perhaps no amino acid 
 in the bacterial photosynthetic reaction 
 center ( rc ; figure 1a ) has been scrutinized 
 more than the tyrosine situated near the dimeric bacteriochlorophyll 
 ( bchl ) primary electron donor ( p ) and the monomeric bchl initial electron 
 acceptor ( ba ) on the a - side of the rc . this residue is 
 tyr m208 in blastochloris viridis and rhodobacter capsulatus and tyr m210 in rhodobacter sphaeroides .
both theoretical work and experiments on mutants have led to consensus that this conserved residue is a significant 
 contributor to extremely rapid and efficient initial a - side charge 
 separation .
interestingly , there are no potential hydrogen bond partners 
 available to tyr m208 , which is unique among the roughly 28 ( depending 
 on species ) tyrosines in the rc .
consistent with this , a unique chemical 
 shift is observed in the c nmr spectrum for the 4-c - carbon 
 ( the carbon bonded to the oh ) of this unique tyr . replacing the phe residue at the c2-symmetry related 
 site , l181 near bb , with
a tyr often has been a design 
 element in eliciting electron transfer ( et ) from p * to the bacteriopheophytin 
 ( bph ) on the b - side ( hb ) . however , p *  phb conversion is at best more than an order of magnitude slower than 
 initial charge separation on the a - side .
the rc et dynamics have long 
 been modeled in the context of the state free - energy relationships 
 shown in figure 1b . here
pba lies between p * and pha in free energy with the analogous b - side 
 states placed somewhat higher and in particular with pbb above p*. these orderings are consistent 
 with a general picture of rapid ( 13 ps ) steps of et from p * 
 to ba and then to ha with details and mechanisms 
 under continuing study .
electrostatic calculations by alden et 
 al . suggested that a specific orientation of the hydroxyl group of 
 tyr m208 having the oh pointing toward ba stabilizes 
 pba .
in addition , this was found to be the strongly preferred orientation 
 over a second , where the oh is rotated 180 about 
 the c  o bond and which , in comparison , destabilizes pba .
the calculations predicted that 
 the stabilization of pba induced by the oh of tyr m208 in the preferred orientation 
 is a significant 200 mev .
note that a possible corollary of 
 this sensitivity is the expectation that the initial et reactions 
 may be inhomogeneous , depending on the timescale of the oh 
 dipole motion , and in fact nonsingle exponential kinetics are commonly 
 observed for these reactions .
sphaeroides x - ray crystal 
 structure ( protein 
 data bank : 2j8c ) .
view of the environment 
 of ba as determined in the wild - type rb . 

note that the numerical values and amino acids of the l and m polypeptides 
 correspond to those of rcs from rb .
sphaeroides ) is a ligand to the m - bchl macrocycle of p ( not shown ) .
the preferred orientation of the oh dipole 
 is not known 
 experimentally given the current resolution of even the best x - ray 
 structures .
therefore , we consider the simulations of alden et al . 
 as providing a working hypothesis for contemplating further protein 
 engineering .
an expanded view of the region around tyr m208 and ba is illustrated in figure 2 in a homology 
 model of the rb .
therefore , we replaced isoleucine m204 with glutamic acid 
 and glutamine to introduce a potential hydrogen bond acceptor for 
 the oh of tyrosine m208 .
as reported previously , to our surprise , 
 the m204q mutation leads to complete loss of ba .
ultrafast measurements on m204q rcs gave the 
 expected results of substantial deactivation of p * to the ground state , 
 no a - side et , and a small yield of b - side charge separation , p *  
 phb , that was increased 
 upon changing phe at l181 to a tyr .
we 
 report here that substitution of m204 with glutamic acid leads to 
 altogether different results .
modeling of glu at m204 using pymol 
 suggests that if a hydrogen bond to the tyr m208 hydroxyl group is 
 formed , the hydroxyl group would be held in an orientation that disfavors 
 p *  pha et , noting , 
 though , that the energetic consequences of a hydrogen - bonded tyrosine 
 oh in this orientation may be different from the nonhydrogen - bonded 
 case of the simulations ( in ref ( 8) ) . with the use of uniformly [ 4-c]-labeled 
 tyr
, the c nmr spectrum reveals that tyr m208 in m204e 
 no longer has the unique chemical shift associated with tyr m208 not 
 being hydrogen bonded , as in wild - type , consistent with the introduction 
 of a hydrogen bond .
ultrafast measurements of m204e rcs reveal that 
 reduction of ha is about a factor of 10 slower than in 
 wild - type and that the room temperature p * decay kinetics are unusually 
 homogeneous .
capsulatus rc was changed to a glutamic acid by site - directed mutagenesis , 
 as described previously , using the quikchange 
 mutagenesis kit ( stratagene ) .
rc isolation and purification followed 
 published procedures utilizing 0.1% deriphat 160-c 
 for protein solubilization for transient absorption or 0.1% n , n - dimethyldodecylamine n - oxide ( ldao ) 
 for nmr and mass spectrometry .
measurements were conducted on rcs 
 in 10 mm tris ( ph 7.8 ) and 0.1% deriphat 160-c , unless noted otherwise . 

