EVENTS SURROUNDING THE EARLY DEVELOPMENT OF EUGLENA CHLOROPLASTS. 12. SPECTROSCOPIC EXAMINATION OF THE PROTOCHLOROPHYLL(IDE) PHOTOTRANSFORMATION IN INTACT CELLS*

Abstract— Absorption measurements in the 600–720 nm region of dark-grown cells of Euglena gracilis Klebs var. bacillaris Cori were made in vivo at room temperature using computer-assisted spectrophotometry. Dark-grown wild-type cells have a prominent absorption maximum at 634 nm due to protochlorophyll(ide) absorption. Upon illumination, the absorption at 634nm decreases and a peak appears at 674nm, representing the phototransformation of protochlorophyll(ide) to chlorophyll(ide). Using difference spectroscopy, the resynthesis in the dark of protochlorophyll(ide) by previously-illuminated wild-type dark-grown cells was found to begin at about 10min after illumination and reached completion by about 25 min, the amount of protochlorophyll(ide) resynthesized being equivalent to that of dark-grown cells. Resynthesis of protochlorophyll(ide) following a second illumination follows the same kinetics, indicating that protochlorophyll(ide) resynthesis is under tight regulation, possibly via feedback control. Cells of dark-grown wild-type and W₃BUL, a mutant lacking protochlorophyll(ide) contain a component absorbing at 658 nm which does not undergo phototransformation when examined by difference spectroscopy at room and liquid N₂ temperatures. Following the phototransformation of protochlorophyll(ide) 634 to chlorophyll(ide) 674, the chlorophyll(ide) 674 shifts to shorter wavelengths, ultimately to 671 nm. Possible relationships among the various spectroscopic forms of protochlorophyll(ide) and chlorophyll(ide) at room temperature and liquid N₂ temperature in Euglena and higher plants are presented. It is concluded that Euglena, unlike older, etiolated higher plants, contains only protochlorophyll(ide) 634, making it an excellent system in which to examine the phototransformation of this pigment species in the absence of other forms.     

NEW SPECTRAL COMPONENTS IN HIGH RESOLUTION ABSORPTION SPECTRA OF GREEN BACTERIAL REACTION CENTER COMPLEXES AT 5K*

Abstract—absorption spcctra of reaction center Complexes I and II from Chlorobium limicola f. thiosul-fatophilum were taken from 760 and 860 nm at 5 K. Fourth and eighth derivatives of the spectra were calculated from the digital data. Light-minus-dark difference spectra were taken, also at 5 K, with 590 nm actinic light. A shoulder not visible at 77 K appears on the long wavelength side of the 834 nm peak in Complex I. In Complex II, which is derived by guanidine HCI treatment of I, the shoulder is much more pronounced; derivative peaks appear at 834 and 838 nm. In the difference spectra, there are troughs at 832 and 838 nm. The latter trough is the first instance in green bacteria of a wavelength coincidence between a light-induced bleaching and a peak in (derivative) absorbance. There is also a nearly symmetrical pair of features, a trough at 814 nm and a peak at 818 nm, that appear to represent a light-induced bathochromic shift of the absorbance at 816 nm, a peak which occurs in both complexes as well as the photochemically inert bacteriochlorophyll a (Bchl a) protein. Other features in the absorption spcctra of both Complexes occur at virtually the same wavelengths as the peaks in purified Bchl a-protein trimer. We conclude that a large fraction of the Bchl a in Complex II is in a conformation similar to that of a single subunit of Bchl a-protein.     

OPTICAL ABSORPTION AND ELECTRON SPIN RESONANCE SPECTRA OF CATION RADICALS OF DIMERIC CHLOROPHYLL a IN LOW-TEMPERATURE MATRICES

Abstract— A chlorophyll (Chl) a solution in 3-methylpentane at 77 K exhibits an absorption spectrum with a distinct peak at 706 nm in the red-band region. The formation of the 706 nm absorbing species (S706) was reversible with respect to temperature change; no chemical change was observed. γ-Irradiation of the rigid 3-methylpentane solution at 77 K yields an absorption spectrum which can be ascribed to S706⁺ and S706⁻. When carbon tetrachloride, an electron scavenger, was added to the solution, the absorption of S706⁺ survived, which has peaks at 850 and 956 nm. It is assumed that the S706 is hydrogen-bonded dimeric Chi a , which may be regarded as a model of P700 in photosynthesis. Cation radicals of monomeric Chi a were formed in a γ-irradiated sec -butyl chloride solution at 77 K, and an absorption spectrum with peaks at 730 and 813 nm was recorded. ESR spectra of the cation radicals of S706 and monomeric Chi a are of a similar shape but their linewidths are 7.5 and 11.0 Gauss, respectively. The linewidth narrowing observed for S706⁺ is clear evidence for the assumption that S706 is dimeric Chi a. Comparison was made of the absorption spectrum of S706⁺ with the light-induced spectrum of P700 reported earlier.     

