the competition for solar photons among 
 aquatic photosynthetic 
 organisms striving to maintain viability at various depths in the 
 water column is fierce , often requiring adaptation of the species 
 for survival . a prime example of the development of adaptive traits 
 is found in the purple photosynthetic bacterium , rhodoblastus ( rbl . ) acidophilus ( formerly rhodopseudomonas acidophila ) strain 7050 . this bacterium 
 is able to alter its number and size of photosynthetic units as well 
 as its pigment composition and light absorption properties of the 
 major light harvesting ii ( lh2 ) antenna pigment  protein complex 
 in response to changes in illumination conditions . if the bacterium is grown under high light , the well - characterized 
 b800 - 850 lh2 complex is formed having bacteriochlorophyll ( bchl ) absorption 
 bands near 800 and 850 nm , and rhodopin and rhodopin glucoside are 
 the primary carotenoid pigments . under low - light conditions , genes 
 that code for a variant lh2 denoted b800 - 820 ( also sometimes referred 
 to as lh3 in the literature ) are activated , the bchl qy absorption band near 850 nm shifts to 820 nm , and 
 the organism accumulates rhodopinal and rhodopinal glucoside in the 
 lh2 complex as primary carotenoid pigments ( figures 1a and 1b ) . this response to changes in ambient light is controlled 
 by a combination of a classical two component regulatory system and 
 bacteriophytochromes that regulate the synthesis of the photosynthetic 
 apparatus . structures of ( a ) all - trans - rhodopin 
 glucoside 
 and 13-cis - rhodopinal glucoside and ( b ) one - third 
 portion of the lh2 b800 - 820 ring complex from rbl . acidophilus strain 7050 ( pdb 1ijd ) showing the protein - bound bchls ( green ) and carotenoids ( purple ) . the shift of the bchl qy band from 850 nm to 
 820 nm that occurs at low light is due to alterations in the 
 amino acid sequence of the apoproteins that are assembled in the variant 
 lh2 pigment  protein complex . results from x - ray crystallography ( figure 1b ) , site - directed mutagenesis , and resonance raman spectroscopy indicate that h - bonding residues 44 ( tyr ) 
 and 45 ( trp ) in the b800 - 850 lh2 prevent rotation of the c3-acetyl 
 group of the b850 bchl and fix the functional group so that its c = o 
 -electron bond resides in a planar orientation relative to 
 the porphyrin macrocycle . the conversion 
 of these h - bonding residues to non - h - bonding 44 ( phe ) and 45 
 ( leu ) in the b800 - 820 lh2 leads to a rotation of the c3-acetyl group 
 out of the plane of the porphyrin ring , thereby inhibiting delocalization 
 of the -electron conjugation to the acetyl carbonyl , resulting 
 in more restricted -electron delocalization and consequently 
 a blue shift of the qy band from 850 nm to 820 
 nm . accompanying the shift of the bchl qy absorption 
 band 
 in the lh2 complex is a change in the absorption spectrum of the carotenoid . 
 under low - light growth conditions , rhodopin and rhodopin glucoside are enzymatically converted to rhodopinal and rhodopinal glucoside 
 as an aldehyde group replaces the methyl group at carbon c20 in the 
 carotenoid structures ( figure 1a ) . the spectral origin ( 00 ) vibronic band of rhodopin glucoside 
 in methanol appears at 500 nm , whereas for rhodopinal glucoside , 
 the band is less resolved spectrally , and it is located at 540 
 nm in the same solvent ( figure 2 ) . previous 
 workers compared the carotenoid - to - bchl energy transfer properties 
 of lh2 complexes isolated from cells of rbl . acidophilus strain 7050 grown under different illumination conditions and found 
 that there was an increase in the energy transfer efficiency from 
 between 50 and 55% for the b800 - 850 complex to between 70 and 75% 
 for the b800 - 820 complex . however , the 
 previous investigation did not address the specific reasons for the 
 increase , i.e. , whether changes in the bchl absorption spectra , or 
 the conversion of rhodopin to rhodopinal in the protein complex , or 
 both factors , were responsible for the enhanced ability of the lh2 
 complex to effectively harvest photons in the region of carotenoid 
 absorption . moreover , the previous work and subsequent ultrafast spectroscopic 
 experiments carried out on the b800 - 820 lh2 complex from rbl . acidophilus strain 7050 did 
 not assign specific values to the energy transfer efficiencies of 
 the individual carotenoids bound in the complexes , nor has there been 
 any direct comparison of the spectra and dynamics of the excited states 
 of rhodopin and rhodopinal either in solution or in the lh2 complexes . these data are important for addressing the specific mechanism of 
 how these alterations in bchl and carotenoid structures and spectra 
 increase the carotenoid - to - bchl energy transfer efficiency and , as 
 a consequence , enhance the viability of the photosynthetic bacterial 
 organism . normalized steady - state absorption spectra of ( a ) rhodopin glucoside 
 and ( b ) rhodopinal glucoside in carbon disulfide , benzyl alcohol , 
 methanol , and acetonitrile recorded in 2 mm path length cuvettes at 
 room temperature . energy transfer from 
 carotenoids involves at least two excited 
 singlet states that can act as donors of absorbed light energy to 
 bchl . these are the s1 ( 2ag ) and s2 ( 1bu ) states whose properties are strikingly distinct . a one - photon 
 transition from the ground s0 ( 1ag ) state to the s1 ( 2ag ) state is forbidden by symmetry , whereas 
 a transition to the s2 ( 1bu ) state is strongly allowed . the s0 ( 1ag )  s2 ( 1bu ) 
 transition is responsible for the vibrant coloration of carotenoids 
 in nature . motivated by the landmark 
 report of the x - ray crystal structure of the lh2 complex from rbl . acidophilus strain 10050 , several investigators sought to understand the role of the s1 ( 2ag ) and s2 ( 1bu ) excited singlet 
 states in the mechanism of energy transfer to bchl in this pigment used 
 ultrafast time - resolved optical spectroscopy applied to the lh2 complex 
 prepared from rbl . acidophilus strain 10050 and reported 
 that the s2 ( 1bu ) state 
 of rhodopin glucoside dominated the pathway for energy transfer to 
 bchl despite its extremely short intrinsic lifetime of 120 
 fs in solution . the s1 ( 2ag ) state of rhodopin glucoside , which has a much longer 
 lifetime of 4 ps in solution , was reported to make only a 
 minor contribution to the overall energy transfer efficiency . in another 
 study using steady - state and ultrafast time - resolved spectroscopy 
 to elucidate the carotenoid - to - bchl energy transfer mechanism in the 
 lh2 complex from rbl . acidophilus 10050 , cong et 
 al . reported the partitioning of energy 
 transfer to be 23  7% from the s1 ( 2ag ) state and 63  10% from the s2 ( 1bu ) state . a recent 
 broadband 2d electronic spectroscopic investigation of lh2 complexes 
 from rbl . acidophilus 10050 and rhodobacter 
 sphaeroides strain 2.4.1 provided convincing evidence for 
 the additional involvement of a dark intermediate state in the carotenoid - to - bchl 
 energy transfer pathway . the present 
 work provides a detailed experimental and computational 
 comparison of the excited state energy levels , spectra , and dynamics 
 of rhodopin , rhodopinal , and their associated glucosides in various 
 solvents and in their respective lh2 complexes isolated from rbl . the use of steady - state absorption , fluorescence and fluorescence 
 excitation spectroscopy and ultrafast time - resolved transient absorption 
 spectroscopy in the visible spectral region have revealed the rates 
 and efficiencies of carotenoid - to - bchl energy transfer for the individual 
 carotenoids . the data address the questions of how and why photosynthetic 
 organisms alter their pigment composition and light - harvesting characteristics 
 to ensure survival under the challenging , light - deprived environmental 
 conditions in which they are sometimes found . acidophilus 10050 and 7050 cultures were 
 grown anaerobically in the light using 
 pfennig s medium . normal growth 
 conditions ( hereafter referred to as high - light ( hl ) conditions ) used 
 continuous illumination at an intensity of 30 mol s m. the rbl . acidophilus 7050 
 culture was also grown at a lower light intensity ( hereafter denoted 
 low - light ( ll ) ) ranging from 3.7 to 5 mol s m. cells were harvested by centrifugation at 
 4000 g in a beckman model j-6b centrifuge . the resulting 
 pellets were resuspended in 1 l of 20 mm mes ph 6.8 buffer , containing 
 100 mm kcl , and centrifuged again to remove any residual media . extraction of the carotenoids 
 from whole cells of the bacteria was accomplished by mixing 24 
 g of thawed cells with 30 ml of methanol at room temperature and stirring 
 in the dark for 15 min . the mixture was then centrifuged at 3000 g using an ss-34 rotor at 4 c in a sorvall rc-5b centrifuge . the pellet was then mixed with 30 ml of fresh methanol and centrifuged 
 repeatedly until the cells appeared gray , signifying that all the 
 pigments had been extracted . the supernatant from the second and subsequent 
 extractions contained primarily carotenoids as evidenced by absorption 
 spectra recorded using a varian cary 50 uv / visible spectrometer . the dried extracts were dissolved in acetonitrile / methanol ( 6:4 , 
 v / v ) and analyzed using a waters 600e/600s hplc system equipped with 
 a waters atlantis t3 obd preparative column having dimensions of 19 
  100 mm . the mobile phase consisted of acetonitrile / methanol 