wild - type and m204e rcs were extracted 
 using acetone / meoh ( 7:2 v / v ) and the bchl : bph pigment ratio determined 
 spectroscopically , as according to van der rest et al .
the pigment content was also assayed by hplc 
 because of the unusual results reported below . for this
, the acetone / meoh 
 extract was dried under a stream of nitrogen and dissolved in acetonitrile / ethyl 
 acetate / meoh / water ( 24:20:47:9 v / v ) .
after filtration through a 0.22 
 m filter , the pigments were analyzed using an agilent infinity 
 1260 hplc setup equipped with an analytical c18 column ( spherisorb 
 c18 , 4.6  250 mm , 5 m ) and a multiple wavelength detector 
 ( 1260 mwd vl ) .
pigments were detected at 770 nm using isochratic elution 
 with acetonitrile / ethyl acetate / meoh / water ( 24:20:47:9 v / v ) at a flow 
 rate of 1 ml / min .
capsulatus cells were grown 
 in a bioreactor ( new 
 brunswick scientific , bioflo3000 , 5 l ) under controlled ph and oxygen 
 levels in the dark to maximize rc yield .
to incorporate labeled [ 4-c]tyr , a modified rcvpy media was used that substituted peptone and yeast 
 extract with a defined amino acid composition , as according to raap 
 et al .
a small amount of antifoam 204 
 ( sigma - aldrich ) was added before inoculation .
the optimal growth conditions 
 were as follows : the culture ph was maintained at 6.8 with a titration 
 solution containing 4 m dl - malic acid with an agitation rate 
 of 500 rpm at a temperature of 34 c .
the atmosphere was controlled 
 using a nitrogen / air mixture ( 95:5 ) , and the dissolved oxygen level 
 was monitored using an oxyprobe .
the oxyprobe was calibrated using 
 a two - point calibration with 100% air and 100% nitrogen as reference 
 values .
the incorporation of labeled [ 4-c]tyr ( cambridge isotopes ) was quantitated by performing 
 lc 
samples were subjected 
 to in - solution digestion using urea and proteasemax ( promega ) and 
 a combination of trypsin/-chymotrypsin to cut out the peptide 
 of interest that contains tyrosine m208 ( fragment m200-m208 ) .
briefly , 
 50 g protein was precipitated with 4 volumes of 20 
 c acetone and incubated at 80 c for 20 min .
after 
 centrifugation , the white pellet ( blue - green pellet in case of m204e ) 
 was rinsed with 300 l acetone and allowed to air - dry for 5 
 min .
then , 15 l of 8 m urea and 20 l of 0.2% proteasemax 
 were added , and the solution was vortexed for 5 min to solubilize 
 the pellet .
the mixture was placed in a shaker at 25 c and 140 
 rpm for 1 h. after solubilization , the sample was reduced with dithiothreitol , 
 alkylated with iodoacetamide , and digested using trypsin/-chymotrypsin 
 ( 1.8 g in total ) and proteasemax at 37 c for 3 h. the 
 digestion was stopped by addition of 2 l 25% trifluoroacetic 
 acid . to remove particles ,
peptides were purified using a stage - tip c18 column . in 
 the first step ,
the column was washed with acetonitrile and in the 
 second step with water/0.1% formic acid .
mass spectra were 
 obtained on a thermo ltq - orbitrap velos mass spectrometer equipped 
 with a nanohplc ( c18 , 100 m i.d .
the peptides were eluted using a gradient running 
 from water ( containing 0.1% formic acid ) to acetonitrile over the 
 course of 60 min ( 340% for 40 min , 4065% for 10 min , 
 6595% for 5 min , and 953% for 5 min ) .
the mass accuracy 
 of the orbitrap and of the ion trap are below 2 ppm and around 250 
 ppm , respectively . labeled 
 rcs ( 25 mg )
were 
 reconstituted into lipids and lyophilized to ensure a homogeneous 
 environment and a dry sample following described procedures.l--phosphatidylcholine ( egg 
 pc ) was dissolved in 10 mm tris ph 8 buffer containing 0.58% n - dodecyl -  - d - maltoside .
twenty - five milligrams rc was added and the total 
 volume was 5 ml ( 1:100 mol % of protein / lipid ) .
biobeads - sm2 were 
 washed vigorously with meoh , water , and finally 10 mm tris , ph 8 . 

the beads were added ( 110 mg , 5-fold w / w excess over detergent ) , 
 and the solution was stirred overnight in the dark at 4 c to 
 remove detergent .
the next day , another 110 mg of biobeads - sm2 were 
 added and the solution was stirred for another 3 h. the beads were 
 removed by filtration , trehalose was added as cryoprotectant to a 
 final concentration of 25 mm , and the solution was stirred at 4 c 
 for 15 min .
the sample was slowly frozen at 20 c , and 
 then transferred to 80 c and finally frozen in liquid 
 nitrogen .
solid - state nmr experiments were performed with an 89 mm wide - bore 
 varian / agilent magnet at 11.7 t ( 125.49 mhz for c ) , varian 
 console , and a home - built four - frequency transmission - line probe with 
 a 13.6 mm long , 6 mm inner - diameter sample coil and a revolution nmr 
 mas vespel stator .
samples were spun at 8000  2 hz , using a 
 varian mas control unit and cooled with 30 c nitrogen 
 gas .
the proton - carbon matched cross - polarization transfer was performed 
 at 83 khz , and the recycle delay was 2 s. the chemical shift scale 
 was referenced to external adamantane .
ultrafast transient absorption 
 ( ta ) measurements were carried out on an apparatus that utilizes 120 
 fs excitation and white - light probe flashes delivered at a repetition 
 rate of 10 hz . in order to ensure that fresh sample was interrogated 
 on each excitation flash ,
rcs were flowed from a small reservoir through 
 a 2 mm path length cell used for the pump / probe interrogation .
the 
 rcs were kept at 10 c by use of an ice bath to cool 
 the reservoir .
the ta data were analyzed 
 using origin ( microcal ) and surface explore ( ultrafast systems ) .
the p / p midpoint potential was obtained using a sec - c 
 thin layer quartz 
 glass cell ( basi ef-1358 ) , platinum counter electrode ( basi ef-1356 ) , 
 a ag / agcl double junction reference electrode ( basi mf-2079 , mf-2030 ) 
 and ferricyanide as the oxidizing species . the reference electrode 
 was calibrated using standard solutions .
the titration starting conditions were 750 l of 20 
 m rcs and 4 mm potassium ferrocyanide ( to ensure initial fully 
 reduced rcs ) in 10 mm tris , ph 7.6 , and 0.1% deriphat .
small increments 
 ( starting with 0.25 l and gradually increasing to 10 
 l as the titration proceeded ) of stock 1 m potassium ferricyanide 
 in 10 mm tris , ph 7.6 , and 0.1% deriphat were added directly to the 
 sample in the cell and mixed by gentle pipetting several times over 
 the course of 10 min .
the absorbance at 865 nm was then measured 
 ( shimadzu uv-1800 ) and the potential ( voltage ) recorded .
the titrations 
 were stopped when addition of ferricyanide no longer altered the measured 
 potential or when the absorption of the ferricyanide began to interfere 
 with reading the rc absorbance ( generally when approaching 50 
 l total ferricyanide stock added ) .
in order to work with the minimal amount 
 of [ 4-c]tyr - labeled media , cell growth of rb .
viridis have shown that rc gene expression can be enhanced by growing cells 
 under microaerophilic conditions in the dark .
a series of experiments determined the optimum dissolved oxygen 
 level for maximum protein expression . holding the dissolved oxygen 
 level constant around 2% for the last few hours before harvesting 
 cells around od660  5 gave good protein expression 
 ( figure s1 of the supporting information ) , about a 3-fold increase in protein yield ( > 60 mg purified protein 
 per 4 l growth medium ) . to determine the 
 amount of [ 4-c]tyr at position m208 incorporated 
 under our conditions , 
 lc 
ms / ms was performed on trypsin/-chymotrypsin - digested 
 rc . to our knowledge ,
mass spectrometry of fragments of bacterial 
 rc proteins has not been previously reported and a more detailed analysis 
 can be found elsewhere .
one problem is 
 the hydrophobicity of large stretches the rc , which makes proteolytic 
 digestion difficult in the absence of detergent . in previous work 
 on rcs ,
detergent was removed by precipitation , and the pellet was 
 treated with 8 m urea .
digestion with 
 trypsin or -chymotrypsin yielded only low sequence coverage 
 for h , l , and m subunits ( 54% h , 14% m , and 10% l with trypsin ; 15% 
 h , 15% m , and 18% l with -chymotrypsin ) . in the present study , 

