carotenoids are present 
 in all phototrophic organisms and play 
 important roles in various light - harvesting complexes as accessory 
 light absorbers and photoprotectors of numerous types of ( bacterio)chlorophylls . in light - harvesting play a structural role and serve as auxiliary 
 light absorbers , harvesting light at wavelengths not covered by bacteriochlorophyll a ( bchl a ) . as the lowest lying excited 
 state s1 is optically forbidden , absorption of carotenoids , responsible for a bright yellow or orange 
 coloration of the pigments , is associated with the strongly allowed 
 s0  s2 electronic transition . in the 
 lh2 complex , this harvested energy is transferred to both the b800 
 and b850 spectral forms of bchls ( figure 1 ) . finally , carotenoids are also excellent 
 photoprotectors , efficiently quenching bchl a triplet 
 states , harmful reactive singlet oxygen , and in some 
 cases also bchl a excited singlet state . steady - state absorption spectra of two batches of lh2 
 complex from 
 the crtc mutant of rba . the excitation wavelength used 
 for time - resolved studies is indicated by an arrow . the standard three - state s0s1s2 model satisfactorily accounts for the 
 typical spectroscopic 
 properties of carotenoids in solvents and in protein environments . 
 however , experimental results from femtosecond time - resolved absorption 
 spectroscopy , first applied to photophysical studies of carotenoids 
 in the 1990s , demonstrated that additional 
 optically silent electronic states may lie in the vicinity of the 
 s1 and s2 states . the most controversial state , 
 s * , was first introduced to explain ambiguous spectral features in 
 transient absorption ( ta ) spectra of the open - chain carotenoid spirilloxanthin , although similar spectral features had been 
 observed previously for long - chain homologues of -carotene . in the case of spirilloxanthin , the s * feature 
 was observed in organic solvents as well as in the lh1 protein complex , 
 and it was natural to use the same term in both cases . two hypotheses were introduced to explain the 
 nature s * state of carotenoids dissolved in solvents . one assumed 
 that the s * is an electronic excited state , resembling the s1 state of a carotenoid with a twisted geometry , whereas the second argued that it is a highly vibrationally excited 
 (  hot  ) ground state , s0 , populated via impulsive 
 stimulated raman scattering or intramolecular 
 vibrational redistribution . most recent 
 investigations suggest that the s * feature can be associated with 
 vibronic transition from either s1 or from vibrationally 
 excited levels of s0 state , depending on the carotenoid 
 conjugation . with respect to protein - bound 
 carotenoids the s * feature ta band 
 was initially observed in the lh1 complex from r. rubrum containing spirilloxanthin , a carotenoid with a 
 long conjugated carbon  carbon double bond system ( n = 13 ) . subsequently , ta signals were assigned as an ambiguous s * 
 state in numerous lh2 antenna complexes with carotenoids such as neurosporene 
 ( n = 9 ) , spheroidene ( n = 10 ) , and 
 rhodopin glucoside ( n = 11 ) . some studies 
 on lh2 suggested that s * , although very minor , is an energy donor 
 to bchl a being consistent with s * as an excited 
 state . it was also argued that the  s * state  
 is a precursor in the ultrafast formation of a carotenoid triplet , 
 further supporting the excited state hypothesis . however , the assumption that the s * 
 feature in lh2 is associated 
 with a distinct excited state of a carotenoid has a major drawback . comparison of lifetimes of the s * feature , obtained for various carotenoids 
 in several lh complexes , did not reveal the expected dependence on 
 the carotenoid conjugation length , n , in apparent contradiction of the energy gap law . this problem could be disregarded for solvents 
 in which the s*-to - s0 internal conversion depends on the 
 rate of preceding conformational change of molecule ; however , this 
 argument does not hold within a protein environment in which the carotenoid 
 geometry is much more rigid due to pigment  protein interactions 
 and steric considerations . interestingly , explanations of the 
 origin of the carotenoid s * 
 feature in lh2 complexes have not taken into account the possibility 
 that it may be somehow associated with the short - lived carotenoid 
 radical cation  b800 anion pair ( carb800 ) that is formed after direct excitation of a carotenoid , 
 during excitation relaxation from its s2 state . ultrafast 
 formation of the carb800 pair 
 in lh2 complexes was not known at the time when s * was introduced , and the potential relation between the carb800 pair and the s * feature in 