 ( 6:4 , v / v ) delivered isocratically at a flow rate of 7.0 ml / min . individual 
 peaks were collected and identified by mass spectrometry using a fisons 
 quattro ii instrument employing atmospheric pressure chemical ionization 
 ( apci ) in negative mode with the following conditions : corona voltage , 
 2.5 v ; cone voltage , 25 v ; source temperature , 120 c ; probe 
 temperature , 300 c , mobile phase , acetonitrile . all solvents 
 were hplc - grade and were purchased from sigma - aldrich corp . briefly , 5 g of pelleted whole cells 
 of the bacteria were suspended in 30 ml of 20 mm tris buffer 
 adjusted to ph 8.0 using 6 m hcl ( hereafter referred to as tris buffer ) , 
 and 2050 mg of dnase and a few grains of mgcl2 were added to degrade the released dna during cellular disruption . the sample was briefly homogenized using a glass tissue homogenizer 
 to ensure smooth passage through the french press operating at 15,000 
 psi . cells were passed through the press three times to ensure complete 
 disruption , and the resulting suspension was centrifuged for 2 h at 
 4 c in a type 70 ti rotor spinning at 180000 g in a beckman l8 - 55 m ultracentrifuge . the resultant pellet 
 containing membrane fragments was diluted using tris buffer to an 
 od of 50 measured in a 1 cm cuvette at the bchl qy absorption 
 band maximum ( 800 nm for ll grown rbl . the membranes 
 were then solubilized by adding 30% lauryldimethylamine oxide ( ldao ) 
 dropwise to a final concentration of 1.0% and allowing the sample 
 to incubate for 60 min while being stirred at room temperature in 
 the dark . the sample was then centrifuged for 30 min at 27000 g in the sorvall rc-5b centrifuge to remove denatured protein 
 and any nonsolubilized material . the supernatant containing the solubilized 
 proteins was then collected for further treatment using sucrose density 
 gradient ultracentrifugation . solutions of 0.2 , 0.4 , 0.6 , and 0.8 m sucrose 
 were prepared using tris buffer containing 0.1% ldao and were carefully 
 layered to form discontinuous gradients in the tubes by using the 
 following amounts : 0.8 m , 5.0 ml ; 0.6 m , 6.5 ml ; 0.4 m , 6.5 ml ; and 
 0.2 m , 5.0 ml . the tubes were then placed in a ti70 rotor and spun at 160000 g for 12 h at 4 c in the beckman l8 - 55 m ultracentrifuge . the procedure effectively separates any free pigments present in the 
 0.2 m sucrose layer , from the lh2 complex appearing in the 0.4 m sucrose 
 layer , from the lh1-rc  core  complexes that appear 
 at the interface of the 0.6 and 0.8 m sucrose solutions . the 
 0.4 m sucrose solution layer containing the lh2 complex was 
 carefully removed from each tube and pooled . the complex was then 
 purified further by column chromatography using de52 anion exchange 
 resin ( 15 g , whatman scientific ) packed into a 5 cm diameter  
 30 cm long solid phase , sintered glass column pre - equilibrated with 
 several bed volumes of tris buffer . after loading the sample onto 
 the column , several bed volumes of tl buffer ( 0.1% ldao , 20 mm tris 
 ph 8.0 ) were applied to remove the sucrose . the lh2 complex was then 
 eluted using tl buffer containing increasing concentrations of nacl 
 in 30 mm increments starting with 10 mm . acidophilus 10050 and 7050 cells eluted between 10 
 and 20 mm nacl , whereas that isolated from ll rbl . the purity of the eluting 
 fractions was monitored by absorption spectroscopy using a shimadzu 
 uv-1700 pharmaspec spectrometer . fractions from hl samples exhibiting 
 spectra with an 850 nm to 280 nm ratio of > 3.0 were pooled for 
 further 
 purification . for the ll sample , fractions exhibiting spectra with 
 an 800 nm to 280 nm absorbance ratio of > 2.5 were pooled for further 
 purification . the pooled sample was reduced to a volume of < 1 
 ml by centrifugation 
 using vivaspin 4 50 k mw cutoff concentrators ( sartorius stedim biotech ) 
 placed in an 11390 rotor and spun at 3250 g in a sigma 
 3k30 centrifuge . the concentrated lh2 was further purified using an 
 akta primeplus ( ge healthcare ) automated chromatography system equipped 
 with an xk-16 long gel filtration column filled with superdex-200 . 0.5 ml fractions were collected of the lh2 band and subsequently assayed 
 using the ratio of the absorbance maximum of the bchl qy band to the protein absorbance at 280 nm . fractions having an 800 
 nm to 280 nm ( from the ll - grown rbl . acidophilus 7050 ) 
 ratio of > 2.7 or an 850 nm to 280 nm ( from the hl - grown rbl . acidophilus cells ) ratio of 3.3 were pooled and concentrated 
 using the vivaspin 4 50 k mw cutoff concentrators to an od of 100 
 measured at the absorbance maximum of the bchl qy band 
 in a 1 cm cuvette . the sample was then divided into 30 l aliquots , 
 placed in pcr tubes , flash frozen in liquid nitrogen , and stored in 
 a 80 c freezer . a 10 l aliquot 
 of the frozen rbl . acidophilus 7050 lh2 complex was 
 thawed on ice , and the liquid was evaporated 
 to dryness using nitrogen gas . the remaining residue was then redissolved 
 in 2 ml of methanol to release the pigments . this extract was then 
 centrifuged at 13600 g for 2 min at room temperature 
 in a fisher scientific 235c benchtop microcentrifuge . the supernatant 
 was then analyzed using a waters 600e/600s hplc system equipped with 
 a waters atlantis t3 5 m analytical column having dimensions 
 of 4.6  250 mm . the mobile phase consisted of an isocratic delivery 
 of acetonitrile / methanol ( 6:4 , v / v ) at a rate of 2.0 ml / min . acidophilus 10050 
 lh2 complex was dried under nitrogen gas and denatured with 2 ml of 
 acetone . following centrifugation , the supernatant was removed and 
 2 ml of fresh acetone was added to the remaining pellet , and the mixture 
 was centrifuged again , this time resulting in a colorless pellet . the sample was taken up in 1 ml of acetonitrile / methanol 
 ( 6:4 , v / v ) and analyzed on the same hplc system with a waters atlantis 
 t3 obd 5 m preparative column . the mobile phase was the same 
 as described above , but with the flow rate increased to 7.0 ml / min . the molar percentages of the carotenoids were calculated using 
 the area of each hplc peak detected at the wavelength of maximum absorption 
 divided by the extinction coefficient of the carotenoid , and then determining the percentage of each pigment 
 relative to the total carotenoid content . peaks that could not be 
 conclusively identified by mass spectrometry remain unidentified , 
 but are presumed to be isomers formed during the extraction procedure . all steady - state absorption and 
 fluorescence emission and excitation spectroscopic measurements were 
 carried out in 1 cm square cuvettes at room temperature unless otherwise 
 stated . rhodopin glucoside and rhodopinal glucoside were dissolved 
 in spectroscopic grade carbon disulfide ( acros organics ) , benzyl alcohol 
 ( sigma - aldrich ) , methanol ( sigma - aldrich ) , or acetonitrile ( sigma - aldrich ) . steady - state absorption spectra of the carotenoids in various 
 solvents and in the lh2 complexes were obtained using either a varian 
 cary 50 or a cary 5000 uv  visible spectrophotometer . fluorescence 
 emission and excitation spectra were recorded using a jobin - yvon horiba 
 fl3 - 22 fluorimeter equipped with double excitation and emission monochromators 
 having 1200 grooves / mm gratings , a 450 w osram xbo xenon arc lamp , 
 and a hamamatsu r928p photomultiplier tube detector . emission 
 spectra of the lh2 complexes were recorded using samples having an 
 od between 0.025 and 0.1 in a 1 cm path cuvette at the bchl qx band at 591 nm , which was also the excitation wavelength . the excitation and emission slit widths corresponded to bandpasses 
 of 6 and 3 nm , respectively for experiments on the lh2 complex from rbl . acidophilus 7050 ( hl and ll ) lh2 complexes were obtained 
 using excitation and emission slit widths corresponding to bandpasses 
 of 12 and 6 nm , respectively . all emission spectra were corrected 
 using an emission correction factor file generated by taking the ratio 
 of the spectral response of a calibrated 200 w quartz tungsten - halogen 
 filament lamp and the instrument detection system . fluorescence 
 excitation spectra were recorded by monitoring the 
 bchl emission at its maximum wavelength ( 870 nm for the b800 - 850 complexes 
 and 860 nm for the b800 - 820 complex ) using samples with an od of 0.025 
 in a 1 cm path cuvette at the bchl qx band . emission was 
 detected at a right angle relative to the excitation with bandpasses 
 corresponding to 6 nm ( 10050 lh2 ) , 7 nm ( 7050 hl ) , and 4 nm ( 7050 
 ll ) for the excitation monochromator , and 12 nm ( 10050 lh2 ) and 14 
 nm ( 7050 hl and ll ) for the emission monochromator . an excitation 
 correction factor file was used to correct for the wavelength variability 
 of the source lamp and excitation monochromator . this file was generated 
 using a photodiode calibration kit consisting of a photodiode assembly 
 and a dm303-p module that was rented from horiba . probe 
 ultrafast transient absorption spectroscopy was 
 carried out using a helios femtosecond transient absorption spectrometer 
 ( ultrafast systems llc , sarasota , fl , usa ) coupled to a laser setup 
 that has been previously described . surface xplorer 
 pro 1.2.2.26 ( ultrafast systems llc , sarasota , fl , usa ) was used to 
 correct for the dispersion in the transient absorption spectra . samples 
 having an od between 0.2 and 0.5 in a 2 mm path cuvette at the carotenoid 
 spectral origin ( 00 ) band were mixed continuously using a 