the pellet was solubilized after detergent removal using proteasemax , 
 which solubilizes during proteolytic digestion , resulting in much 
 higher sequence coverage , in particular for the hydrophobic l and 
 m subunits ( 60% h , 54% m , and 41% l ) . for our purpose , a combination 
 of trypsin/-chymotrypsin was used because this treatment releases 
 a peptide corresponding to residues m200-m208 ( sequence hglsiaaly ) . 
 both labeled and unlabeled wild - type rcs were digested and are compared 
 in figure s2 of the supporting information .
the peptide hglsiaaly has a m / z value of 473.76 for the doubly charged ion .
comparison 
 with the [ 4-c]tyr labeled wild - type shows that 
 the whole isotope pattern is shifted by m / z = 0.5 , indicating > 99% incorporation of label ( the 
 mass 
 accuracy of orbitrap mass analyzers is < 2 ppm ) .
sphaeroides rcs , where a high concentration of [ 4-c]tyr 
 in the media was found to be necessary to compete with tyrosine biosynthesis 
 and ensure quantitative incorporation of label into the rc . in the previous studies , total hydrolysis of the rc was performed 
 to quantify total [ 4-c]tyr incorporation , whereas our approach provides a direct method 
 to quantify site - specific labels using lc  ms / ms .
nmr spectroscopy is a 
 useful technique to monitor the protonation and hydrogen bond status 
 of the tyrosine hydroxyl group using the chemical shift of [ 4-c]tyr , as shown in numerous studies .
figure 3 shows the c cpmas nmr spectrum of [ 4-c]tyr - labeled 
 wild - type and m204e rcs obtained at 230 k. the rb . 

capsulatus rc contains 31 tyrosines , and this accounts 
 for the major and unresolved [ 4-c]tyr peak between 
 154 and 158 ppm , typical for protonated and hydrogen - bonded tyr . in 
 wild - type , a small , well - resolved peak
this spectrum is similar to that found for [ 4-c]tyr - labeled wild - type rcs from rb .
sphaeroides , where this peak could be attributed to tyr m210 ( equivalent to 
 m208 in rb .
notably , tyr m208/m210 is the only tyrosine that is not 
 hydrogen bonded and the upfield shift is typical of this effect . the x - ray structure of rb .
sphaeroides rcs confirms that there is no hydrogen 
 bond acceptor in close proximity to the tyr m210 hydroxyl group .
note that there may also be some contribution 
 to this shift from the ring current of ba .
c cpmas 
 solid - state nmr of [ 4-c]tyr labeled rcs at t = 230 k. the unique resolved 
 peak at 153 ppm (  ) in wt ( wild - type ) rcs is assigned to the 
 4-c carbon ( * ) of tyr m208 .
it is shifted back 
 into the poorly resolved broad band corresponding to all the other 
 hydrogen bonded tyrosines in the m204e mutant .
the c nmr spectrum of m204e with [ 4-c]tyr ( figure 3 ) clearly shows a significant 
 change compared with wild - type .
the unique upfield - shifted peak at 
 153 ppm is absent in m204e , apparently shifted back into the unresolved 
 peak containing all the other hydrogen - bonded tyrosine residues in 
 the rc .
this result suggests that the introduced glutamic acid at 
 m204 creates a hydrogen bond to the hydroxyl group of tyr m208 as 
 designed . as noted above , 
 the m204q mutant assembles without ba , as reflected in 
 the absorption spectrum and determined by analysis of the pigment 
 content . as shown in figure 4 , the 77 k absorption spectrum of m204e rcs has 
 two bands near 800 nm that are assigned to ba and bb . in the wild - type spectrum in figure 4 ,
the qy bands of ba and bb are 
 not resolved ; however , different detergent or buffer conditions and 
 some mutations also lead to partial resolution of these bands .
the 
 rc pigment contents of wild - type and m204e rcs were assayed via the 
 standard spectroscopic method . following 
 extraction of rcs with acetone : meoh ( 7:2 , v / v ) ,
the solution is filtered 
 before acquiring the uv / vis / nir spectrum in order to minimize the 
 contribution of protein light - scattering .
bchl / bph ratios of 1.4 for 
 m204e and 1.9 for wild - type were determined , indicating extraction 
 of only 3 bchls from the m204e rc .
the same results were obtained 
 if instead of filtering the extraction solution it was centrifuged 
 for 1 min in a benchtop unit .
confirming incomplete extraction of 
 pigment from m204e rcs , the pelleted residue from m204e is blue - green , 
 whereas for wild - type it is pale yellow ( figure s3 of the supporting information ) .
attempts to extract 
 a ( presumed ) bchl pigment from the m204e pellet using a variety of 
 solvents were not successful .
low - temperature uv / vis / nir spectra obtained 
 at 77 k. the spectra 
 are normalized at 366 nm for comparison .
fresh acetone / meoh extract was dried under a stream of nitrogen and 
 dissolved in the solvent mixture used for hplc analysis ( see experimental section ) .
figure s4a of the supporting information shows hplc traces recorded 
 at 770 nm . to make an absolute comparison ,
the analysis was performed 
 on the same concentrations of wild - type , m204e , and a 1:1 mixture 
 of both .
the traces are very similar , and there is no difference in 
 retention times between wild - type and mutant .
the only difference 
 is the intensity of the band around 7 min , which can be attributed 
 to bchl .
the ratio of the integrated peak areas for wild - type , m204e , 
 and the 1:1 mixture is 4:2.7:3.3 , again suggesting incomplete extraction 
 of bchl .
the amount of extracted bph is the same for wild - type , mutant , 
 and mix ( at 16 min ) .
extraction methods using different solvents or 
 solvent mixtures did not change the observation that one bchl is not 
 extracted from m204e . in an earlier study on a rc mutant of rb .
sphaeroides , in which isoleucine l177 ( c2-symmetry partner to m204 ) 
 was substituted by histidine , it was proposed that one bchl might 
 be covalently attached to the protein based on a colored polypeptide 
 band in sds  page . for the m204e 