 lh2 complexes has not been further evaluated . as formation of 
 a carb800 pair reduces the 
 initial pool of carotenoids in the excited state , 
 spectral features of car , visible as transient 
 absorption band in the nir spectral range , should also be accompanied 
 by bleaching of a ground state absorption of a carotenoid s 
 neutral form in the vis range . this fact has not been taken under 
 consideration in fitting models of transient absorption data of carotenoids 
 in lh2 complexes . the ta data show that an s * feature always comprises 
 negative bands that more or less correspond to carotenoid ground state 
 bleaching . in addition , an s * feature also consists of an adjacent 
 positive band that has been interpreted as a transient absorption 
 band of the ambiguous s * state . however , the s * feature in the 
 lh2 complexes may have an alternative 
 origin . it seems apparent that the short - lived carb800 pair will temporarily form quite a strong 
 electric dipole that will provide additional polarization of the nearest 
 environment . it is not difficult to imagine that this temporal polarization 
 brought by the local electrostatic field of a carb800 pair will lead to an electrochromic response 
 of neighboring pigments ( like carotenoid ) . in such a scenario , ta 
 spectra of the lh2 complex ( upon carotenoid excitation ) recorded in 
 the range of the carotenoid band should also consist of a spectral 
 feature that will be a mixture of two elements : ground state bleaching 
 ( associated with portion of carotenoids converted to car ) and electrochromic spectrum of a neutral carotenoid nearby to a 
 carb800 pair . the temporal 
 characteristics of this spectral feature should be completely independent 
 from the conjugation of an involved carotenoid ; its decay will be 
 coupled to recombination of car back to a carotenoid 
 neutral form . as shown below , this hypothetical temporal spectral 
 feature fits quite well to the spectral and temporal properties of 
 the s * feature in lh2 complexes . to demonstrate that the s * 
 feature is indeed associated with the 
 carb800 pair , as suggested 
 above , we have studied two neurosporene - containing lh2 complexes from 
 a rba . sphaeroides mutant strain , either with a native or a severely attenuated b800 bchl a absorption band . these preparations will be referred to 
 as ldao - tris - lh2 and ddm - hep - lh2 where ldao and ddm are abbreviations 
 for the detergents used during purification processes : n , n - dimethyldodecylamine n - oxide and -dodecyl 
 maltoside , respectively . time - resolved absorption spectroscopy 
 studies combined with simple 
 modeling of changes in a local electric field brought by the carb800 pair show that the transient 
 spectral feature previously assigned to an s * state can be very adequately 
 modeled as combination of a bleaching of ground state absorption of 
 the fraction of the excited carotenoids that had been converted to 
 radical cations and electrochromic response of the neighbor neutral 
 carotenoid . in order to experimentally test this hypothesis , it is known that in this type of lh2 a carb800 pair can not be formed ; consequently , an s * feature should also be absent in ta spectra . sphaeroides crtc , neurosporene - containing mutant were grown according to the protocol 
 described previously . the lh2 complexes 
 were prepared using two different protocols , yielding preparations 
 with a well - developed or severely depleted b800 bchl a absorption band . the lh2 complexes with an intense b800 absorption 
 band were prepared using a protocol published previously . the lh2 preparation with a reduced b800 band 
 was obtained according to the protocol published in ref ( 28 ) . the final sample buffer 
 ( ph = 7.5 ) consisted of 20 mm hepes and 0.03% -dodecyl maltoside 
 ( -ddm ) . as shown in ref ( 30 ) , this protocol and buffer are perfect for simultaneous 
 preparation of lh2 and lh1 complexes , although prolonged storage of 
 lh2 complex leads to a slow oxidation of b800 bchl a to 3-acetyl - chl a , probably due to the oxidative 
 properties of hepes . transient absorption experiments were carried 
 out using a helios femtosecond time - resolved and eos , nanosecond time - resolved 
 pump  probe absorption spectrometers ( ultrafast systems lcc , 
 sarasota , fl ) coupled to a spectra - physics femtosecond laser system 
 described previously ( spectra - physics , santa clara , ca ) . the excitation wavelength was preferentially 
 set to excite the first vibronic band of the s0  
 s2 carotenoid absorption at 490 nm . the energy of the excitation 
 beam was kept between 200 and 400 nj , corresponding to an intensity 