IDENTIFICATION OF THE PROTON ACCEPTOR OF SCHIFF BASE DEPROTONATION IN BACTERIORHODOPSIN: A FOURIER-TRANSFORM-INFRARED STUDY OF THE MUTANT ASP85 → GLU IN ITS NATURAL LIPID ENVIRONMENT

Abstract— In order to assign the proton acceptor for Schiff base deprotonation in bacteriorhodopsin to a specific Asp residue, the photoreaction of the Asp85 → Glu mutant, as expressed in Halobacterium sp . GRB, was investigated by static low-temperature and time-resolved infrared difference spec-troscopy. Measurements were also performed on the mutant protein labeled with [4-¹³C]Asp which allowed discrimination between Asp and Glu residues. 14,15-di¹³C-retinal was incorporated to distinguish amide-II absorbance changes from changes of the ethylenic mode of the chromophore. In agreement with earlier UV-VIS measurements, our data show that from both the 540 and 610 nm species present in a pH-dependent equilibrium, intermediates similar to K and L can be formed. The 14 ms time-resolved spectrum of the 540 nm species shows that a glutamic acid becomes protonated in the M-like intermediate, whereas the comparable difference spectrum of the 610 nm species demonstrates that in the initial state a glutamic acid is already protonated. In conjunction with earlier observations of protonation of an Asp residue in wild-type M, the data provide direct evidence that the proton acceptor in the deprotonation reaction of the Schiff base is Asp85.     

PHOTOBIOLOGICAL CHARACTERIZATION OF A SPORE GERMINATION MUTANT dkgl WITH REVERSED PHOTOREGULATION IN THE FERN Ceratopteris richardii

Abstract— This paper describes the mutant dkgl in the fern Ceratopteris richardii, which shows rapid germination in darkness but is markedly inhibited by white light. Action spectra plotted at 10 nm intervals from 400 to 800 nm are presented for germination responses of wild-type and mutant spores to photon flux densities of 0.004, 0.04 and 0.4 jtmol/mVs. The action spectra for wild-type spores exhibit a sharp phytochrome-mediated peak at 660 nm, a broad peak from 670 to 740 nm resulting from an apparent high irradiance response and no germination below 560 nm. In the corresponding action spectra for mutant spores, the blue region displays rather complex fine structure with prominent minima at 450 and 470 nm, which suggests that cryptochrome is unaltered in these spores. The region from 550 to 640 nm shows the greatest inhibition of spore germination, but this region exhibits no obvious fine structure, which argues rather strongly against the possibility of a unique photoreceptor being active in mutant spores. The mutant spectra resemble the wild-type spectra in the region from 650 to 800 nm, and thus phytochrome seems normal in the mutant spores. The dkgl mutation appears to act late in the phytochrome transduction pathway where a hypothetical coupling protein may regulate the light-sensitive step in spore germination.     

LIGHT-INDUCED PERTURBATION OF AROMATIC RESIDUES IN BOVINE RHODOPSIN AND BACTERIORHODOPSIN*

Light-induced changes in the UV absorption spectrum of bovine rod outer segment membranes were measured by conventional difference spectroscopy and by flash photolysis methods. Different thermal intermediates of rhodopsin (lumirhodopsin, metarhodopsin I, metarhodopsin II, and meta-rhodopsin III) have absorption spectra in the ultraviolet which differ from the rhodopsin spectrum and from each other. The spectra associated with metarhodopsin I, metarhodopsin II, and metarhodopsin III are characteristic of perturbation of a small number of tyr. and/or trp residues, most likely one trp residue. These aromatic residues are in the neighborhood of the retinal Schiff base and undergo coordinated changes of interaction with retinal during the bleaching sequence. At the metarhodopsin II stage, the magnitude of the UV spectral changes is consistent with the exposure of a previously shielded trp residue to an aqueous environment. The present results are consistent with previous spectral studies which limit the extent of light-induced conformational changes to regions of the protein in the neighborhood of the retinal Schiff base. An analogous study was made on light-adapted purple membranes of Halobacterium halobium. The UV absorption spectrum associated with the deprotonated Schiff base intermediate of the trans-bacteriorhodopsin cycle is indicative, in part, of aromatic residue perturbation. However, significant changes in the secondary and tertiary structures of the bacterio-rhodopsin protein characteristic of a delocalized conformational change are unlikely at this intermediate stage.     