mutant , no colored polypeptide band is seen in sds  page and 
 no chromophore absorption was observed in uv / vis / nir spectra from 
 parts of the gel that were cut out ( figure s4b of the supporting information ) .
lc  ms / ms analysis 
 of m204e and wild - type was also used to investigate possible covalent 
 attachment of a bchl .
the mass spectra of peptide m200-m208 ( hglseaaly ) 
 show no evidence for covalent attachment of bchl ( figure s5 of the supporting information ) . however , the blue - green 
 pellets for m204e ( compared to pale yellow for wild - type ) obtained 
 during the course of processing rcs for both the uv / vis / nir spectroscopic , 
 and the mass spectral pigment assays provide visual evidence that 
 a chromophore is not being solubilized , consistent with the low bchl 
 content obtained from the acetone : methanol extraction method .
the 
 time - resolved measurements presented in the next section give no indication 
 of the absence of a pigment from m204e rcs and should be sensitive 
 to such a deficiency . in order to quantify 
 redox potentials 
 of the special pair ( p )
the values obtained are 490  9 mv and 
 498  3 mv for wt and m204e , respectively .
this indicates that 
 the free energies of the charge - separated states ( e.g. , pba ) in m204e are not altered due to 
 a change in the redox potential of the bchl dimer p. figures 5 and 6 show the ta spectra of m204e 
 rcs in the near - infrared and visible regions and kinetic data and 
 fits at select key wavelengths . the data in figures 5a and 6 ( panels a and b ) were obtained 
 with direct excitation of p at 865 nm , and the data in figure 5b were acquired using excitation at 595 nm .
the 
 0.50.6 ps spectra in figures 5 and 6 are that of p * and are identical to p * spectra 
 reported previously . on the red side of the bleaching of the 865 nm 
 band of p ,
the ta spectra are dominated by stimulated emission from 
 p * with only the far red - edge ( 900920 nm ) of p bleaching 
 observed in the spectra acquired at 100 ps and longer times 
 in figure 5a .
decay of the stimulated emission 
 averaged over the 10 nm interval 920930 nm is shown in the 
 inset of figure 5a .
this interval spans an 
 925 nm isosbestic point in the p bleaching spectrum ( see e.g. , 
 figure 5b inset ) .
the solid line in figure 5a inset is the fit to the convolution of the instrument 
 response plus one exponential plus a constant ( fit returns zero for 
 the constant ) , giving a p * lifetime of 38  2 ps .
there is no 
 indication of a second longer component to the p * stimulated emission 
 decay at this key wavelength interval ( or of a shorter component either ) . 
 to this point , fitting the data to a function with two exponentials 
 does not yield meaningful results , giving , for example , 38 ps for 
 the values of both components
global fitting of the data encompassing 
 the entire spectral region shown in figure 5a returns 36  2 ps for the p * lifetime .
a transient absorption 
 band at 1017 nm that would indicate formation of a bchl anion ( e.g. , 
 ba ) is not resolved to within a few 
 percent yield .
ta spectra and kinetics acquired with 120 fs 
 excitation flashes 
 at ( a ) 865 nm or ( b ) at 595 nm . in both ( a and b ) , the kinetic data 
 (  ) are the absorption changes averaged over the 920930 
 nm interval of the spectra shown in that panel . the solid lines through 
 the kinetic data
are fits to the convolution of the instrument response 
 plus one exponential plus a constant .
the time constant ( p * lifetime ) 
 measured in the 920930 nm interval is 38  2 ps ( panel 
 a , inset ) , 38  1 ps ( panel b ) , and 36  2 ps from global 
 analysis of the spectral evolution at all wavelengths shown .
( a ) ta spectra of m204e acquired at the times 
 indicated following 
 120 fs excitation flashes at 865 nm .
( b ) kinetics averaged between 
 539 and 545 nm (  ) and fit to the instrument response plus 
 three exponentials plus a constant .
the resultant time constants are 
 35  2 ps , 238  10 ps , and 3 ns ( fixed ) .
figure 5b inset shows time - resolved 
 ta spectra 
 acquired using 595 nm excitation flashes which allow probing the entire 
 long - wavelength ( qy ) absorption band of p. the decay kinetics 
 in the same 920930 nm interval are shown in the main panel 
 of figure 5b .
the solid line is again a fit 
 to the convolution of the instrument response plus one exponential 
 plus a constant .
the p * lifetime returned from the fit is 36  
 1 ps and again to within a few percent , there is no indication of 
 a second longer component to the decay kinetics at this key isosbestic 
 point .
the spectra in figure 5b inset show , 
 on the blue - side of the p bleaching where stimulated emission contributes 
 minimally , that 2025% of p bleaching has decayed at 3 ns . 

