Regular paper

Electron transfer dynamics in Rhodobacter sphaeroides reaction center

mutants with a modified ligand for the monomer bacteriochlorophyll

on the active side

Evaldas Katilius1,*, Jennie L. Babendure1,2, Su Lin1 & Neal W. Woodbury11Department of Chemistry and Biochemistry, Arizona Biodesign Institute and the Center for the Study ofEarly Events in Photosynthesis, Arizona State University, Tempe, AZ 85287-1604, USA; 2Present address:Department of Biological Science, University of California, San Diego, La Jolla, CA, 92093-0673, USA;*Author for correspondence (e-mail: evaldas@asu.edu; fax: +1-480-965-2747)

Received 8 July 2003; accepted in revised form 11 March 2004

Key words: electron transfer, P-less mutants, site-directed mutagenesis

Abstract

The histidine ligand of the monomer bacteriochlorophyll molecule on the active side (BA) of the photo-synthetic reaction center from Rhodobacter sphaeroides was mutated to a number of other amino acids.Histidine (H) at the position L153 was replaced with aspartic acid (D), glutamic acid (E), glutamine (Q),glycine (G), leucine (L), phenylalanine (F), serine (S), valine (V) and tyrosine (Y). These mutations werecreated to investigate how the alteration of the ligand residue affects the properties of the BA molecule andchanges the dynamics of the primary charge separation in reaction centers. In all of the mutants, thechanges in the ligand result in a blue-shift of the BA absorption spectrum, in both visible and near-infraredspectral regions, with the size of the shift varying between mutants. The primary electron transfer timeconstants in these mutant reaction centers range from 4.5 to 18 ps, being substantially slower than the wild-type value of 3 ps. The decrease in the electron transfer rate is interpreted as being due to an increase in thefree energy of the initial charge-separated state P+B�

A.

Abbreviations: BA,B – monomer bacteriochlorophyll; BChl – bacteriochlorophyll; Bl – Blastochloris; BPheo– bacteriopheophytin; DAS – decay-associated spectra; EDTA – ethylenediaminetetracetic acid; HA,B –bacteriopheophytin; LDAO – lauryldimethylamine N-oxide; P – initial electron donar; QA,B – quinonemolecules RC(s) – reaction center(s); Rb – Rhodobacter; SADS – species-associated decay spectra; Tris –tris(hydroxymethyl)aminomethane; WT – wild type

Introduction

The primary energy conversion process in photo-synthesis occurs in a pigment–protein complexcalled the reaction center (RC), where light energydrives electron transfer across the photosyntheticmembrane. The reaction center from purple non-sulfur photosynthetic bacteria is probably thebest–studied example (for reviews see Parson 1996;Hoff and Deisenhofer 1997; van Brederode and

Jones 2000). The RC from Rhodobacter (Rb.)sphaeroides consists of 3 protein subunits L, M andH, which coordinate 10 cofactors: 4 bacteriochlo-rophyll (BChl), 2 bacteriopheophytin (BPheo),2 ubiquinone molecules, a carotenoid moleculeand a nonheme iron. The BChl, BPheo andquinone cofactors are arranged in two, nearly C2

symmetrical branches. Because of this symmetry,there are two potential pathways for photosyn-thetic electron transfer (usually labeled A and B),

Photosynthesis Research 81: 165–180, 2004.� 2004 Kluwer Academic Publishers. Printed in the Netherlands.

165

both starting with the initial electron donor, P (apair of bacteriochlorophylls), and then proceedingthrough a monomer bacteriochlorophyll (eitherBA or BB) and a bacteriopheophytin (either HA orHB) to a ubiquinone (either QA or QB).