ACTION SPECTRA FOR ACCUMULATION OF INORGANIC CARBON IN THE CYANOBACTERIUM, Anabaena variabilis

Abstract— Action spectra for accumulation of inorganic carbon were obtained for Anabaena variabilis , strainM–2, in the presence and absence of photosynthetic CO₂ fixation. The action spectrum for inorganic carbon accumulation in the presence of CO₂ fixation showed a peak around 684 nm, corresponding to chlorophyll a absorption in PS 1, while that for CO₂ fixation showed a peak around 630 nm, corresponding to phycocyanin absorption in PS 2. The action spectra obtained in the presence of iodoacetamide or diuron, which inhibit CO₂ fixation, showed two peaks, one at about 684 nm and the other at 630 nm, with the 630 nm peak height 80 to 90% of the 684 nm peak. These results indicate that inorganic carbon transport in A. variabilis can be driven with near equal efficiency by energy derived from absorption in photosystem 1 alone and with energy transferred to PS 1 after absorption by PS 2.     

Core mutations of Synechococcus sp. PCC 7002 phycobilisomes: a spectroscopic study.

Three cyanobacterial strains harboring mutations affecting phycobilisome (PBS) cores were studied using steady state absorption and fluorescence and time-resolved fluorescence. The apcF mutant, missing beta 18, and the apcDF mutant, missing both alpha APB and beta 18, showed only small spectroscopic differences from the wild-type strain; their PBS emission was blue shifted by 10 nm, whereas their absorption spectra and time-resolved fluorescence kinetics were virtually unchanged. The third mutant studied was the apcE/C186S mutant in which the chromophore-binding cysteine-186 in the LCM99 polypeptide has been substituted with serine. The apcE/C186S mutant contained a modified chromophore which significantly changed the spectroscopic properties of the PBS complex. The apcE/C186S PBS absorbed more than the wild-type strain at 705 nm, and the emission spectrum gave two peaks at 660 nm and 715 nm. The time-resolved kinetics of the apcE/C186S mutant PBS were also significantly altered from those of the wild-type strain.     

A NEW GLOW PEAK, IN RHODOPSEUDOMONAS SPHAEROIDES

Abstract— A new glow peak at 120 K has been observed in Rhodopseudomanas sphaeroides and in its carotenoidless green mutant. This peak (labelled Z_n ), which is composed of two peaks at 120 and 150 K, appears when the bacteria are illuminated with white light while being cooled to 77 K and then warmed in darkness at a heating rate of 10 K per min. Delayed light emission and prompt fluorescence spectra show peaks around 530, 610 and 660 nm. The action spectra of light emission show a major peak at 410 nm and a smaller peak around 545 nm. The pigment responsible for the light emission is also leached out in the suspension medium. The chromophore responsible for the light emission appears to be magnesium protoporphyrin IX, not bacteriochlorophyll.     

ACTION SPECTRA FOR HYPHAL AGGREGATION,THE FIRST STAGE OF FRUITING,IN THE BASIDIOMYCETE Pleurotus ostreatus*

Abstract. Action spectra were determined for hyphal aggregation in Pleurotus ostreatus at wavelengths between 360 and 600 nm. The action spectrum for a 50% response had two maxima, one at 370 nm in the near-UV and the other a broad peak at 440–450 nm in the blue. Both were approximately of the same magnitude. A minimum was present at 400 nm and wavelengths greater than 530 nm invoked no response. Action spectra for a range of responses, 10–90%, were also determined which showed gradual changes in the peaks in the blue region. It was concluded that hyphal aggregation in P. ostreatus is under the control of a cryptochrome-like photoreceptor system.     