 of 0.51  10 photons / cm per pulse , low enough to prevent singlet group velocity dispersion 
 in ta data sets was corrected using surface xplorer software provided 
 by ultrafast systems by building a dispersion correction curve from 
 a set of initial times of transient signals obtained from single wavelength 
 fits of representative kinetics . target analysis , a directed kinetic 
 modeling of ta results , was performed using carpetview , data viewing 
 and analysis software for ultrafast spectroscopy measurements ( light 
 conversion ltd . the fitting employed the kinetic 
 models with anticipated realistic branches mimicking the true decay 
 pathways following excitation of the carotenoid . if the underlying 
 assumptions are correct , targeted kinetic analysis separates spectral 
 components such as excited state absorption ( esa ) of the specific 
 excited state of molecule , etc . the results are commonly abbreviated 
 as sads or sas , species associated ( decay ) spectra . the instrument temporal response function was assumed to 
 have a gaussian - like shape with the full width at half - maximum ( fwhm ) 
 of 200 fs and was used as a fixed parameter in the fitting 
 procedure . steady - state absorption spectra 
 of the ldao - tris - lh2 and ddm - hep - lh2 complexes containing neurosporene 
 are given in figure 1 . the spectra consist of features associated with electronic 
 transitions of bchl a : soret bands with maxima at 
 370 nm , qx bands at 590 nm and 
 two qy bands , b800 ( 800 nm ) and 
 b850 ( 850 nm ) associated with monomeric and excitonically 
 coupled forms of bchl a , respectively . the vibronic 
 peaks of the neurosporene s0  s2 band 
 appear between 400 and 500 nm . the absorption spectrum of the ddm - hep - lh2 
 shows a substantial reduction in intensity of the b800 peak and the 
 presence of a new weak band at 690 nm , indicating that the 
 b800 bchls a are not released from the protein but 
 were oxidized to 3-acetyl - chlorophyll a ( ac - chl a ) . this structural modification of the pigments in the 
 b800 ring only marginally affects the spectroscopic properties of 
 the carotenoid , which are manifested as a small , 2 nm , hypsochromic 
 shift of the absorption band . a similar but bathochromic shift of 
 the b850 band in respect to their counterpart from the ldao - tris - lh2 
 is also observed . the overall integrity of the ddm - hep - lh2 complex 
 is preserved , including carotenoid - b850 bchl a pigment 
 interactions , as shown in previous studies . because there is a mismatch in the excited state energies between 
 neurosporene and ac - chl a transient 
 absorption results taken in the vis and nir spectral ranges of the 
 ldao - tris - lh2 excited at the neurosporene absorption band are given 
 in figure 2 . figure 2a , b shows 3d - pseudocolor 
 contour maps of ta within 8 ps after excitation in vis and nir spectral 
 ranges , respectively . transient absorption data of the ldao - tris - lh2 complex recorded 
 after excitation into the ( 00 ) vibronic band of carotenoid 
 neurosporene ; ( a , b ) ta contour maps highlighting esa bands and bleaching 
 of ground state absorption ( gsb ) of either carotenoid ( car ) or bchls 
 present within 8 ps after excitation . the states from which the transition 
 occurs are indicated . ( c , d ) exemplary transient absorption spectra 
 taken at various delay times after excitation . the main esa bands associated with the carotenoid neurosporene 
 are indicated along with spectral features corresponding to bleaching 
 of ground state absorption ( gsb ) of either carotenoid or bchls . in 
 the ldao - tris - lh2 sample , the s1  sn esa band with maximum at 540 nm is instantaneously 
 populated and then fades away within 3 ps . the remaining spectrally 
 broad signal with a maximum at 520 nm represents the esa associated 
 with an ambiguous  s * state  . another noticeable band 
 present in the nir range appearing only within a few hundred femtoseconds 
 after excitation ( figure 2b , deep - red color ; figure 2d , black line ) is associated with the short - lived neurosporene 
 s2 state . previous experimental and theoretical evaluations 
 of that band demonstrated that it is associated with the s2  sn transition . simultaneously with the s * feature , another spectral band 
 is clearly present in the nir range between 920 and 1000 nm . this 
 band is associated with d0  d3 absorption 
 of carotenoid radical cation that is formed shortly after 
 excitation via interaction with b800 bchl a that 
 leads to ultrafast formation of the temporarily existing neub800 pair . simultaneous appearance and decay of 
 both transient bands ( d0  d3 and s * ) 
 suggests that those may have common temporal characteristics . transient absorption results taken in vis and nir spectral ranges 