some of this occurs as p * decays , indicating internal conversion of 
 p * to the ground state , and some of the p bleaching decay occurs on 
 the nanosecond timescale and is associated with decay of phb , as will be described below .
the ta spectra in the visible region acquired using 865 nm flashes 
 are shown in figure 6a .
the spectrum of p * 
 at 0.5 ps is identical to that reported previously for wild - type and 
 numerous mutants .
the spectrum at 95 ps ( nearly three 3 multiples 
 of the p * lifetime ) displays features associated with a mix of pha and phb .
the expected wavelengths for bleaching of the 
 qx absorption bands are 542/543 nm ( ha ) and 
 527/528 nm ( hb ) .
both such bleaching features are in evidence 
 in the spectrum at 95 ps , with ha bleaching dominating . 
 the peaks of the broad anion bands of ha and hb are known to occur at 665 nm ( ha ) and 640 
 nm ( hb ) .
again , the 95 ps spectrum displays anion absorption 
 indicative of a mix of species but dominated by ha . at 3 ns , the spectrum has largely the characteristics 
 of pqa ( and pqb ) .
the data averaged between 
 539 and 545 nm , encompassing the maximum 
 of the ha bleaching , and fit to the convolution of the 
 instrument response plus three well - separated exponentials plus a 
 constant are shown in figure 6b .
this fit returns 
 values of 35  2 ps ( assigned to p * decay ) and 238  10 
 ps ( assigned to pha decay ) , 
 with a 3 ns component fixed for phb decay .
( previous work with mutant rcs where phb is formed and where there are no mutations in 
 the immediate hb and qb sites has found that 
 the phb lifetime is 2 
 or 4 ns in the presence or absence of qb , respectively . fixing this value in the present work at either 
 2 or 4 ns
does not significantly alter the fit results ; the 3 ns average 
 value was chosen for convenience and since the qb occupancy 
 is unknown . ) a global fit of the data between 480 and 720 nm to the 
 same function returns values of 36  2 ps and 230  15 ps 
 with , again , the third exponential ( of small amplitude ) fixed at 3 
 ns .
the 230 ps lifetime of pha is identical to that measured for pha  pqa et in wild - type rcs in deriphat , and this assignment 
 is made here .
analysis of the p bleaching decay kinetics on the blue 
 side of the qy band ( e.g. , 840850 nm ) that includes 
 a 230 ps component does not improve the fits .
we can not rule out that 
 the yield of pha  
 pqa et might be slightly 
 less than 100% . in wild - type rcs ,
pha forms in near 100% yield and bleaching 
 of the qx band 
 of ha reaches essentially the same magnitude as the bleaching 
 of the qx band of p at 600 nm . for the m204e mutant ,
the 
 maximal bleaching of ha at 543 nm reaches a much smaller 
 maximum amplitude between 90 and 100 ps ( figure 6 ) .
this is a consequence of the long 35 ps p * lifetime , the 
 reduced yield of pha , and 
 the 230 ps lifetime of pha . to understand this in detail ,
kinetic simulations were performed 
 and ranges of values explored for the rate constants for p * internal 
 conversion and et to ha and hb that would simultaneously 
 reproduce the measured p * lifetime in m204e and the measured yields 
 of charge - separated states .
the scheme 
 in figure 7 shows a composite model that reproduces 
 the measured p * lifetime of 35 ps and the measured yields 
 of ground state and charge - separated states formed from p*. a number 
 of studies have consistently found that the internal conversion time 
 constant ( 1/kic ) for ( unperturbed ) p * 
 in rb .
capsulatus rcs in deriphat - tris 
 buffer at 295 k falls within a range of 180 to 220 ps ( although 
 this value is sensitive to the environment of p , for example , being 
 100 ps in ldao - tris buffer ) . 

mutations that perturb p of course would be expected to affect the 
 time constant for p * internal conversion . however , modeling with much 
 smaller or larger time constants for p * internal conversion would 
 result in a respectively larger or smaller yield of ground state recovery 
 from p * than the measured experimental value .
photochemical model for 
 m204e rcs . as discussed in the text , the 
 values of the time constants for the wild - type rb . 

capsulatus rc ( and detergent ) are nominally the same 
 as those shown for the m204e rc , except the first a - side steps in 
 wt are p *
 pba  
 pha in 3 and 1 ps , respectively , 
 with unity yield . from previous work , and with no mutations introduced 
 around bb , the time constant ( 1/kpb ) for ( presumably 
 superexchange mediated ) p *  phb et is expected to be similar to that of p * internal 
 conversion , 200 ps . the measured 1520% yields for 
 both p  ground state and p *  phb for m204e are consistent with this , and thus 
 the simulations explored a relatively small range ( 150 to 220 ps ) 
 of values for 1/kic and 1/kpb . again , smaller / larger values for 1/kpb and 1/kic would result 
 in higher / lower yields of phb and ground state recovery , inconsistent with the experimentally 
 observed yields .
in addition to reproducing the yields of the p * decay 
 products , the values used for kic , kpa , and kpb must 
 reproduce the measured 35 ps lifetime of p * [ i.e. , 35 ps = 
 1/(kic + kpb + kpa ) ] . from this analysis 1/kpa
is determined to be 50 ps and the 
 yield of pha is 65% . in 
 the simulations ,
the maximal transient buildup of pha is only 4045% and this occurs 
 at 90100 ps .
this is in excellent agreement with the experimentally 
 observed time course of ha bleaching , attaining maximal 
 amplitude between 90 and 100 ps ( figure 6b ) . 