THE WHITE MEMBRANE OF CRYSTALLINE BACTERIOOPSIN IN HALOBACTERIUM HALOBIUM STRAIN R1mW AND ITS CONVERSION INTO PURPLE MEMBRANE BY EXOGENOUS RETINAL

Abstract— A new strain isolated from Halobacterium halobium designated R₁mW, contained negligible amounts of isoprenoid pigments, had a yellowish white color due to respiratory pigments and showed no proton movement in response to light. However, addition of all-trans-retinal converted R₁mW into purple cells. Formation of both halorhodopsin and bacteriorhodopsin was indicated by induction of light-dependent proton uptake and release, respectively. Both haloopsin and bacterioopsin were thus postulated to be present in R₁mW. Electron micrographs of freeze-fractured cytoplasmic membranes revealed patches in a hexagonal array of trimeric particles, comparable to the purple membrane structure. These white membrane patches were isolated by procedures similar to those for the purple membrane. The white membrane's buoyant density was about 1.18 g/m/, and its main component migrated on sodium dodecylsulfate polyacrylamide gels at the same rate as bacteriorhodopsin. The white membrane showed only a small absorption peak at ～410nm due to contaminating respiratory pigments and a strong absorption at around 275 nm and shorter wavelengths. The white membrane was thus considered to be mainly composed of bacterioopsin, which was readily converted into bacteriorhodopsin by an addition of all-trans-retinal. The absorption and CD spectra of the white membrane were measured before and after addition of retinal. The molar extinction coefficient of dark-adapted bacteriorhodopsin formed was determined to be 53000M^?1 cm^?1 at 560 nm from retinal binding studies. The CD spectrum of the white membrane was negligible in the visible region but showed several bands assigned to aromatic and backbone structures in the UV region. Retinal addition caused considerable changes in the spectrum, yielding the CD spectrum of crystalline purple membrane bacteriorhodopsin. The white membrane thus appears to be a preparation suitable for structure-function studies of bacteriorhodopsin.     

ENERGY TRANSFER FROM PHYCOBILINS TO CHLOROPHYLL a IN HEAT-STRESSED CELLS OF Anacystis nidulans: CHARACTERIZATION OF THE LOW TEMPERATURE 683 nm FLUORESCENCE EMISSION BAND*

Abstract— Heat-induced changes of the characteristics of fluorescence spectra of Anacystis nidulans cells were studied after 39°C-grown cells were heated at 55°C. Heat-treatment of the cells induced no changes in the absorption properties or photosystem I-catalyzed cytochrome oxidation, but induced a dramatic change in the fluorescence characteristics of the cells. The low temperature fluorescence emission spectra of heated cells showed a large increase of fluorescence emission at683–685 nm (F683) and at 695 nm, while the bands at 660 nm (allophycocyanin) and at 718 nm (chlorophyll a of photosystem I) were not affected when the cells were excited with light absorbed by phycobilins. When the cells were heated for various periods, a progressive increase of the intensity of F683 occurred with the loss in oxygen evolution capacity. The increase of the F683 band was observed prior to the increase of the F695 band. Quenching of emission spectra by the addition of quinones indicates that the F683 band emanated mainly from a long wavelength form of allophycocyanin. Excitation spectra of heated cells measured at 77 K showed that light absorbed by phycobilins was effective in exciting F685, F695, and F715 emission. A possible energy distribution pathway in Anacystis nidulans is discussed.     

FLUORESCENCE OF PHYTOCHROME IN THE CELLS OF DARK-GROWN PLANTS AND ITS CONNECTION WITH THE PHOTOTRANSFORMATIONS OF THE PIGMENT

Abstract Fluorescence of phytochrome is found in the cells of etiolated monocotyledonous and dicotyledonous plants. The red light-absorbing form of phytochrome (P_r) fluoresces at 77 K with a yield 0.3±0.1 and maxima at 672–673 nm and 684–686 nm in the excitation and emission spectra, respectively. The emission is characterized by the sharp temperature dependence of its intensity, its high (～ 40%) polarization, and the violation of the mirror symmetry rule. Connection of the fluorescence with P_r photoreactions is followed in the interval 77–293 K. A P, photoproduct, lumi-R, is fluorescent with maxima at 696 nm and 705 nm in the excitation and emission spectra; the far-red light absorbing form of phytochrome (P_fr) is practically nonfluorescent. Three isochromic emitting P_r species are present differing in their photochemical properties: P_r1 and P_r2 which phototransform irreversibly and reversibly at T 170 K into lumi-R, and lumi-R₂, respectively, and P_r3 which undergoes photoconversion only at T > 240 K. The activation energies of P_r2 and P_r3 photoreactions are evaluated to be 2.9–3.3 kJ/mol and 26 kJ/mol. Complex dynamics of changes of P_r fluorescence and of the extent of its decrease in the photoconversion P_r? P_fr in germinating pea and bean seeds suggests the existence of two P_r pools one of which is incapable of P_r? P_fr phototransformation. Thus, the developed fluorescent method of phytochrome assay and investigation in the cell revealing multiplicity of phytochrome states in vivo proves to be very sensitive (about 1 ng) and informative.     