 of the ddm - hep - lh2 excited in the neurosporene absorption band are 
 given in figure 3 . transient 
 absorption data of the ddm - hep - lh2 complex recorded after 
 excitation into the ( 00 ) vibronic band of carotenoid neurosporene . ( a , b ) ta contour maps highlighting esa bands and bleaching of ground 
 state absorption ( gsb ) of either the carotenoid ( car ) or bchls present 
 within 10 ps after excitation . ( c , d ) exemplary transient absorption spectra 
 taken at various delay times after excitation . note that the d0  d3 absorption band of neu has disappeared and the s * spectral feature is replaced by a noticeably 
 sharper and blue - shifted transient absorption band . contour maps of ta shown within the first 10 ps 
 after excitation 
 ( figure 3a , b ) demonstrate 
 few significant changes in respect to lh2 with a fully developed b800 
 absorption band . the neurosporene s1  sn esa band appears at 535 nm and persists for a much 
 longer time indicating weakened energetic coupling with ( b)chls . at 
 the very early delay times , the s1  sn esa band is also red - shifted , representing vibrational 
 equilibration of the s1 state . most importantly , the broad 
 spectral feature characteristic of  s *  has substantially 
 changed . it has been replaced by a sharp and slightly blue - shifted 
 positive transient band with a maximum at 507 nm . in the nir spectral range the band associated with absorption of neu completely disappeared , demonstrating that neurosporene is not able 
 to form the cation further spectroscopic 
 evaluation by measurements in the submicrosecond time regime demonstrated 
 that this transient band is identical to triplet  triplet ( t1  tn ) esa of lh2-bound 
 neurosporene , as demonstrated in figure 4 . it shows the ta spectrum of the ldao - tris - lh2 
 taken at 7 ps , corresponding to so - called s * spectral feature overlaid 
 with equivalent ta spectrum of the ddm - hep - lh2 ( because neurosporene 
 decays longer in this lh2 batch it was taken at 30 ps ) . comparison 
 of transient absorption spectra taken for both lh2 batches 
 ( ldao and ddm preps ) at time delays at which the main s1  sn esa of neurosporene decayed . 
 for the ldao - tris - lh2 , the ta spectrum taken at 7 ps corresponds to 
 s * spectral feature . for comparison , also ta spectrum of the ddm - hep - lh2 
 at the same delay time the 30 ps spectrum ( free of s1  sn esa band ) is identical to the triplet - minus - singlet 
 ( t  s ) spectrum of neurosporene taken at 50 ns after excitation . the fourth spectrum corresponds to ta of the ddm - hep - lh2 
 taken 
 at 50 ns , and due to the long time delay , it can be only associated 
 with the so - called triplet - minus - singlet ( t  s ) spectrum of 
 neurosporene . it is apparent that both 30 ps and 50 ns ta spectra 
 are essentially identical but are very different from the spectral 
 envelope of the s * feature . it is worth noting that , thus , the triplet 
 t1  tn band is already 
 clearly resolved in the ta spectrum at 1 ps ( figure 3c ) . experimental results shown above clearly 
 suggest a connection between the neub800 pair and the s * transient spectral feature . as we have hypothesized 
 that the s * band can be associated with an electrochromic response 
 of neutral carotenoid to the electric field temporarily induced by 
 the carb800 pair , it would 
 be particularly interesting to know what level of electric field the 
 neighboring neutral carotenoid may sense from the cation sphaeroides lh2 is not known , the results should be interpreted rather as  qualitative evidence used to support the concept . because the pigments are not 
 infinitesimally small , in order to make this simple modeling feasible 
 it was necessary to approximate them by points that lie on the surface 
 that is designated by the plane of the b800 macrocycle . with these 
 assumptions , the arrangement of the pigments of interest can be represented 
 as in figure 5 . the 
 point symbolizing b800 bchl a has been placed in 
 the center of the pigment s macrocycle . to simplify calculations , the frame of reference was placed in the midpoint between b800 and car . simplified arrangement of the carb800 pair and the nearest neutral 
 carotenoid ( carsens ) based 
 on pigment coordinates from rps . point symbolizing b800 bchl a is placed in the center of the pigment s macrocycle . the electric field that the nearest 
 neutral carotenoid senses from 
 the electric dipole formed by the carb800 pair can be described by following equation:1 the parameters d , r , and  
 are provided in figure 5 ; q is the electron charge , and p is the relative dielectric constant of the surrounding protein medium . 