 magnetic microstirrer to avoid photodegradation . the pump laser had 
 an energy of 1 j / pulse focused on a 1 mm diameter spot , which 
 corresponds to a laser intensity between 3.2 and 3.9  10 photons / cm . the integrity of the samples was 
 assayed by taking steady - state absorption spectra before and after 
 laser excitation . global fitting of the transient absorption 
 data sets was performed 
 in asufit 3.0 provided by dr . evaldas katilius at arizona state university 
 and carried out according to a sequential excited state decay model 
 which yielded evolution associated difference spectra ( eads ) . reconstruction of the 1-t and fluorescence excitation 
 spectra was done using origin software version 9 . ground state geometries were 
 generated using b3lyp/6 - 31g(d ) methods as implemented 
 within gaussian 09 . excited state geometries 
 were generated using single - configuration interaction ( cis ) methods 
 and full single ci . the effect of the 
 solvent environment was simulated by using the polarizable continuum 
 model ( pcm ) . spectroscopic 
 properties were calculated using mndo - psdci and equation 
 of motion coupled - cluster singles and doubles ( eom - ccsd ) methods . the mndo - psdci methods are semiempirical and have been 
 used successfully to study long - chain polyenes and carotenoids . the eom - ccsd calculations were carried out using 
 a d95 double - zeta basis set , but the size of the target systems limited 
 the use of this method to only rhodopin . the steady - state absorption spectra of rhodopin 
 glucoside and rhodopinal 
 glucoside recorded in carbon disulfide , benzyl alcohol , methanol , 
 and acetonitrile are shown in figure 2 . the 
 primary bands in these spectra represent an electronic transition 
 from the ground s0 ( 1ag ) state to the s2 ( 1bu ) state . in any particular solvent , the spectrum of rhodopinal glucoside 
 is shifted to longer wavelength by 3040 nm compared to that 
 of rhodopin glucoside . the spectrum of rhodopin glucoside displays 
 well - resolved vibronic bands in all of the solvents , whereas the spectrum 
 of rhodopinal glucoside is much less structured , except in carbon 
 disulfide , where a shoulder on the long - wavelength side of the primary 
 absorption band is observed . also , the high polarizability of carbon 
 disulfide ( p( ) = 0.354 ) results in a 3040 
 nm red shift of the spectra of rhodopin glucoside and rhodopinal glucoside 
 compared to their spectra recorded in the less polarizable solvents , 
 acetonitrile ( p( ) = 0.210 ) and methanol ( p( ) = 0.202 ) . benzyl alcohol , which has a polarizability 
 value ( p( ) = 0.314 ) between those of carbon 
 disulfide and methanol or acetonitrile , red shifts the spectrum of 
 rhodopinal glucoside more than for rhodopin glucoside ( figure 2 ) . this is undoubtedly due to the presence of the 
 aldehyde group on rhodopinal glucoside , which interacts more strongly 
 with this solvent than the methyl group in the same position on rhodopin 
 glucoside . except for a small change in relative intensities of the 
 vibronic bands of rhodopin glucoside , the spectra are not significantly 
 affected by changing the solvent from a protic ( methanol ) to a nonprotic 
 ( acetonitrile ) polar solvent . it should be mentioned that the glucoside 
 moiety has no effect on the positions and intensities of the absorption 
 bands . the spectra of rhodopin glucoside and rhodopinal glucoside 
 are indistinguishable from the corresponding spectra of rhodopin and 
 rhodopinal . acidophilus 10050 , 
 7050 hl , and 7050 ll recorded at room temperature are shown in figure 3 . all spectra show the strong s0 ( 1ag )  s2 ( 1bu ) transition characteristic of carotenoids 
 in the 400550 nm region . also , the positions of the bchl soret 
 band at 375 nm and the bchl qx band at 590 
 nm are nearly identical for all of the complexes . however , one bchl 
 qy band of the lh2 complex is located at 800 nm , and the 
 other is at 859 nm for strain 10050 , at 855 nm for 7050 hl , and at 
 823 nm for 7050 ll . in addition , compared to the spectrum of the lh2 
 complex from strain 10050 , the spectra of the lh2 complexes from 7050 
 hl and 7050 ll show more absorption in the region between 550 and 
 600 nm where the spectrum of the carotenoid partially overlaps with 
 the bchl qx band at 590 nm . this is due to the 
 presence of rhodopinal and rhodopinal glucoside in the lh2 complexes 
 from strain 7050 . acidophilus 7050 will always result in a very small amount of the b800 - 820 lh2 
 complex in hl cells due to the effect of light shading in the culture 
 media . likewise , a small amount of b800 - 850 lh2 complex will be present 
 in the ll cells due to the fact that there is a limit to how low the 
 light intensity can be adjusted to ensure a realistic amount of bacterial 
 growth . a spectral analysis of the bchl absorption bands in the qy region ( figure s1 in the supporting information ) shows that this amount is less than 20% . moreover , it should be 
 noted that the regulatory pathways for the switch from rhodopin glucoside 
 to rhodopinal glucoside and from b800 - 850 to b800 - 820 are independent 
 of each other . during growth at progressively decreasing light intensity , the carotenoid pathway switch is activated before , i.e. , at a higher 
 light intensity than the switch that controls the type of complex 
 present . therefore , it is possible to 
 obtain a b800 - 850 complex that contains a significant amount of rhodopinal 
 glucoside as is the case for lh2 complex isolated from 7050 hl cells 
 ( table 1 ) . the pronounced similarity in the 
 structures of the b800 - 850 and b800 - 820 protein complexes precludes 
 complete separation by chromatographic techniques . however , by a judicious 
 choice of excitation and detection wavelengths in the steady - state 
 and transient absorption spectroscopic experiments , the properties 
 of the individual pigment acidophilus 10050 , 7050 ll , and 7050 hl 
 recorded in 2 mm path length cuvettes at room temperature . uncertainties in the values , 
 based on standard deviations from the mean , were equal to or less 
 than two percentage points . the carotenoid 
 composition of the different lh2 complexes was determined 
 by hplc analyses carried out as illustrated in figure 4 . the molar percentages of the carotenoids 
 in the different lh2 complexes are given in table 1 . acidophilus 7050 cells display a significant 
 increase in total rhodopinal ( defined in this context as rhodopinal 
 plus rhodopinal glucoside ) concurrent with a decrease in total rhodopin 
 ( defined here as rhodopin plus rhodopin glucoside ) compared to that 
 found in the bacterium grown under hl conditions . total 
 rhodopinal in the lh2 complexes increased from 32% in the 7050 hl 
 sample to 65% ( 58% + 7% ) in the 7050 ll sample ( table 1 ) . concurrently , total rhodopin in the lh2 complexes decreased 
 from 56% ( 28% + 28% ) to 15% ( 5% + 10% ) when cells were grown using 
 ll ( table 1 ) . as previously reported and confirmed by the present work , the lh2 complex from rbl . acidophilus 7050 lh2 complexes prepared from cells 
 grown under ll ( top trace ) 
 and hl ( bottom trace ) conditions . the major pigments were identified as follows : 1 , rhodopinal 
 glucoside ; 2 , rhodopinal ; 3 , rhodopin glucoside ; 4 , bchl a ; 5 , rhodopin ; and 6 , lycopene . the fluorescence spectra of the lh2 complexes recorded using 
 591 
 nm excitation , which excites the bchl qx band , are shown 
 in figure 5 ( blue traces ) . the maximum wavelength 
 of emission occurs at 870 nm for the lh2 complexes from 10050 and 
 7050 hl , and at 860 nm for the lh2 complex from 7050 ll . fluorescence 
 spectra were also recorded using excitation wavelengths of 560 , 570 , 
 580 , and 590 nm , but no changes in the position or shape of the resulting 
 emission spectra were observed . fluorescence excitation spectra of 
 the lh2 complexes are also shown in figure 5 ( red traces ) . these spectra were recorded by detecting the fluorescence 
 from the samples at the maximum wavelength of bchl emission , but identical 
 lineshapes were observed using any detection wavelength between 820 
 and 880 nm ( figure s2 in the supporting information ) . emission ( blue ) , excitation ( red ) , and 1-t ( black ) spectra of lh2 
 complexes obtained from rbl . the green line shows the ratio of the normalized 
 excitation and 1-t spectra and in the region of carotenoid absorption 
 gives a quantitative measurement of the carotenoid - to - bchl energy 
 transfer efficiency . it is somewhat surprising that the fluorescence maximum of 
 the 
 b800 - 820 complex occurs at 860 nm rather than closer to the 820 nm 
 qy band . however , as mentioned above , previous work detailing 
 the effect of light intensity and temperature on cell growth and the 
 formation of the b800 - 820 complex indicated that the conversion of 
 rhodopin to rhodopinal occurs prior to the conversion of b800 - 850 
 to b800 - 820 . therefore , it is likely that 
 the fraction of b800 - 850 complex in our ll sample ( see figure s1 in 
 the supporting information ) contains rhodopinal 
 and represents the main emission component . this interpretation rationalizes 
 the appearance of the fluorescence maximum at 860 nm and the fluorescence 
 excitation spectra not being affected by changing the detection wavelength . t is transmittance 
 ( black traces ) , and the excitation spectra ( red traces ) were normalized 
 at the bchl qx and qy bands , and the ratio of 
 the excitation and 1-t spectral amplitudes were determined in the 