ACTION SPECTRA FOR THE trans TO cis PHOTOISOMERISATION OF UROCANIC ACID in vitro and IN MOUSE SKIN

Urocanic acid (UCA) is a major UV chromophore in the upper layers of the skin where it is found predominantly as the trans isomer. UV irradiation induces photoisomerisation of trans-UCA to cis-UCA which has been shown to mimic some of the immunosuppressive properties of UV exposure. We examined the wavelength dependence for trans-UCA to cis-UCA photoisomerisation in vitro and in mouse skin in vivo over the spectral range270–340 nm. The resulting action spectra were very similar with maximal effectiveness at300–315 nm and equal activity at 270 nm and325–330 nm, demonstrating that UVA-II radiation (320–340nm) is efficient at UCA photoisomerisation. These action spectra differed markedly from the trans-UCA absorption spectrum in vitro and also the reported action spectrum for UV suppression of contact hypersensitivity in mice. These findings suggest that the relationship between cis-UCA formation in skin and UV-induced immunosuppression may be complex.     

BASF 13.338 INDUCED CHANGES IN STRUCTURE and FUNCTION OF THE PHOTOSYNTHETIC APPARATUS OF WHEAT SEEDLINGS

Abstract— Growing wheat seedlings in the presence of BASF 13.338 [4-chloro-5-dimethylamino-2-phenyl-3(2H)pyridazinone], a PS II inhibitor of the pyridazinone group, brought about notable changes in the structure and functioning of photosynthetic apparatus. In BASF 13.338 treated plants, there was a decrease in the ratio of Chi a/Chl b, an increase in xanthophyll/carotene ratio and an increase in the content of Cyt b 559 (HP + LP). Chl/p700 ratio increased when measured with the isolated chloroplasts but not with the isolated PS I particles of the treated plants. The SDS-PAGE pattern of chloroplast preparations showed an increase in the CPII/CP I ratio. The F685/F740 ratio in the emission spectrum of chloroplasts at -196°C increased. The difference absorption spectrum of chloroplasts between the control and the treated plants showed a relative increase of a chlorophyll component with a peak absorption at 676 nm and a relative decrease of a chlorophyll component with a peak absorption at 692 nm for the treated plants. The excitation spectra of these chloroplast preparations were similar. Chloroplasts from the treated plants exhibited a greater degree of grana stacking as measured by the chlorophyll content in the 10 K pellet. The rate of electron transfer through photosystem II at saturating light intensity in chloroplast thylakoids isolated from the treated plants increased (by 50%) optimally at treatment of 125 μM BASF 13.338 as compared to the control. This increase was accompanied by an increase in (a) I₅₀ value of DCMU inhibition of photosystem II electron transfer; (b) the relative quantum yield of photosystem II electron transfer; (c) the magnitude of C550 absorbance change; and (d) the rate of carotenoid photobleaching. These observations were interpreted in terms of preferential synthesis of photosystem II in the treated plants. The rate of electron transfer through photosystems I and through the whole chain (H₂O → methyl viologen) also increased, due to an additional effect of BASF 13.338, namely, an increase in the rate of electron transfer through the rate limiting step (between plastoquinol and cytochrome f). This was linked to an enhanced level of functional cytochrome f. The increase in the overall rate of electron transfer occurred in spite of a decrease in the content of photosystem I relative to photosystem II. Treatment with higher concentrations (> 125 μM) of BASF 13.338 caused a further increase in the level of cytochrome f, but the rate of electron transfer was no greater than in the control. This was due to an inhibition of electron transfer at several sites in the chain.     

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.     

PHOTOSYNTHETIC EFFICIENCY OF A PHYCOCYANIN-LESS MUTANT OF CYANIDIUM*

Abstract— 1. R-II is a u.v. induced mutant of cyanidium caldarium which lacks the major accessory pigment phycocyanin. 2. Loss of phycocyanin does not impair photosynthesis or the Hill reaction. 3. The action spectrum for the quantum yield indicates an efficiently operating photosynthetic mechanism which is shifted in peak efficiency toward longer wavelengths when compared to the wild-type spectrum. The red drop is also shifted toward the far red in R-II. 4. Although 30 per cent enhancement was obtained in wild-type Cyanidium, no enhancement was observed when phycocyanin was absent. 5. A pigment absorbing at 720 nm has been observed and may possibly represent a chlorophyll-phycocyanin complex.     