 its value has been estimated for various lh2 complexes in the 1.21.3 
 range , and a value of 1.2 has been used 
 in this model . calculations show that the nearest neutral carotenoid 
 ( carsens ) senses an electric field of 4.4 mv / cm . previous studies of electrochromic responses of carotenoids bound 
 to lh2 suggested that the strong bathochromic shift of carotenoid 
 absorption observed in the lh2 protein , with respect to low polarizable 
 solvents like n - hexane , is caused by a local electric 
 field of 4 mv / cm that is most likely brought by protein residues like 
 arg-10 . if those two above - mentioned 
 factors , a static local electric field from protein residue ( eloc ) and a temporal electric field brought by 
 carb800 pair ( et ) , will simultaneously perturb the carotenoid s2 state , the overall temporal electrochromic shift of the carotenoid 
 absorption spectrum (  , in wavenumbers ) can be 
 calculated on the basis of the following equation for a stark shift:2here , eloc and et are electric field vectors ;  and  are changes in the vector of dipole moment and polarizability tensor , 
 respectively , of the carotenoid between its s0 and s2 states . for simplicity ,  is 
 assumed to be a scalar with value of  = 1350  that was previously calculated for structurally 
 similar open - chain carotenoid lycopene ( 1  = 1.113  10 c m v ) . the values of electric vectors are eloc = 4 mv / cm , et = 4.4 mv / cm , and 1/hc = 0.5 
  10 c j m. it was also assumed that the first term in eq 2 is negligible . according to the simplistic 
 model in figure 5 , et and  vectors would be orthogonal to each other (  will be essentially in line with carotenoid conjugation 
 that crosses the graph surface while et will 
 lie on the graph surface ) . then , if et and eloc are mutually oriented such that the dot product 
 is maximized and positive ( similar orientation , parallel ) , the second 
 term will give an electrochromic shift of 1330 cm while the third one will be 730 cm and an overall 
 red - shift of 2060 cm in respect to absorption 
 in a low polarizable medium like n - hexane . as in n - hexane , the ( 00 ) vibronic band of neurosporene 
 appears at 467 nm ( 21  410 cm ) , so that the shifted spectrum should appear at 
 19  350 cm , which corresponds to 517 nm . we 
 have shown that this carotenoid may temporarily experience additional 
 a 26 nm red - shift ( 1000 cm ) of its steady - state 
 absorption in lh2 ( normally the ( 00 ) band is at 491 nm ) . as 
 demonstrated in figure 6 , this ultrafast stark shift is strong enough to very adequately 
 reconstruct the s * transient spectral feature . figure 6a shows the expected transient stark spectrum 
 of the neurosporene , induced by the electric field of the neub800 pair ( red line ) . the spectrum 
 is a spectral difference of shifted ( dashed black line ) and nonshifted 
 ( blue solid line ) absorption spectra of neurosporene with spectral 
 shift of 26 nm . the pure transient stark spectrum is similar to the 
 s * transient feature ; however , it does not mimic it correctly , as 
 demonstrated in figure 6b . this suggests that other factor(s ) could influence its spectral 
 shape . in order to be sure that raw s * feature spectrum is not significantly 
 distorted by coexisting transient bands of bchls , it was done by subtracting a corresponding transient 
