slow bi-exponential return to the initial intensity at pH 7.4 (mean t(1/2)=2.3-23 min). No correlation was found between the time of fluorescence recovery and that of reactivation. The acid-treated serpins are proteolyzed at neutral pH by their target proteinases. alpha(1)-Proteinase inhibitor was studied in more detail. Its acidification at pH 3 has a mild effect on its secondary structure, strongly disorders its tertiary structure, changes the microenvironment of Cys(232) and causes a very fast change in ellipticity at 225 nm (t(1/2)=1.6s). Neutralization of the acid-treated alpha(1)-proteinase inhibitor is an exothermic phenomenon. It leads to a much faster recovery of activity (t(1/2)=4+/-1 min) than of fluorescence intensity (t(1/2)=23+/-19 min), ellipticity (t(1/2)=32+/-4 min) and change in total energy, indicating that the inhibitory activity of alpha(1)-proteinase inhibitor does not require a fully native structure.     

Formation of a long-lived photoproduct with a deprotonated Schiff base in proteorhodopsin, and its enhancement by mutation of Asp227

Proteorhodopsin, a retinal protein of marine proteobacteria similar to bacteriorhodopsin of the archaea, is a light-driven proton pump. Absorption of a light quantum initiates a reaction cycle (turnover time of ca. 50 ms), which includes photoisomerization of the retinal from the all-trans to the 13-cis form and transient deprotonation of the retinal Schiff base, followed by recovery of the initial state. We report here that in addition to this fast cyclic conversion, illumination at high pH results in accumulation of a long-lived photoproduct absorbing at 362 nm. This photoconversion is much more efficient in the D227N mutant in which the anionic Asp227, which together with Asp97 constitutes the Schiff base counterion, is replaced with a neutral residue. Upon illumination at pH 8.5, most of the D227N pigment is converted to the 362 nm species, with a quantum efficiency of ca. 0.2. The pK(a) for this transition in the wild type is 9.6, but decreased to 7.5 after mutation of Asp227. The short wavelength of the absorption maximum of the photoproduct indicates that it has a deprotonated Schiff base. In the dark, this photoproduct is converted back to the initial pigment with a time constant of 30 min (in D227N, at pH 8.5), but it can be reconverted more rapidly by illumination with near-UV light. Experiments with "locked" retinal analogues which selectively exclude rotation around either the C9=C10, C11=C12, or C13=C14 bond show that formation of the 362 nm species involves isomerization around the C13=C14 bond. In agreement with this, retinal extraction indicates that the 362 nm photoproduct is 13-cis whereas the initial state is predominantly all-trans. A rapid shift of the pH from 8.5 to 4 greatly accelerates thermal reconversion of the 362 nm species to the initial pigment, suggesting that its recovery involving the thermal isomerization of the chromophore is controlled by ionizable residues, primarily the Schiff base and Asp97. The transformation to the long-lived 362 nm photoproduct is apparently a side reaction of the photocycle, a response to high pH, caused by alteration of the normal reprotonation and reisomerization pathway of the Schiff base.