Since the determination of the reaction centercrystal structure (Deisenhofer et al. 1984; Allenet al. 1987), the function of the monomer bacte-riochlorophylls has been the target of many stud-ies. The earliest kinetic studies revealed electrontransfer from the initial electron donor P to thebacteriopheophytin acceptor on the active branchof the cofactors (HA) (Kirmaier et al. 1985a, b). Itwas proposed initially that the monomer BChlmolecule (BA), which is situated between P andHA, acts primarily as a bridge in a super–exchangeelectron transfer process between P and HA. Later,the transient population of the charge–separatedstate P+B�

A was detected (Holzapfel et al. 1989;Arlt et al. 1993, 1990), which led to the conclusionthat this state is a real intermediate in the electrontransfer process.

The crystal structure of the Rb. sphaeroides RCshows that monomer BChl molecules, BA and BB,are ligated by histidines L153 and M182, respec-tively (Allen et al. 1987; El-Kabbani et al. 1991;Ermler et al. 1994). One of the early site-directedmutagenesis studies of Rb. capsulatus RCs hasshown that these histidines can be replaced byleucine (L), serine (S) or threonine (T) and stillresult in functional reaction centers, while muta-tion of the histidine M182 to arginine (R) led to aphotosynthetically incompetent mutant (Bylinaet al. 1990). Later, the histidine to leucine muta-tion at the position M182 was again created inRb. capsulatus and Rb. sphaeroides RCs (Gallo1994; Katilius et al. 1999). It was determined thatthe BB molecule in this mutant was replaced with abacteriopheophytin. The exchange of the pigmentsalso caused an alteration of RC photochemicalproperties, as electron transfer to the normallyinactive B-side was observed (Katilius et al. 1999,2002).

Histidine at the position L153 has been mu-tated to cysteine (C), glutamic acid (E) and leucine(L) in reaction centers from Blastochloris (Bl.)viridis (Arlt et al. 1996). Changing histidine tocysteine did not significantly alter the RC proper-ties, while mutation to glutamic acid caused aroughly threefold decrease in the rate of P* decay.The mutation of histidine to leucine resulted in the

replacement of the BA molecule with a BPheo.This caused significant changes in the electrontransfer dynamics, as the energetics of the charge–separated states were affected by the pigment ex-change (Arlt et al. 1996).

In this report, we describe a series of mutationsof histidine L153 in the Rb. sphaeroides RC. Ninemutations were constructed; histidine (H) was re-placed with aspartic acid (D), glutamic acid (E),glutamine (Q), glycine (G), leucine (L), phenylal-anine (F), serine (S), tyrosine (Y) and valine (V)(the mutants are referred to as HX(L153), where Xis any of the above nine amino acids). The maingoal of this study is to characterize how the natureof the ligand affects the spectral and electrochem-ical properties of the BA molecule in Rb. sphaero-ides RCs and, in turn, how these properties affectthe electron transfer reactions. The variety of dif-ferent amino acids inserted at L153 should alsoprovide more information concerning how theenergetics of the initial charge separated stateP+B�

A affects the electron transfer dynamics in thereaction centers of purple bacteria.

Materials and methods

Mutations were created using the Chameleon orQuikChange mutagenesis kits (Stratagene,La Jolla, California). The mutagenesis reactionswere performed on the pUCAH plasmid, whichwas made up of pUC19 with the Asp718I-HindIIIfragment of the L-subunit gene. The mutagenesisreactions were performed according to the kitmanuals, and the mutations were verified by DNAsequencing of the whole L-subunit gene fragment.For each mutation, the Asp718I-HindIII fragmentof pUCAH was then subcloned into the plasmidpRKSCH7H, which contained the rest of theL-subunit and subunit genes and included sevenCAC codons at the end of the M-subunit geneencoding a hepta-histidine tag. Those plasmidswere then transferred by conjugation into theRb. sphaeroides pufLM deletion strain DLM1.1,which lacked the reaction center gene, and cellswere grown under semiaerobic conditions (Lin etal. 1994). Cells were harvested by centrifugation at10,000 · g, the pellet was washed and resuspendedin 10 mM Tris-HCl (pH 8) buffer. Cells were thenbroken using a french pressure cell at 18000–20000psi, and the lysate was incubated with DNAseI