 region from 300 to 650 nm ( green traces ) . these traces represent the 
 efficiency of excitation energy transfer ( eet ) to bchl , which was 
 found in the carotenoid absorption region between 425 and 550 nm to 
 be in the range of 55% to 61% for the 10050 lh2 complex consistent 
 with previously published results . the lh2 complexes 
 from 7050 hl and 7050 ll displayed higher carotenoid - to - bchl eet efficiencies 
 and were in the range of 63% to 71% and 76% to 86% , respectively . hplc analysis ( table 1 ) revealed that the 
 lh2 samples from the 7050 samples contain both rhodopin and rhodopinal 
 ( and their associated glucosides ) in different amounts . therefore , 
 in order to examine the contributions of these individual chromophores 
 to the spectra of the pigment  protein complexes , reconstructions 
 of the 1-t ( left panel , figure 6 ) and fluorescence 
 excitation ( right panel , figure 6 ) spectra 
 were carried out based on the absorption spectra of the hplc - purified 
 carotenoids recorded in benzyl alcohol . the spectra of the carotenoids 
 in the lh2 protein ( figure 3 ) are well - reproduced 
 by their spectra in benzyl alcohol ( figure 2 ) due to the fact that this solvent has a polarity and polarizability 
 similar to the average of those of the protein in the binding environment 
 of the carotenoid . the 
 spectral reconstructions yielded the specific carotenoid - to - bchl eet 
 efficiencies for rhodopin and rhodopinal from the ratios of the individual 
 fluorescence excitation and 1-t bands in the profiles . ( because rhodopin 
 and rhodopinal have identical absorption ( and 1-t ) spectra as their 
 corresponding glucoside derivatives , in 
 this context and unless explicitly noted otherwise , any statements 
 about the spectra of rhodopin or rhodopinal should be taken to mean 
 the combined contribution from the chromophores associated with both 
 the glucoside and non - glucoside molecules . ) the eet efficiency for 
 rhodopin in the lh2 complex from the 10050 sample becomes evident 
 from a side - by - side , horizontal comparison of the amplitudes of the 
 1-t and fluorescence excitation spectra ( figures 6a and 6b ) in the region of carotenoid 
 absorption , and was found to be 56  1% , consistent with previous 
 reports . a similar analysis was carried out to obtain 
 the values for the individual carotenoid - to - bchl eet efficiencies 
 in the lh2 complex from the 7050 ll sample ( figures 6c and 6d ) , which were found from the 
 spectral reconstruction to be 54  5% for rhodopin ( orange traces ) 
 and 97  2% for rhodopinal ( purple traces ) . the computed individual 
 carotenoid - to - bchl eet efficiencies for the lh2 complex prepared from 
 7050 hl cells ( figures 6e and 6f ) were 50  3% for rhodopin ( orange traces ) and 98  
 2% for rhodopinal ( purple traces ) , consistent with the values determined 
 for the lh2 complex from 7050 ll . these results 
 are summarized in table 2 and reveal the remarkable 
 finding that rhodopinal in the lh2 complex from rbl . acidophilus 7050 transfers essentially all of its excited state energy to bchl 
 with minimal loss through internal conversion to the ground state . this stands in striking contrast to rhodopin in the lh2 complexes 
 from all rbl . reconstruction of the 
 ( a , c , e ) 1-t and ( b , d , f ) fluorescence 
 excitation spectra ( black traces ) of the lh2 complexes from rbl . the 1-t spectra 
 of purified rhodopin glucoside ( orange traces ) and rhodopinal glucoside 
 ( purple traces ) were recorded in benzyl alcohol and summed to generate 
 the reconstructed spectra ( red traces ) . the bchl bands in the soret 
 region between 300 and 400 nm and in the qx region near 
 600 nm were modeled using gaussian functions ( green lines ) for simplicity . the uncertainties 
 in the amplitudes 
 were obtained by comparing the reconstructed spectra with those experimentally 
 recorded . those values figure 7 shows ultrafast 
 time - resolved transient 
 absorption spectra of rhodopin glucoside ( left panels ) and rhodopinal 
 glucoside ( right panels ) recorded in carbon disulfide , benzyl alcohol , 
 methanol , or acetonitrile at various delay times after laser excitation . 
 upon excitation , an immediate onset of bleaching of the strongly allowed 
 s0 ( 1ag )  
 s2 ( 1bu ) transition 
 occurs , resulting in a negative signal in the 450550 nm region . 
 in addition , a strong positive signal in the 520700 nm region 
 appears that can be attributed to excited state absorption ( esa ) associated 
 with the s1 ( 2ag )  sn transition . this suggests that the decay 
 of the s2 state via internal conversion to populate the 
 s1 state is occurring on the same time scale as the instrument 
 response time of 100 fs . similar to the steady - state absorption 
 spectra of the carotenoids , the excited state absorption bands shift 
 to longer wavelength and are broader as the polarizability of the 
 solvent increases . the spectra are broadest and most red - shifted in 
 the highly polarizable solvent , carbon disulfide , compared to the 
 spectra recorded in the other solvents . a similar effect is evident 
 in the spectrum of rhodopinal glucoside in carbon disulfide and benzyl 
 alcohol ( figure 7 , right panels ) , where it 
 is also clear that the spectral bands are broader overall than the 
 spectra of rhodopin glucoside in the same solvents ( figure 7 , left panels ) . also , an additional peak at 740 
 nm , which is not found in the spectra of rhodopin glucoside , is seen 
 in the transient absorption spectra from rhodopinal glucoside . unlike 
 the main s1 ( 2ag )  sn transient absorption band , this feature 
 is insensitive to the polarizability of the solvent , and therefore 
 appears more separated from the main esa feature in methanol and acetonitrile 
 compared to in carbon disulfide . transient absorption spectra of rhodopin 
 glucoside and rhodopinal 
 glucoside in carbon disulfide , benzyl alcohol , methanol , and acetonitrile 
 recorded at room temperature using the indicated excitation wavelengths . in order to obtain detailed information 
 regarding the dynamics 
 of the excited states of the carotenoids , global fitting according 
 to a sequential decay model , resulting in evolution associated difference 
 spectra ( eads ) , was carried out on the transient absorption data sets . for both rhodopin glucoside and rhodopinal glucoside 
 in all four solvents , the first eads has a very short lifetime ranging 
 from < 100 to 120 fs , and the profile of this component shows bleaching 
 of the ground state s0 ( 1ag )  s2 ( 1bu ) absorption and stimulated emission from the s2 ( 1bu ) state . it is important 
 to point out here that although the best global fitting results are 
 achieved in some cases using time constants smaller than 100 fs , these 
 very small values can not be taken factually because the instrument 
 response time of the laser spectrometer is on the order of 100 
 fs . hence , these values are specified as < 100 fs . more precise 
 values in this time domain were reported by macpherson et al . who used fluorescence upconversion spectroscopy 
 and found the s2 ( 1bu ) lifetime of rhodopin glucoside in benzyl alcohol to be 124  
 8 fs . the second eads component for both molecules has a time constant 
 that ranges from 330 to 530 fs in the different solvents . this component 
 can be assigned to a transition from a vibronically hot s1 ( 2ag )  sn excited singlet state due to the fact that its broad esa peak narrows 
 and shifts to shorter wavelength upon decaying to form the third eads 
 component . the second and third eads for rhodopinal glucoside in all solvents 
 also show the additional peak at 740 nm alluded to above , 
 which is not found in the spectra of rhodopin glucoside . the fact 
 that this feature does not depend on solvent polarity indicates that 
 it is not associated with the formation of an intramolecular charge 
 transfer ( ict ) state in rhodopinal glucoside . evolution 
 associated difference spectra ( eads ) obtained from globally 
 fitting the transient absorption data sets from rhodopin glucoside 
 and rhodopinal glucoside in carbon disulfide , benzyl alcohol , methanol , 
 and acetonitrile given in figure 7 . the band profile of the third eads component for 
 both molecules 
 is very strong and well - known to be associated with esa from the relaxed 
 s1 ( 2ag ) state 
 making a transition to a higher excited singlet state . the lifetime 
 of this component for rhodopin glucoside in all solvents was found 
 to be in the narrow range of 3.8 to 4.3 ps with no obvious effect 
 of solvent polarity on the value . however , the s1 ( 2ag ) lifetime for rhodopinal 
 glucoside in the polar solvents methanol and acetonitrile was 3.7 
 and 3.9 ps respectively , which are slightly shorter than the 4.4 and 
 4.7 ps values found for the molecule in the less polar solvents , benzyl 
 alcohol and carbon disulfide , respectively . the fact that the lifetime 
 of this component is reasonably similar for both rhodopin glucoside 
 and rhodopinal glucoside indicates that neither the configuration , 
 which is 13-cis for rhodopinal glucoside in solution 
 compared to all - trans for rhodopin glucoside ( figure 1a ) , nor the presence of the aldehyde group at carbon 
 c20 on rhodopinal glucoside compared to the methyl group at the same 
 position on rhodopin glucoside results in any significant change in 
 the dynamics of the s1 state that would impact its role 
 in light - harvesting . the fourth eads component of both molecules 
 in all solvents has 