INTRAMOLECULAR ENERGY TRANSFER IN NATIVE tRNA AT ROOM TEMPERATURE

Abstract— The odd nucleoside 4-thiouridine, which is present in position 8 of 70% of E. coli tRNAs, possesses unusual spectroscopic properties which make it suitable for intramolecular energy transfer studies. Both its luminescence excitation spectrum and the action spectrum (230–380 nm) for the 8–13 link formation have been established in native E. coli tRNA at room temperature. The spectra are identical and present a new unexpected peak around 260 nm. At this wavelength, they are amplified by a factor of nine as compared with the absorption and excitation spectra of the free nucleoside in aqueous solution.
The origin of this new peak is discussed and it is concluded that energy transfer does occur from the common nucleosides to the 4-thiouridine residue. Using the values of the nucleosides to 4-thiouridine distances inferred from the sets of atomic coordinates obtained on yeast tRNA^phe crystals, a satisfactory account of our finding can be obtained assuming singlet-singlet energy transfer. The efficiency of the mechanism is probably favoured by a good overlap between the emission spectra of the common nucleosides and the absorption spectrum of 4-thiouridine.     

AGGREGATION OF MONOVINYL- and DIVINYL-PROTOCHLOROPHYLLIDE IN ORGANICSOLVENTS*

Abstract— Two different protochlorophyllides (PChlide), PChlide 629/433 (absorption data in methanol) and PChlide 630/441 (the monovinyl (MV) and divinyl (DV) forms) were isolated from the pigment mutant C-2A'of Scenedesmus obliquus. Their spectroscopic behaviour in several organic solvents and their aggregation in toluene was investigated. In polar solvents such as ether, acetonitril or acetone, absorption maxima similar to those in methanol were observed, while in solvents such as tetrahydrofuran and pyridine a bathochromic shift of the blue absorption band compared to the spectra in methanol occurred. The absorption maxima of MV-PChlide shifted from 629 nm and 433 nm in methanol (monomeric form), to 631 nm and 443 nm in toluene (aggregated form). The absorption maxima of DV-PChlide shifted from 630 nm and 441 nm in methanol to 655 nm and 483 nm in toluene (aggregated form). The fluorescence excitation and emission spectra of the two protochlorophyllides yielded the according results. The aggregation process was faster for DV-PChlide than for MV-PChlide and was reversible upon addition of small amounts of polar solvents. The similarity of the spectral characteristics of the aggregated forms of the different protochlorophyllides after toluene treatment with those reported for “active”-PChlide in vivo are discussed.     

COOPERATIVITY OF THE DEHYDRATION BLUE-SHIFT OF BACTERIORHODOPSIN*

Abstract– Dehydration of purple membrane (PM) causes a hlue-shift of the absorbance maximum from 570 nm to about 530 nm [Lazarev and Terpugov (1980) Biochim. Biophys. Acta 590 .324–338; Hildebrandt and Stockburger (1984) Biochemistry 23 ,5539–5548]. The absorbance spectra of PM dried in films at pH 0, 7 and 11 were measured at controlled relative humidities (RH). At pH 7, a blue-shift was observed similar to that previously reported. At pH 0(1M H₂SO₄) a reversible transition was observed from the “acid blue membrane” (maximum near 600 nm at 100% RH) to a blue-shifted dehydrated pigment (maximum near 578 nm at 50% RH), with isosbestic points at 592 and 710 nm. At pH 11 (NaOH) the absorbance maximum shifted to 530 nm, similar to the dehydrated form at pH 7. The fraction of hydrated chromophore, X_h, was calculated (assuming only two chromophore states, hydrated and dehydrated) as a function of humidity and pH. The resulting curve at pH 7 showed a steep decline in X_h below 20% RH. Near this hydration level, water clusters on protein surfaces break up, causing side-chain pK reversals. The Hill coefficient for the transition was about 2, indicating the minimum number of water molecules involved in a cooperative transition. The results suggest that as few as two water molecules are coordinated to the protonated retinal Schiff base of bacteriorhodopsin. A mechanism for the pH 7 dehydration blue-shift is proposed, involving a pK reversal of the protonated Schiff base and a nearby carboxyl side chain. At pH 0, a sharp decline in X_h occurs between 100 and 70% RH. Near this hydration level, complete protein surface coverage by a water monolayer occurs. The Hill coefficient is about 20, suggesting involvement of a large region of the surface.