other points of agreement between the experimental data , the simulations , 
 and composite model in figure 7 include the 
 amount of p * that decays to the ground state by internal conversion 
 ( mentioned above ) and the magnitude of p bleaching observed at 34 
 ns . as a further check of the model in figure 7 , we set the challenge to reproduce the experimentally observed 
 ta 
 spectrum at 100 ps using the known basis spectra of the species involved . 

the ta spectrum of each individual state p * , pha , and pqa , the latter two both at 100% yield with respect to p * , are 
 known with high fidelity from extensive studies on wild - type rcs . 

the ta spectrum of state phb is also known , in this case from a mutant in which this state forms 
 in 70% yield as the sole et product of p * decay ( 200 ps internal conversion 
 accounting for the remaining 30% p * decay pathway ) .
thus , we have the basis ta spectra needed to reproduce 
 the ta spectrum at 100 ps ( or at any other time ) .
figure 8 compares the experimental ( red ) and calculated 
 ( simulated ) ta spectrum at 100 ps , and the agreement is excellent . 

the calculated spectrum in blue is the following sum of the known 
 ta basis spectra : 9% p * + 48% pha + 15% phb + 15% pqa + 1% pqb + 12% ground state ( no ta change ) .
the percentages 
 of these species present at 100 ps were based on the kinetic simulations 
 ( figure s6 of the supporting information ) .
beer s law holds here , requiring that the simulation quantitatively 
 reproduce the ta changes referenced to the initial concentration of 
 p * produced upon excitation . in other words ,
the simulation must replicate 
 not only shapes of spectral features but also the absolute magnitude 
 of the absorption changes and such are achieved in figure 8 .
experimental ta spectrum of m204e rcs acquired 100 ps 
 following 
 excitation with 120 excitation flashes at 865 nm ( red ) .
the 
 blue ta spectrum at 100 ps is calculated from the basis set of the 
 ta spectra of p * and the charge separated states .
in 
 the following , we place the unusually straightforward and homogeneous 
 kinetics observed for the m204e mutant in the context of many attempts 
 to understand the connection between energetics and kinetics in rcs , 
 which in most cases are considerably more complex .
glutamic acid at 
 m204 introduces into the environment of the conserved tyr m208 a residue 
 with the potential to hydrogen bond to the tyr hydroxyl group and 
 change its positioning with respect to ba .
theoretical 
 work predicts that reorienting the tyr oh can significantly 
 raise the free energy of pba and impede a - side charge separation .
first , an nmr signal 
 analogous to one previously assigned in studies of wild - type rb .
sphaeroides rcs to a nonhydrogen bonded tyr m210 
 residue is present in the nmr spectrum of wild - type rb .
hydrogen bonded or not , the nmr 
 data clearly indicate that the introduced glutamic acid has significantly 
 affected the environment of tyr m208 .
ultrafast measurements reveal 
 that this single amino acid change has substantial effects on charge 
 separation .
electron transfer from p * to ha is about a 
 factor of 10 slower than in wild - type and pha no longer forms in 100% yield .
specifically , 
 the p * lifetime is 35 ps and p * decays via 50 ps et 
 to ha ( 65% yield ) and via et to hb and 
 internal conversion to the ground state with about equal time constants 
 ( 200 ps ) and yields ( 1718% ) for both , as in figure 7 . in broad view , the m204e mutant has parallels 
 to two rb .
sphaeroides mutants that 
 bear a single amino 
 acid change and result in a similar p * lifetime . replacing the conserved 
 tyr m210 in rb .
sphaeroides with trp 
 results in p * having an 40 ps lifetime but heterogeneous kinetics . replacing the native gly at m203 
 with leu in rb .
the 
 crystal structure of the m203l mutant reveals steric exclusion of a water molecule , denoted water - a , that 
 in the wild - type crystal structures ( figure 2 ) is positioned such that it could form a hydrogen - bond bridge between 
 his m202 ( in rb .
sphaeroides the axial 
 ligand to the mg of one macrocycle of p ) and the ring - v keto group 
 of ba .
these three mutants provide glimpses 
 into potential multiple molecular interactions that may stabilize 
 and fine - tune the free energy of pba : the presence of a tyr near ba , optimal orientation of 
 the tyr hydroxyl group with respect to ba , and a hydrogen 
 bond to the ring - v keto group of ba .
for all three mutants , 
 pba is suggested to be sufficiently 
 destabilized that it is above p * , thereby lengthening by 10-fold the 
 p * lifetime .
electron transfer in the m204e mutant is consistent 
 with prior 
 work on a still relatively small family of rb .
capsulatus mutants in which p * decay is partitioned fairly straightforwardly 
 between significant yields ( more than just a few percent ) of internal 
 conversion and et to hb along with a - side et .
many , but 
 not all , of these mutant rcs employ a tyr at l181 ( which presumably 
 lowers the free energy of pbb ) and a phe at m208 .
however , in m204e , the native tyr m208 and phe 
 l181 are unchanged , as they were in the first two mutants in which 
 et to the b - side was observed , namely the  dh  and  kdh  
 mutants [ where dh = g(m201)d+l(m212)h and kdh = 
 s(l178)k+dh ] . for ease of observing hb bleaching unambiguously , 
 in many of the  wrong way  mutants the leu at m212 is 
 changed to a his , which results in a bchl , denoted  , in place 
 of ha . however , this is not 
 the case in m204e or the  yf  mutant ( swap of l181 phe to tyr and of m208 tyr to phe ) or in 
 mutants in which there is no pigment in the ha site ( dll mutants ) or no pigment in the ba site ( m204q mutants ) . among these mutants ,
specifically , deriphat 160-c and ldao impart different 
 photophysical properties to p * and the rates of its decay pathways . 