166

(Sigma, St. Louis, Missouri) and 100 lM Ca2+

and Mg2+ for 15 min to digest chromosomalDNA. This lysate was centrifuged at 16000 · g for10 min to separate cellular debris and unbrokencells from soluble photosynthetic membranes.Chromatophores were incubated at 26 �C in thepresence of 0.65% LDAO detergent for about 15–20 min. The solubilized RCs were then centri-fuged at 32000 g for 30 min to separate themfrom unsolubilized membranes. To improve thespe-cific binding of the solubilized RCs tothe metal-chelating Ni-NTA column (Qiagen,Valencia, California), 150 mM NaCl was added tothe solution before loading it on the Ni-NTAcolumn as well as to the washing buffer. This im-proved the specific binding of the poly-His taggedRCs to the column and resulted in faster purifi-cation of the RCs. Imidazole was omitted from thewashing buffer during isolation of the mutant RCsand the RC elution from the Ni-NTA column wasperformed with a buffer containing 50 mM EDTAinstead of 50 mM imidazole, because addition ofimidazole previously showed variability in prepa-rations of RCs from the HL(M182) mutant(Katilius et al. 2002). Eluted RCs were dialyzedovernight and additionally purified via ion ex-change chromatography on Toyopearl DEAE-650M (diethylaminoethyl) column (Supelco,St. Louis, Missouri) (Lin et al. 1994). This alteredprocedure was previously shown to yield samplesof the poly-His tagged HL(M182) mutant with thesame pigment ratio as measured in the isolatedHL(M182) mutant RCs without the poly-His tag(Katilius et al. 2002). However, additional purifi-cation using an ion exchange column revealedsignificant heterogeneity in some of the mutant RCpreparations. For most of the mutants, two reac-tion center-containing fractions were collectedfrom this column. The first fraction usuallyshowed very little or no absorbance at around865 nm; the second reaction center fraction, whicheluted off the column a few minutes later, had aP-band absorbance at around 865 nm, although itsamplitude was usually reduced compared to theWT RC spectrum. The spectra of the P-containingfractions are presented in Figures 1 and 2 for allmutants, except for HQ(L153), in which a reactioncenter fraction containing the P band was notisolated. The stability of the P-containing andP-less fractions was assessed by keeping the sam-ples at room temperature for several days and

measuring the absorption spectra every severalhours. Under these conditions, P-containing frac-tions showed a decrease in the P absorbanceoccurring within a few days, which indicated thatdegradation of the protein was occurring on longtime scales. However, no significant spectralchanges were observed after performing transientabsorbance measurements, which usually tookseveral hours (spectra of the samples were mea-sured before and after the measurements, resultsnot shown). No significant degradation of the P-containing fractions of the mutant RCs was ob-served upon exchange of the LDAO detergent forTriton X-100, which was done on an ion exchangecolumn according to a previously published pro-tocol (Peloquin et al. 1994). Investigation of P-lessfractions showed that their spectra did not changeover the course of several days at room tempera-ture, which would indicate no further degradationof the protein. To test for possible oxidation of P(which could give rise to the apparently P-lessfractions and to the decreased intensity of theP-band in some of the P-containing fractions), upto 50 mM of sodium ascorbate was added to thesamples, however, no recovery of the P-band wasobserved. Pigment extractions of the P-containingfractions as well as P-less fractions fromHL(L153), HQ(L153) and HY(L153) mutantswere performed in 7:2 (v/v) acetone methanolmixture as previously described in (Van der Restand Gingras 1974).

The experimental setup for the transient absor-bance measurements has been described previously(Katilius et al. 1999, 2002). For these measure-ments, RCs were suspended in either 15 mM Tris-HCl (pH 8.0), 0.025% LDAO and 1 mM EDTA(unreduced samples) or 15 mM Tris-HCl (pH 8.0),0.05% Triton X-100 and 1 mM EDTA (reducedsamples). The addition of 100 lM ortho-phenan-throline was used to block QA to QB electrontransfer in the unreduced samples. In order to blockthe electron transfer from HA to QA, 1 mM sodiumdithionite was used to chemically reduce QA.Measurements of RCs with intact QA were per-formed in a rotating circular cell with a 2.5 mmoptical path containing about 4 mls of sample. Thecell rotation allowed complete sample removalfrom the excitation region between laser pulses.The samples with reduced QA were measured in anairtight, 2-mm pathlength glass cuvette with con-stant stirring. All measurements were performed at