 a lifetime that ranges from 5.9 to 15.3 ps . this component is rather 
 weak , but displays negative features associated with ground state 
 bleaching as well as a positive feature on the short wavelength side 
 of the main s1  sn absorption profile . the negative features indicate that some fraction of the carotenoid 
 population is still in an excited state . the short wavelength band 
 is reminiscent of the s * state initially proposed to be an intermediate 
 state between the s1 ( 2ag ) and s2 ( 1bu ) excited 
 singlet states that is involved in both s2 ( 1bu ) depopulation and carotenoid triplet state 
 formation in light harvesting complexes . subsequent 
 work on several open - chain carotenoids 
 has shown that the s * yield is larger for molecules with longer -electron 
 conjugation suggestive of it being associated with a twisted molecular 
 conformation of the carotenoid in the s1 ( 2ag ) state . finally , a very weak , infinitely long - lived component was necessary 
 to obtain a completely satisfactory fit to the data sets . the lh2 complexes 
 were excited in the region of carotenoid absorption at either 525 
 or 570 nm , and then transient absorption spectra were recorded at 
 various delay times after the pump laser pulse ( figure 9 ) . acidophilus 10050 
 displayed transient absorption profiles ( top panel of figure 9 ) that were similar to previously reported spectra 
 and include the instantaneous onset of bleaching of the carotenoid 
 ground state spectrum and broad positive esa at intermediate times , 
 followed by the appearance of a strong narrow s1  
 sn transition at 580 nm at later times . the hplc pigment analysis of the lh2 complex 
 from strain 10050 ( table 1 ) revealed that the 
 carotenoid composition consists of roughly equal amounts of rhodopin 
 and rhodopin glucoside with a small amount of lycopene , all of which 
 have identical absorption spectra . using this information regarding 
 the spectra of the carotenoids in the lh2 complex from rbl . acidophilus 10050 as a control , the features attributable 
 to rhodopin and rhodopinal can be distinguished in the transient spectra 
 of the lh2 complexes prepared from 7050 ll and 7050 hl cells , which 
 contain a mixture of these carotenoids ( table 1 ) . ( recall that any reference to rhodopin or rhodopinal in this context 
 should be taken to mean the combined spectral properties of the molecules 
 and their respective glucosides , which are indistinguishable . ) laser 
 excitation at 525 nm of the 7050 ll or 7050 hl lh2 complexes results 
 in a combination of transient absorption signals from rhodopin and 
 rhodopinal because both molecules absorb at this wavelength . selective 
 excitation of rhodopinal can be achieved by tuning the pump laser 
 to 570 nm , which is a wavelength where rhodopin does not absorb . acidophilus 10050 , 7050 ll , and 7050 hl recorded at room 
 temperature using the indicated excitation wavelengths . transient absorption spectra from the 7050 ll and 
 hl lh2 complexes 
 excited at 525 nm ( combined rhodopin plus rhodopinal excitation ) or 
 at 570 nm ( selective rhodopinal excitation ) are shown in figure 9 . similar to the transient absorption spectra from 
 strain 10050 , the transient spectra recorded for these lh2 complexes 
 at a 50 fs delay time ( black traces in figure 9 ) have negative features corresponding to the bleaching of the carotenoid 
 s0 ( 1ag )  
 s2 ( 1bu ) ground state 
 absorption bands . in addition , there is a positive signal in the 540700 
 nm region which represents an s1 ( 2ag )  sn transition indicating 
 very fast decay of the s2 ( 1bu ) state to populate the s1 ( 2ag ) state of the carotenoid . in the 200 fs 
 delay time spectra ( red traces ) , the ground state bleaching has partially 
 recovered , indicative of energy transfer to bchl , but the strong positive 
 s1 ( 2ag )  
 sn absorption band has gained amplitude and is broader 
 compared to the subsequent 1 ps delay time spectrum ( green traces ) . the gain in amplitude of the feature associated with the s1 ( 2ag )  sn transition indicates that internal conversion from s2 to populate the s1 state is competing effectively with 
 eet from s2 to bchl . a close examination of the 1 
 ps time delay ( green trace ) spectrum 
 recorded for the 7050 ll lh2 excited at 525 nm ( second panel in figure 9 ) shows a narrow positive feature at 580 
 nm and a broader one at 630 nm . comparison of this spectrum 
 with those taken at the same time delay from the 10050 lh2 excited 
 at 525 nm ( selective rhodopin excitation , upper panel of figure 9 ) and the 7050 ll lh2 sample excited at 570 nm ( selective 
 rhodopinal excitation , third panel of figure 9 ) reveals that the narrow peak at 580 nm in the spectrum 
 from the 7050 ll lh2 excited at 525 nm is due to the s1  sn transition of rhodopin whereas the 630 
 nm peak is from rhodopinal . this is expected because excitation of 
 the 7050 ll sample using 525 nm light excites both carotenoids which 
 are present in this lh2 sample ( table 1 ) . this 
 interpretation is supported by the transient absorption spectra resulting 
 from 525 nm excitation of the 7050 hl lh2 complex ( fourth panel of 
 figure 9 ) . note that the narrow positive feature 
 at 580 nm in the 1 ps time delay spectrum ( green trace ) attributable 
 to the s1  sn transition of rhodopin 
 is much more pronounced relative to the broader band at 630 
 nm associated with rhodopinal . this is because the lh2 complex from 
 7050 hl contains 56% total rhodopin compared to the 7050 ll lh2 sample , 
 which has only 15% rhodopin ( table 1 ) . as expected , 
 selective excitation of rhodopinal at 570 nm in the 7050 hl lh2 sample 
 ( bottom panel of figure 9 ) shows no sign of 
 the narrow s1  sn feature belonging 
 to rhodopin . the major positive band in the 1 ps time delay spectrum 
 ( green trace ) is that of the s1  sn esa 
 of rhodopinal . the 5 ps delay time spectra of the 10050 and 
 7050 ll lh2 samples 
 ( blue traces in figure 9 ) show significantly 
 diminished carotenoid ground state bleaching and reduced s1  sn esa indicating that a substantial amount 
 of the carotenoid s1 state excited state population either 
 has been transferred to bchl or has decayed via internal conversion 
 back to the ground state . also in this time frame , an additional peak 
 appears at 560 nm on the short wavelength side of the main 
 esa peak and persists longer than 100 ps ( magenta trace ) . the eads 
 components of the lh2 complexes obtained from a global fitting of 
 the transient absorption data sets are shown in figure 10 . the fitting of the transient absorption spectra required 
 five components , the first of which has a lifetime < 100 fs for 
 all the lh2 complexes and has features associated with the bleaching 
 of the ground state absorption as well as stimulated emission from 
 s2 ( 1bu ) . this is consistent 
 with the value of 57 fs reported by macpherson et al . the 
 second eads component ( red traces in figure 10 ) has a lifetime ranging from 250 to 490 fs and has broad features 
 in the carotenoid absorption region characteristic of vibronically 
 hot s1 ( 2ag ) 
  sn excited singlet state transition . this second 
 eads has weaker negative bands in the carotenoid ground state absorption 
 region than the first eads , indicating that some of the carotenoid 
 molecules have returned to the ground state via eet from the s2 state to bchl . also evident in the second eads is a small 
 negative dip at 590 nm appearing on the broad positive esa 
 spectrum . this is due to the bleaching of the bchl qx band 
 brought about by eet to bchl from the carotenoids . as the second eads 
 component decays into a third ( green lines in figure 10 ) , the line shape narrows considerably for the 10050 lh2 excited 
 at 525 nm and a strong band associated with the s1 ( 2ag )  sn transition 
 is evident at 580 nm . this line narrowing is accompanied by a decrease 
 in the extent of ground state bleaching indicating that more of the 
 carotenoid molecules have returned to the ground state , perhaps by 
 eet from the vibronically hot s1 state . this third component 
 decays in 3.0 ps for the 10050 lh2 and represents the s1 lifetime of rhodopin in the lh2 pigment  protein complex . the peak at 580 nm has much less amplitude in the third eads component 
 from the 7050 ll lh2 sample obtained from data also using 525 nm excitation 
 ( green trace in the second panel of figure 10 ) . this is due to the fact that this sample has much less total rhodopin 
 ( rhodopin plus rhodopin glucoside ) than the 10050 lh2 . in fact , the 
 peak at 580 nm is completely absent in the third eads obtained from 
 data using 570 nm excitation ( green trace in the third panel of figure 10 ) . instead , a broad line shape peaking at 620 
 nm and belonging to rhodopinal is observed . note that the lifetime of 
 this component is 1.2 ps in the 7050 ll lh2 and 1.3 ps in the 7050 
 hl lh2 , which is significantly shorter than the average value of 4.1 
 ps found for rhodopinal in the different solvents and also shorter 
 than the value of 3.0 ps found for rhodopin in the lh2 complex from 
 strain 10050 . this is suggestive of significantly faster eet to bchl 
 from the s1 state of rhodopinal compared to rhodopin . evolution 
 associated difference spectra ( eads ) obtained from globally 
 fitting the transient absorption data sets of lh2 complexes from rbl . the uncertainties 
 in the values 
 were obtained by exploring the region of solution for each parameter 
 according to the goodness of fit and minimization of the residuals . 