not considering mutants aimed specifically at affecting the dimer , 
 the long - wavelength absorption band of p usually occurs near 850 nm 
 for rb .
capsulatus rcs in ldao : tris 
 buffer but usually is at 865 nm in deriphat : tris buffer , the latter 
 essentially the same as found when the ( wild - type ) rc resides in its 
 native membrane . for both wild - type and the l(m212)h (  beta  ) 

mutant , the p * lifetime in deriphat : tris buffer is about double that 
 found for these rcs in ldao - tris buffer .
similarly , the time constant for internal conversion of p * is about 
 a factor of 2 larger for rcs solubilized in deriphat ( 200 ps ) than 
 in ldao ( 100 ps ) .
although this was apparent during early work , it was underscored in the recently revived dll mutant , first made in the youvan lab ca .
1990 , wherein p * decays solely ( 100% ) by internal conversion to the ground 
 state .
the recent work established that the time constant for this 
 process at room temperature is 200 ps for rb . 

capsulatus rcs in deriphat : tris buffer and 100 
 ps for rcs in ldao : tris buffer .
the dll ( ha - less ) mutant has provided basis 
 for understanding a 200 ps component of biexponential p * stimulated 
 emission decay sometimes found in rb .
capsulatus mutants in deriphat : tris buffer where the goal has been to manipulate 
 the free energies of the a- and b - side charge - separated states .
the 
 amplitude of the 200 ps stimulated emission component has generally 
 ranged between 20 and 40% .
meanwhile , a second , and shorter kinetic 
 component is measured for p * stimulated emission decay and measured 
 at wavelengths associated with reduction of electron acceptors ( e.g. , 
 ha/ and hb ) .
however , the 200 
 ps kinetic component is not measured at wavelengths associated with 
 reduction of electron acceptors , rather only at the wavelengths of 
 p * stimulated emission and p bleaching .
these results have indicated 
 that attempts to favor et to the b side ( by lowering the free energy 
 of pbb ) and disfavor et 
 to the a side ( by raising the free energy of pba ) can ( and often do ) give rise to a 2040% 
 population of  inactive  rcs in which charge separation 
 does not occur ( or is substantially impeded ) . for this  inactive  population , 
 p *
has an 200 ps lifetime and decays as in dll by 
 internal conversion to the ground state ( or 100 ps for rcs 
 in the ldao : tris buffer ) .
capsulatus rc in deriphat , 
 having a ( relatively ) 
 long p * lifetime and an absence of a population of rcs in which et 
 does not occur .
capsulatus dll-1 variant ( formerly denoted dll - fylfm in ref ( 52 ) ) , in which p * decays at room temperature with single exponential 
 kinetics ( to within a few percent ) , as discussed further below .
p * 
 populations in which differing rates of et and even differing photochemistry 
 occur are likely rooted in the following inter - related factors .
( i ) 
 in the native rc , the five key electronic states for initial charge 
 separation ( p * , pba , pbb , pha , and phb ) span a modest 0.5 ev in free energy .
( ii ) the mutants designed 
 to influence the primary events aim to shift the free energies of 
 one or more states and likely compress this span by as much as a factor 
 of 2 .
this results in very small spacings between any of these states 
 and especially between p * and pba and pbb .
sphaeroides ) and p is estimated to be 150 to 200 mev higher in free energy 
 than pha , placing it essentially 
 isoenergetic with ( wild - type ) pba .
( iii ) such small energy spacing makes the rates , yields , and directionality 
 of charge separation very sensitive to different rc or p *  conformations  
 or 
populations  that represent a natural landscape 
 of cofactor , protein interactions , static , dynamic , or both .
heterogeneous 
 kinetics in wild - type rcs have been ascribed to pigment / protein conformers 
 or populations and links between protein dynamics 
 and et have been much discussed .
the presence , position , and orientation of water 
 molecules may contribute , such as one (  water - a  ) 
 that may bridge the his ( m202 , equiv of m200 in rb . 