167

room temperature. The OD of the samples wasabout 1.2 at 800 nm. Measurements for all mutantswith the exception of HD(L153) were performedwith both unreduced and reduced samples. In thecase of the HD(L153) mutant, insufficient amountsof the P-containing fraction to perform measure-ments with unreduced QA were obtained, thus onlymeasurements with reduced QA were performed.The data analysis was performed using a locallywritten data analysis program ASUFIT based onMATLAB software (Mathworks).

Results

Absorption spectra at room temperature

Absorption spectra of all mutants at room tem-perature are shown in Figure 1 (the absorbancemaxima of P and the monomer BChls are sum-marized in Table 1). The spectra of most of themutants are quite different from the WT RCspectrum with the exception of the mutantHS(L153). Despite the replacement of histidinewith the much smaller residue serine, the absorp-tion spectrum of HS(L153) mutant RCs is almostidentical to the absorption spectrum of WT RCs.In the spectra of other mutants several majorchanges are observed. First, the amplitude of theP-band at 865 nm significantly decreased relativeto the amplitude at monomer BChl band (800 nm)or BPheo band (760 nm) in most of the mutants.In the HQ(L153) mutant, the reduction in theamplitude of the P-band becomes extreme, as italmost totally disappears. The decrease of theP-band is usually accompanied by a relative in-crease in the H-band at around 760 nm (normal-izing to the peak absorbance near 800 nm), whencompared to the WT spectrum. The extent of theP-band decrease is in general not correlated to thenature of the new ligand. The largest decrease isobserved in the mutants in which the histidine isreplaced with glutamate, glutamine or leucine.While one might conclude from this that the size ofthe residue is an important factor, mutating histi-dine to either tyrosine or phenylalanine results in asimilar decrease in P-band oscillator strength as isobserved in the histidine to glycine or valine mu-tants.

The maximum of the B-band in all of the mu-tants except HS(L153) shifts from 804 nm in WTto about 799 nm. The shape of this band becomesclearly asymmetric; in the HF(L153), HG(L153),HV(L153) or HY(L153) mutants, a shoulder ataround 810 nm is resolved. Shifts and splitting ofthe QX BChl absorption band are also evident inseveral mutants. For example, in the HF(L153)mutant, the QX BChl band blue-shifts by about2 nm. In other mutants the shift is even larger,from 598 nm in WT to 592 nm in HG(L153) andeven to 587 nm in the HD(L153) mutant. In thecase of the HV(L153) mutant, there is an obvioussplitting of the QX BChl band with two bandsappearing at about 576 and 598 nm. In general, it

Figure 1. Absorption spectra of reaction centers from the mu-

tants HD(L153), HE(L153), HF(L153), HG(L153), HL(L153),

HQ(L153), HS(L153), HV(L153), HY(L153) and WT RCs at

room temperature.

168

appears that most mutations of the ligand to BA

result in a blue-shift of both the QX and QY BA

absorption transitions. However, the effects of themutations on the QX spectral band of BA areharder to unambiguously determine, because theabsorbance of P in the mutants is also significantlyaltered and P contributes to the absorbance in the600 nm region.

As mentioned above, the decrease of theP-band observed in most of the mutants isaccompanied by an increase in the BPheoabsorption intensity at around 760 nm. The in-crease in the BPheo absorption is also evident inthe QX transition region around 530–540 nm. Theintensity of absorption at 530 nm in most ofthe mutants increases significantly compared tothe spectra of the HS(L153) mutant or WT RCs.It is not clear whether this increase in absorptionintensity at around 530 nm is due to an increasedamount of BPheo in the samples, or is associatedwith the absorption increase in the 400–500 nmregion, which affects the total absorption inten-sity in the BPheo QX absorbance region. Itshould also be noted, that in the HQ(L153)mutant a broad absorption band with a maxi-mum at around 445 nm can be resolved (seeFigure 1). The origin of this band will be dis-cussed below.