 an infinitely long ( on the time scale of the experiment ) component the uncertainties 
 were obtained 
 by exploring the region of solution for each parameter according to 
 the goodness of fit and minimization of the residuals . an infinitely 
 long ( on the time scale of the experiment ) component was required 
 for a good fit in all cases . the fourth eads component has a lifetime in the range 13 to 27 
 ps for all the data sets and a significantly diminished overall amplitude 
 relative to the preceding eads profiles . the lifetime of this component , 
 along with the clearly evident positive band on the short wavelength 
 side of the feature attributable to the main s1  
 sn absorption band , and the wavy features in the region 
 of the carotenoid ground state absorption are all characteristics 
 of the s * state in accord with previous reports . the fifth and final eads component 
 has an infinitely long lifetime on the time scale of the experiment 
 and was necessary to obtain a satisfactory fit in all cases . the primary 
 spectral feature in this last eads component is the negative amplitude 
 at 590 nm , which corresponds to the bleaching of the bchl qx band that is expected to persist on the order of nanoseconds until 
 the s1 state of bchl relaxes back to the ground state . quantum computations 
 were carried out to augment the experimental results , but in order 
 to make them tractable , the majority were carried out on the model 
 chromophores shown in figures 11c and 11d . the model chromophores include the entire central 
 polyene portion of rhodopin and rhodopinal , but replace the glucoside 
 and aliphatic end groups with methyl groups . the resulting model polyenes 
 have cs or c2h symmetry , and this symmetry , in combination 
 with the smaller size , allows higher quality calculations to be carried 
 out than would otherwise be possible . test calculations on the full 
 and model chromophores indicate that the end groups do not have a 
 significant impact on the atomic charges other than small changes 
 in the charges on the carbon atoms at the ends of the polyene chains . single ci calculations on the full system ( figures 11a and 11b ) and the model systems ( figures 11c and 11d ) demonstrate that 
 the end groups create less than a 0.02 ev shift in the transition 
 energies . this conclusion is consistent with the observation that 
 the absorption spectra of rhodopin and rhodopin glucoside are indistinguishable . b3lyp/6 - 31g(d ) calculated structures of ( a ) trans - rhodopin glucoside , ( b ) trans - rhodopinal glucoside , 
 ( c ) trans - rhodopin , ( d ) trans - rhodopinal , 
 ( e ) the s1 relaxed excited state of trans - rhodopinal , ( f ) 13-cis - rhodopinal , and 13-cis - rhodopinal model . the structures given in panels c , 
 d , e , and g are simplified , higher - symmetry analogues used in the 
 mndo - psdci , eom - ccsd and cas - scf theoretical calculations , which retain 
 the full -system . the calculated vacuum dipole moments ( in 
 debyes ( d ) ) and the dipole moment vectors ( the length is not relevant ) 
 are shown underneath those structures that have a dipole moment . the 
 dashed ellipse in panel d shows the primary repulsive atom atom 
 interaction responsible for making the cis configuration 
 more stable than the trans configuration by 8 
 kj / mol in nonpolar solvent ( n - hexane ) and 8.5 
 kj / mol in polar solvent ( acetonitrile ) . although a 
 primary goal of this work is to understand the photophysical properties 
 of rhodopinal in lh2 complexes , the process is started by identifying 
 the geometry of rhodopinal in solution . previous studies have proposed 
 that rhodopinal acquires a cis configuration in solution . based on density functional theory and an analysis 
 of the absorption spectra , it is demonstrated here that rhodopinal 
 takes on a cis configuration in both nonpolar and 
 polar solvent . the all - trans rhodopinal chromophore 
 exhibits just one significant intramolecular repulsion involving the 
 interaction of the aldehyde hydrogen with the nearest hydrogen atom 
 on the main polyene chain . the repulsion 
 is both electrostatic ( both atoms have positive charge ) and steric 
 ( the atoms are separated by 2  ) . . the aldehyde group can 
 rotate out of plane , or the double bond adjacent to the aldehyde group 
 can rotate 180 to create a cis linkage 
 near the center of the polyene chain . based on b3lyp/6 - 31g(d)/pcm 
 calculations the resulting 13-cis configuration is 
 more stable than the all - trans configuration by 8 
 kj / mol in nonpolar solvent ( n - hexane ) and 8.5 
 kj / mol in polar solvent ( acetonitrile ) . this number is invariant to 
 whether the calculation is done on the smaller model chromophore ( figure 11d ) or the full chromophore ( figure 11b ) . the value in n - hexane increases to 9.3 
 kj / mol when a much larger basis set [ 6 - 311+g(2d , p ) ] is used . experimental support for the 13-cis configuration 
 of rhodopinal in solution is provided by comparing the excited state 
 manifolds calculated for both the cis and the trans configurations with the experimental spectra . note that there is a relatively strong vibronically 
 resolved band at 28 kk ( 28,000 cm ) observed in 
 the experimental spectra ( solid yellow ) . the mndo - psdci calculations 
 indicate that this band is associated with a ag excited state ( top right - hand panel of figure 12 ) , resulting in transition from the ground state 
 that is often called the  cis - band  . it should be mentioned that the viability of 
 the mndo - psdci methods was explored by carrying out an eom - ccsd calculation 
 on rhodopin . the level ordering of the first eight excited singlet 
 states was identical with the exception that the eom - ccsd methods 
 predicted that the bu state 
 is higher in energy than the bu state . the high relative intensity of the  cis - band  band provides spectroscopic evidence for a cis linkage near the center of the polyene chain . the combination of theory and experiment provide 
 strong support for rhodopinal having a cis configuration 
 in both polar and nonpolar solvent . analysis of the excited state manifold 
 responsible for the electronic 
 absorption spectrum of rhodopinal in n - hexane ( solid 
 yellow spectra ) based on mndo - psdci theory . four configurations ( figure 11 ) were investigated relative to experimental observations : trans - rhodopinal ( upper left ) , 13-cis - rhodopinal 
 ( upper right ) , trans - rhodopinal in a corkscrew conformation 
 ( lower left ) , and 13-cis - rhodopinal in a corkscrew 
 conformation ( lower right ) . the corkscrew conformation was generated 
 by adjusting the dihedral angles of the single bonds 10 from 
 planar and the double bonds 5 from planar , with all dihedral 
 distortions in the same direction . the cis configuration 
 involves rotation of the double bond directly connected to the aldehyde 
 group , identified using the torsional arrow in figure 11d . the heights of the bars are proportional to the calculated 
 oscillator strengths of the transitions , and the color reflects the 
 ionic versus covalent character ( figure 13 ) . 
 the approximate symmetry is indicated for selected states , and 1 kilokayser 
 ( kk ) = 1000 cm . the computations suggest , however , that 13-cis - rhodopinal may form a corkscrew conformation to help stabilize the 
 molecule in solution . a corkscrew conformation involves clockwise 
 or counterclockwise dihedral distortion involving both single and 
 double bonds , with a majority of the distortion in the single bonds . the change in geometry costs very little in 
 terms of torsional distortion energy but provides intramolecular electrostatic 
 stabilization in nonpolar solvent and solute  solvent stabilization 
 in polar solvent . a modest corkscrew rotation of 10 in the single 
 bonds and 5 in the double bonds generates improved agreement 
 between experiment and theory ( bottom panels in figure 12 ) . it should be emphasized that the same improvement in simulating 
 the observed spectra is obtained by generating modest , random dihedral 
 distortions , such as would be generated via thermal motions and occupation 
 of low - frequency torsional modes . the crystal structure of 
 the b800 - 820 lh2 complex did not fully 
 resolve the rhodopinal structure , but provided 
 ample evidence that the structure is in an all - trans configuration . given the above results that demonstrate that 13-cis - rhodopinal is more stable than trans - rhodopinal in solution , the question arises as to why an all - trans configuration for rhodopinal exists in the lh2 
 complex . first , the 
 binding site for rhodopinal glucoside , which , as mentioned previously , 
 is formed independently of , and prior to , the conversion of b800 - 850 
 to b800 - 820 , must also serve as the binding site for rhodopin glucoside , 
 whose most stable configuration is undisputedly all - trans . second , the formation of rhodopinal requires enzymatic activity 
 to attach the aldehyde group . the enzyme would likely release trans - rhodopinal glucoside into solution for assembly into 
 the pigment  protein complex , and formation of the 13-cis - rhodopinal glucoside would require either an isomerase 
 or > 30 min for thermal isomerization to generate the equilibrium 
 structure . as shown in figure 7 , there 
 are significant 
 differences in the transient absorption spectra of rhodopinal compared 
 to rhodopin . in particular , the transient absorption maximum is red - shifted 
 and an additional feature at 740 nm is observed for rhodopinal . 