capsulatus ) ligand on pm and ba via hydrogen bonds , and that has been the subject of much recent 
 study . additionally for rb .
capsulatus rcs , 
 p * is 26 mev lower in free energy in deriphat than in ldao , 
 which is 25% of the estimated free - energy difference between 
 p * and pba .
this makes even 
 more plausible the differences found between the p * lifetimes for 
 some rb .
capsulatus rcs in deriphat 
 versus ldao or for differences between membrane - bound rcs versus rcs 
 in detergent micelles .
this mutant was designed to 
 potentially reposition the hydroxyl group of tyr m208 nearly in an 
 orientation postulated to maximally destabilize pba in free energy and thus impede the critical 
 first step of p *  pba  pha primary charge 
 separation . the calculations of alden et al .
indicated that in wild - type rcs , the m208 tyr oh group is 
 confined to an orientation that lowers the free energy of pba by as much as 200 mev ( 40 
 mev of this is an effect on the p oxidation potential ) .
our modeling 
 of possible interactions between a glu at m204 and a tyr at m208 indicates 
 that if a hydrogen bond forms between these residues , the tyr hydroxyl 
 group could be rotated 120 away from the ideal geometry 
 found by alden et al . and only 60 from a geometry that 
 provides the least free energy stabilization of pba . given that pba
is thought to be only 50100 mev below 
 p * in wild - type , it is plausible that this state is higher in free 
 energy than p * in m204e ( figure 7 ) .
( note that 
 the energetic consequences of the tyr - oh dipole being hydrogen bonded 
 to a glutamic acid were not modeled in alden et al . )
we can not know with certainty that the hydroxyl group 
 of tyr m208 is hydrogen bonded to the glutamic acid introduced at 
 m204 , but the nmr spectrum is consistent with this and minimally shows 
 that the oh of tyr m208 ( and/or the entire residue ) experiences 
 a different environment .
in addition or alternatively , depending on 
 the side chain position and ionization state , a glu at m204 could 
 directly or indirectly affect ba , p , or 
the functional 
 consequence remains that p *  pha et has an 50 ps time constant ( and 65% 
 yield ) , allowing the relatively slow native 200 ps time constants 
 for et to hb and internal conversion of p * to the ground 
 state to compete .
additionally , and somewhat unusually , the 
 p * decay kinetics in 
 m204e are quite homogeneous with no discernible contribution of an 
 200 ps ( or other ) component in the p * stimulated emission 
 decay kinetics and no discernible detection wavelength dependence 
 to the p * decay kinetics .
thus , not only is an  inactive  
 population absent , the data indicate only a comparatively narrow ( or 
 no ) distribution of p * functional forms . even for dll rcs , 
 where again p * decays solely by internal conversion , a 3-fold wide 
 distribution of p * decay time constants spanning several hundred picoseconds 
 is found at 77 k in both ldao and deriphat .
the absence of detection - wavelength dependent kinetics in the m204e 
 mutant may indirectly signal , or at least be consistent with , the 
 introduced glutamic acid negating or constraining some pigment or 
 protein native motions ( conformational changes or rotomers ) as might 
 result , for example , upon formation of a hydrogen bond between glu 
 m204 and the oh group of tyr m208 .
some final connections 
 can be made to two other dll variants , ones that led to our making m204e in the first 
 place . in both of these mutants , p *
decay was found to be biexponential , 
 but instead of one photochemically  active 
p * population 
 and an  inactive  p * population with an 200 
 ps lifetime there were two distinct  active  p * populations . 

in one , p * decayed with an 10 ps time constant and gave rise 
 to formation of pba , which 
 state lived for 300 ps and was trapped because ha is absent from all rcs in the dll mutant family . in the 
 second population , p * had an 100 ps lifetime and decayed via 
 a combination of et to hb and internal conversion to the 
 ground state .
the dll motif bears a number of changes around 
 the a - side cofactors , including having a phe at m208 . in the dll variants where pba was trapped ,
m208 was restored to a tyr , and we speculated whether 
 the two populations might reflect orientations of tyr at m208 .
the 
 m204e rc was born of the idea of whether it might be possible to pin 
 down tyr m208 and affect the populations .
the simple scheme 
 shown in figure 7 , where 
 both pba and pbb are placed higher than p * , suggests 
 that in m204e p *  pha et occurs not by a two - step processes wherein pba is a chemical intermediate but rather 
 occurs in a manner posited for p *  phb et .
all cases of p *  phb to date , even aided by a tyr at l181 , 
 are presumed to occur with superexchange assistance of pbb .
depending on the mutations involved , 
 the time constant for p *  phb et has ranged from 70 to 200 ps ; again , these values 
 are for various mutants in deriphat and 40 ps , the smallest 
 time constant obtained , is for a mutant in ldao .
interestingly , the 
 50 ps time constant for p *  pha et in m204e is within this range . on the b - side , 

the smaller values in a 100200 ps range of time constants 
 have been achieved with a tyr residue at l181 with 200 ps the case 
 for the native phe .
electron transfer in the m204e mutant thus 
 conforms well compared 
 to prior observations and analyses .
it appears that in this interesting 
 mutant , the free energy of pba is significantly affected ( likely raised above p * ) , and the balance 
 of et in the rc is disrupted even though the native , key tyr at m208 
 is in place .
even though the p * lifetime is a very long 35 
 ps , pha is still the dominant 
 product of p * decay , though reduced to 65% yield .
the native 
 phe at l181 also is in place , and the time constant for p *  
 phb is unchanged from the 
 200 ps value ( for rb .
capsulatus in deriphat ) that has been repeatedly found for mutants with a ( presumably ) 
  native  b - side but in which et to hb is 
 observed because of impeded a - side et .
similarly , the time constant 
 for p * internal conversion is unchanged from the  native  

capsulatus in deriphat ) . finally , p * decay in m204e appears to occur fairly 
 uniformly in the entire rc or p * population .
this suggests a p *  population  
 in which pba is elevated 
 well out of the small , congested free - energy window normally occupied 
 by all five key electronic states , whose free energy spacing / ordering 
 is highly sensitive to static / dynamic protein effects that drive the 
 relative contributions of the three p * decay pathways , which give 
 et to the a - side , et to the b - side , or no et at all .
 slow , 50 
 ps , p *  p+ha electron 
 transfer is observed in rhodobacter 
 capsulatus reaction centers ( rcs ) bearing the native 
 tyr residue at m208 and the single amino acid change of isoleucine 
 at m204 to glutamic acid . 
 the p * decay kinetics are unusually homogeneous 
 ( single exponential ) at room temperature . 
 comparative solid - state 
 nmr of [ 4-13c]tyr labeled wild - type and m204e rcs 
 show that the chemical shift of tyr m208 is significantly altered 
 in the m204e mutant and in a manner consistent with formation of a 
 hydrogen bond to the tyr m208 hydroxyl group . 
 models based on rc crystal 
 structure coordinates indicate that if such a hydrogen bond is formed 
 between the glu at m204 and the m208 tyr hydroxyl group , the oh 
 would be oriented in a fashion expected ( based on the calculations 
 by alden et al . 
 , j. phys . 
 chem.1996 , 100 , 1676116770 ) to destabilize p+ba in free energy . 
 alteration 
 of the environment of tyr m208 and ba by glu m204 via this 
 putative hydrogen bond has a powerful influence on primary charge 
 separation . 
Introduction
Experimental Section
Results
Discussion