Absorption spectra at 77 K

To better characterize the mutants, absorptionspectra were also measured at 77 K (see Figure 2

and Table 1). In this case, several general trendsare again noticeable. In addition to changes of theP-band amplitude relative to the other two bandsin the near infrared, at low temperature, changesin the P-band position are also evident. Forexample, in the HS(L153) mutant at 77 K theP-band is blue shifted to 873 from 888 nm inWT. Asmaller blue-shift is also observed in the HF(L153)mutant, while in most of the other mutants there isa small (2–3 nm) red-shift of the P-band (see Ta-ble 1). It is also notable that in the HQ(L153)mutant, in which the P-band was practically absentat room temperature, at low temperature theP-band is clearly resolved, though it has a de-creased oscillator strength relative to wild-type, andits peak position is blue shifted to 870 nm.

Significant changes are also visible in thestructure of the QY absorbance band of themonomer BChls in many of the mutants. Whilethe position and width of the band around 800 nmwere quite similar in the HS(L153) and WT spectraat room temperature, at low temperature this bandis less intense at the peak and somewhat broader inHS(L153) than in WT. Due to the band broaden-ing, the shoulder on the longer wavelength side,which in WT is usually associated with the absor-bance of the BB molecule (Kirmaier and Holten1987; Breton 1988; Hoff and Deisenhofer 1997), isnot as well resolved in this mutant. In contrast, thisshoulder becomes very well resolved in theHV(L153) and HY(L153) mutants, as the BA

absorption band peaks at 799 nm. However, themost unusual changes are observed in theHF(L153) and HG(L153) spectra. In the HF(L153)

Table 1. Positions of the P and monomer BChl QY and QX absorption bands at 295 and 77 K

Sample 295 K 77 K

P, nm BChl QY,nm BChl QX, nm P, nm BChl QY, nm BChl QX, nm

HD(L153) 864 799 587 891 798 581, 603

HE(L153) 862 799 589 887 800 583, 602

HF(L153) 866 799 596 876 799 585, 598

HG(L153) 865 798 592 892 789, 812 585, 602

HL(L153) 860 799 586 ND ND ND

HQ(L153) 853 797 589 870 799 588

HS(L153) 866 804 597 873 802 593

HV(L153) 866 800 576, 598 890 798 579, 601

HY(L153) 863 801 598 891 799 580, 601

WT 865 804 598 888 803 598

ND = not determined.

169

mutant, the maximum of the monomer BChl bandin the QY region is at 799 nm, but in addition to theshoulder at around 809 nm, another shoulder at790 nm can be resolved (see Figure 2). On the otherhand, in the spectrum of the HG(L153) mutant, theBA and BB absorption bands at 789 and 812 nm,respectively, are clearly resolved.

At 77 K, the BChl QX band is split in practicallyall mutants. The smallest shift is observed in theHS(L153) spectrum, where at least two transitionscan be resolved peaking at around 592 and 600 nm.

In the HF(L153) and HG(L153) mutants the BChlQX transitions are clearly split into two bands ataround 585 and 600 nm. In the HV(L153) andHY(L153) mutants the two bands are even moreseparated, with their maxima at around 580 and600 nm. The splitting is, however, not observed inthe HQ(L153) mutant, instead there is a broaden-ing and blue-shift of the band to about 588 nm.