 to explore these differences theoretically , mndo - psdci theory was 
 used to calculate the energies and oscillator strengths for excitations 
 from the ground state and the relaxed first excited state ( figure 13 ) . the computations also simulated the transient 
 absorption spectra based on the assumption that the origin states 
 are the fully relaxed lowest - excited s1 ( 2ag ) states generated using full single ci 
 and acetonitrile as the solvent . however , the mndo - psdci calculations 
 are for vacuum conditions , and the effect of solvent is limited to 
 the geometry of the relaxed origin state . the simulated spectra ( figure 14 ) reproduce the key features shown in figure 7 by predicting both the red shift of the transient 
 maximum and the farther red - shifted weaker band observed only for 
 rhodopinal . the calculations of the s1  sn transitions for rhodopin predict a maximum absorbance at 500 
 nm , which corresponds to the experimental band observed at 600 
 nm . this band is associated with a transition from the relaxed s1 ( 2ag ) excited state 
 to the 11th excited ( 5bu ) singlet state ( figure 13 ) . this state also gives rise to a weakly allowed 
 transition from the ground state calculated to be at 340 nm . this ensuing discussion of the transient absorption spectrum of rhodopinal 
 in solution is limited to an analysis of the 13-cis configuration because it was demonstrated above that the 13-cis - isomer dominates in that environment ( figure 12 ) . the strong band observed spectroscopically for 
 rhodopinal at 630 nm ( calculated to be at 520 nm ) 
 is associated with a transition from the relaxed s1 ( 2ag ) excited state to the 11th 
 excited ( 5bu ) singlet state ( figure 13 ) , which is an assignment identical to that for 
 the intense band of rhodopin . the major difference is a lower energy 
 transition calculated to be at 680 nm and experimentally observed 
 at 740 nm . this more red - shifted band is associated with a 
 transition from the 2ag excited 
 state to the eighth excited ( 5ag ) singlet state ( figure 13 ) . reported similar red - shifted transient absorption 
 bands from 8-apo--carotenal and also interpreted the 
 results in terms of a transition from the s1 ( 2ag ) state to a higher ag state . moreover , they proposed that the allowedness 
 for the transition arises due to the carbonyl group introducing asymmetry 
 into the conjugated polyene system . the presence of asymmetry in the 
 molecule would have the effect of relaxing the selection rules that 
 render the transition forbidden in more symmetric , linear carotenoids , 
 e.g. , rhodopin . excited state  * level ordering for trans - rhodopin , trans - rhodopinal , and 13-cis - rhodopinal for excitations from the ground state for 
 the equilibrium 
 ground state conformation ( s0 geom ) and the relaxed first excited 
 singlet conformation ( s1 rlxd ) . the energies and oscillator strengths 
 were calculated based on mndo - psdci theory using a ci basis set of 
 the 10 highest energy filled  orbitals and the 10 lowest energy 
 unfilled  orbitals . the stationary states are represented by 
 rectangles where the height is proportional to the oscillator strength , 
 and the color reflects the ionic versus covalent character of the 
 state ( see inset ) . the values 
 in parentheses index electronic states 8 and 11 discussed in the text . simulation of the s1 to sn spectra of trans - rhodopin ( top ) , trans - rhodopinal 
 ( middle ) , and 13-cis - rhodopinal ( bottom ) based on 
 mndo - psdci theory ( full single and double configuration interaction 
 involving the 11 highest energy filled and 11 lowest energy unfilled 
  orbitals ) . the 
 horizontal axis is linear in energy , where 1 kilokayser ( kk ) = 1000 
 cm , and the corresponding wavelength is marked 
 as an inset in green . although there are higher energy bands calculated to be present 
 only in 13-cis - rhodopinal ( compare the middle and 
 bottom spectra in figure 14 ) , the above - mentioned 
 red - shifted band calculated to be at 680 nm appears to be 
 present in both cis- and trans - rhodopinal . 
 this observation can be traced to the fact that , whereas a transition 
 from the ground s0 ( 1ag ) state to the 5ag state only 
 has intensity in molecules with cis configurations , 
 hence its designation as the  cis - band  , 
 a transition from the s1 ( 2ag ) excited state is strong for both trans- and cis - rhodopinal . therefore , the additional 
 red - shifted band seen only in the transient absorption spectrum of 
 rhodopinal in solution is due to the presence of the aldehyde group 
 on the molecule , and not to the configuration of the carotenoid . reconstruction 
 and comparison of the 1-t and fluorescence excitation spectra from 
 the lh2 complexes ( figures 5 and 6 ) show clearly that rhodopinal is much more efficient than 
 rhodopin at transferring excited state energy to bchl . the precise 
 factors responsible for this effect can be obtained from a consideration 
 of the rate constants for de - excitation of the carotenoid excited 
 states derived from the global fitting analysis . the s1 and s2 states of carotenoids represent important donor 
 excited states for eet to bchl . these routes are represented in figure 15 by the rate constants ket1 and ket2 , respectively . deactivation 
 of these two excited states may also occur via internal conversion , 
 and these processes are indicated in the figure by the rate constants , k10 and k21 . it is 
 important to mention here that broadband 2d electronic spectroscopic 
 results provide compelling evidence for the participation in eet of 
 a dark intermediate state of the carotenoid having an energy in the 
 vicinity of the state associated with the qx transition 
 of bchl . this dark state was reported 
 to be populated in 21 fs and depopulated in 62 fs . both of these times 
 are shorter than the 100 fs time resolution of the laser spectrometer 
 used in the present work . hence , the rate constant ket2 illustrated in figure 15 should 
 be interpreted as including the kinetics of this dark state in addition 
 to those of s2 . the total eet efficiency , et , resulting from 
 both the s1 and s2 pathways is given by the 
 expression1where the 
 rate constants correspond to the 
 processes illustrated in figure 15 . the rate 
 constants can be computed from the lifetimes of the s1 and 
 s2 excited states measured in solution , sisoln , and in the lh2 complexes , silh2 . the relevant expressions are2and3 tables 3 , 4 , and 5 summarize the lifetimes obtained from the global 
 fits and the rate constants and efficiencies obtained either from 
 the data presented here and using eqs 13 or from previous work . rate constants for the lh2 complexes were obtained using laser excitation 
 at 525 nm ( rhodopin excitation ) for the sample prepared from rbl . acidophilus strain 10050 ( which contains only the rhodopin 
 chromophore ) and at 570 nm ( selective rhodopinal excitation ) for the 
 sample prepared from rbl . acidophilus strain 7050 
 ll . determined from the 
 dynamics ( dyn ) 
 of the excited states according to eq 1 . determined from steady - state fluorescence 
 excitation spectroscopy ( fl ) as shown in figures 5 and 6 . computed from eq 2 using the values 
 of 3.0 ps ( 3 in table 4 ) 
 and 4.1 ps measured here for the lifetime of the 
 s1 state of rhodopin in the lh2 complex and as an average 
 value in solution ( 3 in table 3 ) , respectively . computed 
 from eq 2 using the values of 57 and 95 fs for 
 the lifetime of the 
 s2 state in the lh2 complex and in solution , respectively . the 57 fs value was obtained from fluorescence upconversion spectroscopic 
 experiments reported in ref ( 33 ) . the lifetime of the s2 state in solution was 
 treated as an adjustable parameter to achieve agreement between the 
 energy transfer efficiency of 56% computed from the dynamics data 
 and that measured by fluorescence excitation spectroscopy . computed from eq 2 using the values of 1.2 ps ( 3 in table 4 ) and 4.1 ps measured here for the lifetime of the 
 s1 state of rhodopinal in the lh2 complex and as an average 
 value in solution ( 3 in table 3 ) , respectively . computed 
 from eq 2 using the values of 10 and 100 fs 
 for the lifetime of the 
 s2 state in the lh2 complex and in solution , respectively . in fact , the s2 lifetime obtained for rhodopinal glucoside 
 in solution was found here to be < 100 fs , which means that the k21 value of 10 ps based 
 on this lifetime is a lower limit to what the value could be . the 
 lifetime of the s2 state in the complex was treated as 
 an adjustable parameter to achieve agreement between the energy transfer 
 efficiency of 97% computed from the dynamics data and that measured 
 by fluorescence excitation spectroscopy . the key result for rhodopin ( and rhodopin glucoside ) 
 in the lh2 
 complex from rbl . acidophilus 10050 is the extent 
 of partitioning between the s1 and s2 donor 
 states of the efficiency of energy transfer to bchl . the data in table 5 show that et1 = 27% and et2 = 40% , which according to eq 1 leads 
 to an overall energy transfer efficiency of 56% . however , it is important 
 to point out that , in the current analysis , the lifetime of the s2 state of rhodopin in solution was treated as an adjustable 
 parameter and set to 95 fs to achieve agreement between the energy 
 transfer efficiency of 56% computed from the dynamics data , and that 
 measured by fluorescence excitation spectroscopy ( figures 5 and 6 ) . the value of 95 
 fs is in very good agreement with the s2 lifetime of 105 
 fs reported by macpherson et al . for 
 rhodopin glucoside in ethanol , and is consistent with the present 
 findings that the value is < 100 fs . previous workers concur that 
 the s2 pathway is most important in the carotenoid - to - bchl 
 energy transfer mechanism in this lh2 complex ( table 5 ) . when rhodopinal ( and rhodopinal 