 absorption spectrum of the carotenoidless lh2 from rba . s * feature spectrum ( blue line ) 
 still substantially deviates from the stark spectrum , mainly in the 
 range of bleaching of the carotenoid absorption band . however , it 
 should be emphasized that , in addition to the transient stark spectrum , 
 an additional transient band should be also observed . this band will 
 correspond to bleaching of a ground - state absorption ( gsb ) of the 
 carotenoid pool fraction that is involved in ultrafast formation of 
 the neub800 pair . our model 
 of pigment interactions assumed that a single molecule of neu affects a single molecule of the neutral carotenoid , 
 and such stoichiometry should be also reflected in the spectroscopic 
 measurements . therefore , the amplitude of gsb should be equal with 
 the amplitudes of shifted and nonshifted absorption spectra as shown 
 in figure 6a ( green 
 line ) . because both spectral features , gsb and stark spectrum , have 
 identical temporal characteristics , both should appear as one spectro - temporal 
 feature that spectrally will resemble the sum of both features . this 
 spectral - temporal feature is given in figure 6c ( red line ) , along with the  pure  
 transient s * spectrum ( blue line ) . ( a ) spectral 
 components involved in reconstructing the s * transient feature : cyan 
 line , ground - state absorption spectrum of neurosporene in the lh2 ; 
 dashed black line , ground - state absorption spectrum of neurosporene 
 affected by the electric field induced by neub800 pair ; red line , difference spectrum ( stark spectrum ) 
 of the previous two spectra ; green line , ground - state absorption bleaching 
 of neurosporene involved in formation of neub800 pair . ( b ) the s * spectral feature cleaned from contribution 
 of transient absorption of bchls by subtracting complementary spectrum 
 of carotenoidless lh2 . ( c ) pure s * feature overlaid with its anticipated 
 spectrum ( for details refer to the main text ) : ta , transient absorption , 
 gsb , ground - state absorption bleaching . another interesting 
 aspect revealed by this study is the ultrafast formation of the carotenoid 
 triplet state in situations when the carb800 pair can not be formed due to an absence of b800 bchl a. triplet formation within 1 ps after excitation strongly 
 suggests that the carotenoid triplet pool is not populated in the 
 typical way via sensitization by bchls triplets , and therefore , carotenoid 
 singlet fission has to be involved . this process in carotenoids was 
 first suggested after observation of a high quantum yield of carotenoid 
 triplet formation in whole cells of the purple bacterium r. 
 rubrum . it has been suggested 
 that s1 and 1bu , a state without a precise sx designation 
 ( as it may lie below or above s2 depending on length of 
 carotenoid conjugation ) , could be viewed as a singlet - coupled combination 
 of two triplets localized in the different parts of a carotenoid chromophore : 
 1bu = t2t1 and s1 = t1t1 . singlet fission , in which tt decouples 
 into physically independent triplet states , was reported to have very 
 low yields in solvents , but it is high ( up to 30% ) in 
 proteins , suggesting 
 that the decoupling process could be facilitated by a conformational 
 distortion of a carotenoid structure provided by the protein environment . 