BChl:BPheo pigment ratio analysis

The results of the pigment extractions are sum-marized in Table 2. In most of the mutants theBChl to BPheo pigment ratio remained the sameas in WT RCs (1.9 ± 0.1). It is quite surprisingthat in the HF(L153) mutant the pigment ratio isthe same as in WT, while an analogous mutationof either of the histidine ligands to the specialpair induces the replacement of BChl withBPheo, resulting in 1:1 pigment ratio (Bylina andYouvan 1988; Kirmaier et al. 1988; Camara-Artigas et al. 2002). On the other hand, thepigment ratio in the HL(L153) mutant is lowerthan in WT (see Table 2), but not 1, as would beexpected if the BA pigment had been replacedwith a BPheo. A reduced BChl : BPheo pigmentratio is also found in the mutants in which thehistidine at L153 has been mutated to glutamicacid or glutamine. Interestingly, the same re-duced pigment ratio of about 1.5–1.6 is foundfor the P-less fractions isolated from severalmutants (for example HL(L153) and HY(L153)).In each case (HL(L153), HE(L153), HQ(L153)and the P-less reaction center fractions of othermutants), the oscillator strength of P is greatlyreduced. Possible explanations for the differencesin the pigment ratios in various mutants as wellas in P-less fractions of the samples will be dis-cussed below.

Transient absorption results

Photochemical properties of all mutants werecharacterized using transient absorption spectros-copy. Mutants which still have the P-band wereexcited at 860 nm, while the P-less fractions fromthe mutants HL(L153) and HQ(L153) wereinvestigated by exciting them at 795 nm. Theresults of kinetic analyses are summarized in Ta-ble 2 and Figures 3–5. P* stimulated emission de-cay time in all mutants is slower than in WT RCs,

Figure 2. Absorption spectra of reaction centers from the mu-

tants HD(L153), HE(L153), HF(L153), HG(L153), HQ(L153),

HS(L153), HV(L153), HY(L153) and WT RCs at 77 K.

170

and it ranges from about 4.5 ps in the HV(L153)and HY(L153) mutants to about 18 ps inHD(L153). In the HE(L153) and HD(L153) mu-tants, P* decay becomes heterogeneous, and atleast two exponential functions are necessary to fitthe kinetics (see Table 2 for lifetimes). The kineticsof secondary electron transfer from P+H�

A toP+Q�

A (see Table 2) do not significantly change inmost of the mutants compared to that in WT RCs(the rate constant of secondary electron transferhas not been determined in the HE(L153) andHD(L153) mutants). These results suggest that themutations primarily affect the properties of P*and/or P+B�

A and do not dramatically affect theproperties of the charge-separated states P+H�

A orP+Q�

A as will be discussed further below.Transient absorbance kinetics of the samples

with neutral QA were analyzed using the sequentialdecay model:

P*�!k1 PþH�A �!k2 PþQ�

A

Using a sequential model instead of the simplesum of exponential functions to fit the data di-rectly provides interpretation of the data accord-ing to the assumed physical model. The fittingresults in this case contain the rate constants(lifetimes) for electron transfer from one state toanother and the species-associated decay spectra,describing the spectral properties of the differentstates. In the analysis of the data we did not in-clude the state P+B�

A, which is believed to be the

initial charge separation state. In WT RCs it hasbeen shown that the P+B�

A state decays faster thanit is formed (Holzapfel et al. 1989, 1990; Arlt et al.1993; Sporlein et al. 2000), thus the population ofthis state at any given time is small compared tothe populations of P* or P+H�

A. In all our inves-tigated mutants we observe that P* decay is longerthan in WT RCs. At the same time no clearspectral evidence for P+B�

A formation is observed,indicating that the electron transfer from BA to HA

must be as fast as in WT RCs, or at least muchfaster than the determined P* decay. The transientabsorbance kinetics are well described by fittingthe data according to the above sequential modelwhich leaves out an explicit P+B�

A state.Species-associated decay spectra (SADS) ob-

tained from the fits of the transient absorbancekinetics of mutant and WT RCs are presented inFigure 3. In all of the mutants, the spectra corre-sponding to the P* state are essentially the same.In the HS(L153) mutant, SADS corresponding tothe different charge-separated states are basicallythe same as in WT (data not shown, spectra areavailable in supplementary material). The spectralfeatures in the SADS of the other mutants showsome differences from the WT spectra. In the caseof the HF(L153) and HG(L153) mutants, there isan obvious additional bleaching of the bandaround 785 nm in the charge-separated states,while in the HV(L153) and HY(L153) mutants thisbleaching is redshifted towards 795 nm (the SADSof HV(L153) and HY(L153) are nearly identical,