 glucoside ) is present in the lh2 
 complex , as is the case for rbl . acidophilus 7050 
 ll and hl , the overall carotenoid - to - bchl energy transfer efficiency 
 becomes much greater . as shown clearly from the analysis of the fluorescence 
 excitation spectra given in figure 6 , this 
 can be traced directly to the fact that rhodopinal is much more efficient 
 at transferring energy to bchl than rhodopin . the data presented in 
 table 5 reveal that this enhancement is due 
 to a significant increase in both ket1 and ket2 for rhodopinal compared to 
 those obtained for rhodopin . note from the data in table 5 that ket1 increases 
 from 0.089 ps to 0.59 ps and ket2 increases from 7.0 ps to 90 ps in going from rhodopin to rhodopinal 
 as the energy donor to bchl in the lh2 complex from 7050 ll . these 
 combined increases result in a substantial enhancement in the carotenoid - to - bchl 
 eet efficiency from 56% for rhodopin to 97% for rhodopinal . however , 
 as was the case for the s2 lifetime of rhodopin discussed 
 above , owing to the fact that lifetimes of the s2 state 
 of rhodopinal in solution and in the lh2 complex are faster than the 
 time resolution of the laser spectrometer , these values were treated 
 as adjustable parameters to achieve agreement between the energy transfer 
 efficiency computed from the dynamics data and that measured by fluorescence 
 excitation spectroscopy . a consideration of the fundamental 
 quantum mechanical expression 
 for the rate constant for eet4where t is the electronic 
 coupling term and j is the normalized spectral overlap5and fd( ) 
 is the emission spectrum of the donor carotenoid and a( ) is the absorption spectrum of the bchl acceptor , leads 
 to the conclusion that the increase in the values of the rate constants 
 in going from rhodopin to rhodopinal ( table 5 ) can not be attributed solely to the different positions of the energy 
 levels of the carotenoids , and consequently neither to differences 
 in spectral overlap . this is particularly evident for the route involving 
 the s2 ( 1bu ) state of 
 the carotenoid and the state corresponding to the qx band 
 of bchl . taking the s0 ( 1ag )  s2 ( 1bu ) absorption spectrum of rhodopin and rhodopinal reflected 
 about their spectral origins as approximations to their ( very weak ) 
 emission profiles , and overlaying these lineshapes with the qx absorption band of bchl in the lh2 complex ( figure 16 ) , one obtains a rhodopinal - to - rhodopin spectral 
 overlap ratio of 1.2 . this value is significantly different from the 
 rhodopinal - to - rhodopin ratio of ket2 rate 
 constants given in table 5 which indicate a 
 value of ( 90/7.0 ) = 13 . although it is impossible to compute the spectral 
 overlap integrals associated with the s1 ( 2ag ) state of the carotenoid due to 
 the lack of either detectable s0 ( 1ag )  s1 ( 2ag ) absorption or fluorescence from the s1 ( 2ag ) state , the small 
 difference in the s1 lifetimes of rhodopin and rhodopinal 
 in solution ( table 3 ) suggests very similar 
 s1 excited state energies for the two molecules . therefore , 
 a minimal effect of the position of the energy levels , and consequently 
 of spectral overlap , is expected for the ket1 rate constant in the lh2 complexes . however , a significant change 
 in ket1 is evidenced by comparing the 
 s1 lifetime of rhodopin in the lh2 complex from rbl . acidophilus 10050 ( 3.0 ps ) with that of rhodopinal 
 ( selectively excited at 570 nm ) in the lh2 complex from rbl . acidophilus 7050 ll ( 1.2 ps ) . using eq 2 and an average lifetime of 4.1 ps for the carotenoids in the four 
 different solvents , the value of ket1 is 
 shown in table 5 to increase by a factor of 
 ( 0.59/0.089 ) = 6.6 in going from rhodopin to rhodopinal . because only 
 small differences in excited state energies and spectral overlap with 
 the bchl qx and qy absorption bands are evident 
 for both the s1 and s2 states of these carotenoids , 
 stronger electronic coupling between the donor and acceptor electronic 
 states induced by the presence of the aldehyde group on the -electron 
 polyene chain in rhodopinal must be a major factor determining why 
 rhodopinal is much more efficient at carotenoid - to - bchl eet than rhodopin 
 in the lh2 pigment  protein complex . spectral overlap of 
 the hypothetical fluorescence of the carotenoid 
 donor ( orange and purple traces ) and absorption of the bchl acceptor 
 ( black trace ) . the absorption spectra of the carotenoids were reflected 
 about their spectral origins to obtain approximations to the s2 ( 1bu )  s0 fluorescence 
 spectra . although a rigorous analysis of 
 the coulomb coupling terms describing 
 eet from s1 and s2 is beyond the scope of this 
 paper , this issue is addressed for s1 by the level ordering 
 analysis shown in figure 13 . the lowest excited 
 singlet state of both cis- and trans - configurations is calculated to be a  forbidden 2ag state that gains oscillator 
 strength due to dipole - induced coupling with the higher energy , strongly 
 allowed 1bu state . the above kinetics 
 analysis has revealed that energy is transferred from the s1 ( 2ag ) state to the bchl 
 acceptor 6.6 times faster than the corresponding state in rhodopin . the computations predict that the enhanced energy transfer rate is 
 associated in part with a significant increase in the oscillator strength 
 of the s1 state in rhodopinal relative to rhodopin . moreover , 
 the increase in oscillator strength in trans - rhodopinal 
 is 35% larger than observed in 13-cis - rhodopinal 
 ( figure 13 ) . because the frster coupling 
 efficiency is proportional to the allowedness of the donor excited 
 state , the all - trans configuration of rhodopinal 
 provides added value to its light - harvesting function . acidophilus 7050 is grown under ll conditions , two 
 major changes in the spectrum of the lh2 complex occur : a blue shift 
 of the bchl qy band from 850 nm to 820 
 nm , which is caused by a change in the orientation of the c3-acetyl 
 group of the b850 bchl ; and a red shift and broadening of the spectrum 
 of the carotenoid , which is a consequence of the enzymatic substitution 
 of an aldehyde group for a methyl group on rhodopin resulting in its 
 conversion to rhodopinal . an important question is then , which of 
 these factors is most important for enhancing the rate and efficiency 
 of carotenoid - to - bchl eet ? the answer is that the conversion of rhodopin 
 to rhodopinal is more important than the shift of the bchl qy band . data suggestive of this conclusion was provided previously 
 by deinum et al . , who investigated the 
 b800 - 820 lh2 complex from rbl . cells of this strain grown either at low temperatures or at ll do 
 not contain rhodopinal ( or rhodopinal glucoside ) , and 
 the carotenoid - to - bchl eet efficiency of its associated lh2 complex , 
 which contains primarily rhodopin and rhodopin glucoside , was found 
 to be 45  5% . if the change in the 
 position of the bchl qy band from 850 nm to 820 
 nm was important for enhancing the efficiency of eet from the carotenoid 
 to bchl , one would expect that the efficiency for rbl . acidophilus strain 7750 would have a value above 45% or even higher than 56% , 
 which is the value found for rhodopin in the b800 - 850 lh2 complex 
 from strain 10050 ( table 2 ) . the fact that 
 it is not larger than 56% , and in fact is smaller than this value , 
 i.e. , 45  5% , indicates that the change in the position of the 
 qy band of bchl is not a factor in determining the efficiency 
 of carotenoid - to - bchl eet . instead , the conversion of rhodopin to 
 rhodopinal , which incidentally occurs prior to the conversion of b800 - 850 
 to b800 - 820 , is the primary controlling factor leading to more efficient 
 eet in the lh2 complex of rbl . acidophilus 7050 grown under ll 
 conditions results 
 in a significant enhancement in the ability of the organism to transfer 
 absorbed light energy to the reaction center . this is due almost entirely 
 to the fact that rhodopinal , and its corresponding glucoside , are 
 nearly 100% efficient at transferring excited state energy to bchl 
 in the lh2 complex . also , the spectrum of rhodopinal occurs at a longer 
 wavelength compared to that of rhodopin , which allows the bacterium 
 containing this carotenoid to capture light energy in a spectral region 
 where its photosynthetic bacterial competitors do not absorb light . this provides an important advantage for this organism as it competes 
 for life - sustaining photons in the water column . moreover , based on 
 the ket1 and ket2 rate constants given in table 5 , the energy 
 absorbed by rhodopinal is transferred to bchl in the lh2 complex 6.6 
 times faster from the s1 state and > 10 times faster 
 from 
 the s2 state than energy absorbed by rhodopin . more effective 
 electronic coupling between the electronic states of the carbonyl - containing 
 rhodopinal and bchl is the primary reason for the increase in these 
 rates . even though the blue shift of the bchl qy band 
 from 
 850 nm to 820 nm induced by growing rbl . acidophilus 7050 under ll conditions does not have an effect on the rate and 
 efficiency of carotenoid - to - bchl eet , this spectral change has another 
 consequence . singlet 
 annihilation measurements that , compared to the b800 - 850 complex , 
 the b800 - 820 complex provides a more effective energy barrier for 
 preventing back transfer of excitation energy out of the reaction 
 center core complex and into the photosynthetic unit . thus , the combined effects of alterations in carotenoid 
 and bchl structures and spectral properties resulting from the adaptation 
 of the bacterial photosynthetic organism to the challenging environmental 
 condition of limited light availability contribute in a very tangible 
 way to ensure survival of this species .