 the ta data from figure 3 show that t1  tn and 
 s1 sn esa bands appear 
 simultaneously , suggesting that the s1 state can not be 
 a state in which singlet fission takes place . the second candidate , 
 an optically forbidden 1bu state , has been proposed to lie just below the s2 state 
 for carotenoids with n  9 ( and that includes neurosporene ) , and its lifetime could 
 be too short to be temporally resolved from the initially excited 
 s2 state . therefore , the detailed 
 mechanism of carotenoid singlet fission was not further investigated 
 in this study . target model analysis ( figure 7 ) can be used to review the study presented 
 here and propose 
 a new , simplified s*-free carotenoid excitation decay pathway in the 
 lh2 complex from purple bacteria . nir 
 combined ta data set of the 
 ldao - tris - lh2 complex . the yellow compartments in the kinetic models symbolize carotenoid 
 neurosporene ( with exception of the neub800 pair ) while green compartments correspond to bchl a. note that the sads amplitudes do not correspond to actual 
 contribution of the component in the raw ta data and should be weighted 
 by their time - dependent concentrations ; exc = excitation . the model should be seen as data simulation that 
 incorporates numerous 
 known parameters adapted from previous studies of neurosporene in 
 solvents and in lh2 . the so - called s * state  has been 
 replaced by the neub800 pair , 
 evolving from the carotenoid s2 state during its relaxation . it was assumed that the population of bchl a triplets 
 is entirely quenched by neurosporene molecules with time constant 
 of 20 ns , a value obtained from fitting the recovery of bchl a ground state absorption bleaching ( figure s1 ) . neurosporene 
 triplet lifetime was obtained from fitting of the t1  
 tn band decay ( figure s2 ) . the effective lifetime of the b850 bchl a of 1.2 ns was obtained from fitting of the kinetic trace 
 recorded at 850 nm ( figure s3 ) and was 
 decomposed to microscopic decays of 1.4 ns ( s1  
 s0 , radiative and nonradiative ) and 7 ns ( intersystem crossing , 
 isc , s1  t1 , to be in agreement with 
 triplet yield ) . due to the temporal resolution limitation of the spectrometer , 
 the effective lifetime of the carotenoid s2 state in lh2 
 complexes could not be measured and instead was set to 50 
 fs as demonstrated in previous studies . microscopic 
 parameters of neurosporene s2 state depopulation are described 
 with the following details : the s2  s1 decay was assumed to have the same characteristics as in solvent ; 
 the s2  bchls decay was set in order to give an 
 overall carotenoid to bchl a energy transfer efficiency 
 of 87% that is known from previous studies of this complex . for simplicity both spectral forms of bchl a , b800 and b850 , were combined as unity . it was assumed , 
 in agreement with experimental data , that neu is 
 populated with 13% quantum efficiency from the s2 state of the neutral form . it should be clarified that the contribution of the sads component 
 in the raw ta spectra should not be judged on the basis of its relative 
 amplitude but on the basis of the product of sads amplitude and its 
 concentration at a specific delay time . this 
 study clarifies the origin of the s * feature in lh2 complexes 
 from purple bacterium rba . sphaeroides . we propose 
 that the s * transient spectral feature corresponds to a combination 
 of bleaching of the ground - state absorption of carotenoid involved 
 in the carb800 pair and the 
 stark spectrum of the nearest neutral carotenoid . s * state  from existing kinetic schemes 
 of the carotenoid excitation decay pathway in the lh2 complex , and 
 thereby provides a new framework for investigating the excited states 
 of carotenoids in these photosynthetic antenna complexes .
carotenoids 
 are a class of natural pigments present in all phototrophic 
 organisms , mainly in their light - harvesting proteins in which they 
 play roles of accessory light absorbers and photoprotectors 
 . extensive 
 time - resolved spectroscopic studies of these pigments have revealed 
 unexpectedly complex photophysical properties , particularly for carotenoids 
 in light - harvesting lh2 complexes from purple bacteria . 
 an ambiguous , 
 optically forbidden electronic excited state designated as s * has 
 been postulated to be involved in carotenoid excitation relaxation 
 and in an alternative carotenoid - to - bacteriochlorophyll energy transfer 
 pathway , as well as being a precursor of the carotenoid triplet state . 
 however , no definitive and satisfactory origin of the carotenoid s * 
 state in these complexes has been established , despite a wide - ranging 
 series of studies . here 
 , we resolve the ambiguous origin of the carotenoid 
 s * state in lh2 complex from rba . 
 sphaeroides by 
 showing that the s * feature can be seen as a combination of ground 
 state absorption bleaching of the carotenoid pool converted to cations 
 and the stark spectrum of neighbor neutral carotenoids , induced by 
 temporal electric field brought by the carotenoid cation 
 bacteriochlorophyll 
 anion pair . 
 these findings remove the need to assign an s * state , 
 and thereby significantly simplify the photochemistry of carotenoids 
 in these photosynthetic antenna complexes .