Table 2. Properties of L153 mutants reaction centers

Sample Pigment ratio P* decay, ps P+H�A decay, ps

HD(L153) 1.9 ± 0.1 18 ± 1; 120 ± 5 ND

HE(L153) 1.6 ± 0.1 9 ± 1; 65 ± 5 ND

HF(L153) 2 ± 0.1 6 ± 0.5 190 ± 10

HG(L153) 1.9 ± 0.1 6.8 ± 0.2 200 ± 10

HL(L153) 1.6 ±0.1 ND ND

HL(L153) P-less fraction 1.4 ± 0.1 – –

HQ(L153) P-less fraction 1.6 ± 0.1 – –

HS(L153) 1.9 ± 0.1 5.8 ± 0.2 180 ± 10

HV(L153) 1.8 ± 0.1 4.5 ± 0.2 180 ± 10

HY(L153) 1.8 ± 0.1 4.5 ± 0.2 190 ± 10

HY(L153) P-less fraction 1.6 ± 0.1 – –

WT 1.9 ± 0.1 3.1 ± 0.2 200 ± 20

ND = not determined.

171

thus, only the SADS for the HV(L153) are shown;the spectra for HY(L153) are available in supple-mentary material). This bleaching obviously af-fects the spectra corresponding to the P+H�

A andP+Q�

A states, as the positive band present in WT ataround 780 nm is reduced or contains a small dipin case of the HF(L153) mutant. The spectralfeature at around 800 nm in the P+H�

A and P+Q�A

difference spectra of WT is usually associated withthe electrochromic shift of the monomer BChlground state absorbance due to the formation ofthe charge-separated state P+H�

A and, later,P+Q�

A. The changes in the spectral shape of thiselectrochromic shift may be due to the BA ground

state absorbance changes, as observed in theground state absorption spectra both at 77 K andat room temperature.

As already mentioned above, in the HE(L153)and HD(L153) mutants, at least two exponentialfunctions are necessary to describe the decay of P*.This indicates that there is some kind of the het-erogeneity in the sample. Thus, transient absor-bance kinetics in these two mutants were analyzedusing a simple sum of the exponential functions,the decay-associated spectra corresponding to thedifferent components are presented in the Figure 4.

The kinetics of the HE(L153) mutant RCs weremeasured with neutral QA. P* decay in this mutant

(a) (b)

(c) (d)

Figure 3. Species-associated decay spectra (SADS) for (a) HF(L153), (b) HG(L153), (c) HV(L153) mutant RCs and (d) WT RCs. The

spectra were obtained from global fitting of transient absorbance data measured over 1-ns time-scale using the sequential model

described in the text. Lifetimes of the states P* and P+H�A are presented in Table 2. The state P+Q�

A is nondecaying on the time scale of

measurements, its lifetime was fixed at 10 ns during fitting.

172

can be described by two exponential componentswith the lifetimes of 9 ps and 60 ps. The decay-associated spectrum of the 9-ps componentcontains the prominent spectral features corre-sponding to P* stimulated emission decay centeredat about 900 nm, and very little ground staterecovery of P. However, the spectrum of the 60-pscomponent shows both P* stimulated emissiondecay as well as a significant recovery of the

P-ground-state bleaching on this time scale (seeFigure 4a). The spectrum of the longest component(non-decaying on the time scale of our measure-ments) corresponds well to the spectrum of P+Q�

A,as observed in WT RCs (Figure 3). From the ratioof the initial bleaching at 865 nm and the longestcomponent amplitude at this wavelength, it can beconcluded that P+Q�

A is formed with about 65%yield in this mutant.

(a) (b)

Figure 4. Decay-associated spectra of HE(L153) and HD(L153) mutant RCs. The spectra were obtained from global fitting of

transient absorbance data measured over 1-ns time-scale at room temperature. The lifetime of the longest component was fixed at 10 ns

during fitting.

Figure 5. Transient absorbance spectra of the HQ(L153) mutant RCs recorded 1, 20 and 660 ps after the excitation of the sample at

795 nm at room temperature.

173

Primary photochemistry of the HD(L153) mu-tant was characterized only in the QA prereducedsample (the measurements were not performedwith the intact QA due to limited amounts ofsample). P* decay lifetimes in this mutant are 18and 120 ps. The decay-associated spectrum of the18-ps component is similar to the spectrum of the9-ps component in the HE(L153) mutant, whilethe decay-associated spectrum of the second, 120-ps component again shows that there is a signifi-cant recovery of the P-ground-state bleaching onthe 120-ps timescale. The spectrum of the long-lived component does not correspond very well tothe spectrum of the state P+H�

A in WT RC. Thespectrum of this component, however, correlateswell to the SADS of the P+H�

A state from theHF(L153) or HG(L153) mutant RCs. The changesin the spectrum of the long-lived component inHD(L153) might be also due to the BA groundstate absorption band-shift, as observed in theground state absorption spectrum. From the de-crease of the P-bleaching intensity, it can be con-cluded that the state P+H�

A in the HD(L153)mutant is formed with approximately 60% yield.The possible reasons for the observed heteroge-neous decay of P* in these two mutants will bediscussed below.

As mentioned above, a P-less fraction of thesamples can be isolated in the mutants HQ(L153),HL(L153), HY(L153) and in smaller amountsfrom other mutants. Upon excitation of the P-lessreaction centers isolated from the HQ(L153) mu-tant at 795 nm, the bleaching of the band at799 nm decays non-exponentially, which can bebest described by a sum of exponential functionswith the lifetimes of 5, 200 ps and several ns.Surprisingly, the transient spectra in the visibleregion of the spectrum show that in addition to thebleaching of BChl QX band around 585 nm, thereis also bleaching of BPheo QX band at around530 nm, as well as a broad absorbance band ataround 630–640 nm (see Figure 5). The bleachingof the BPheo band is unexpected, as excitation ofBChl molecules in the RC should not result in theenergy transfer to the BPheo. At the same time, theband around 630–640 nm was previously associ-ated with the formation of a BPheo anion, inparticular H�

B (Kellogg et al. 1989; Heller et al.1995). Thus, these spectral changes imply thatthere is formation of a charge-separated stateinvolving the bacteriopheophytin HB and bacte-

riochlorophyll. Previously, transient formation ofthe state with the same spectral characteristics wasobserved when WT RCs were excited with blue(390 nm) light (Lin et al. 2001) or in ND(L170)/ND(M199) mutant RCs upon excitation at800 nm (Haffa et al. 2003). In that case, the spec-tral evolution was interpreted as formation of thecharge separated state Bþ

BH�B ; thus, we can also

speculate that the state formed in the P-less sampleupon excitation at 795 nm could be Bþ

BH�B . The

putative charge separated state BþBH

�B apparently

lives at least several hundred picoseconds (seeFigure 5), which is similar to a long lived stateobserved in ND(L170)/ND(M199) or HE(L168)/ND(L170) mutant RCs where the stabilization ofthe charge separated state Bþ

BH�B was attributed to

introduction of potentially negatively chargedamino acids in the vicinity of P and the monomerBChls (Haffa et al. 2003, 2004). Of course, thepossibility cannot be excluded that the spectralproperties of BA and HA and their respective cat-ion/anion states become altered in the P-less frac-tions such that the spectral properties of the anionband associated with HA shifts, mimicking thenormal position of H�

B . However, it is interestingto note that the same transient spectral changeswere also detected in the P-less fraction isolatedfrom the HL(L153) mutant. This indicates that thetransient charge separation between monomerBChl and BPheo may be a general characteristic ofP-less samples.

Discussion

A series of mutants was created by replacing thehistidine ligand of the reaction center BA moleculewith a number of other amino acids. The mutantswere created to investigate how the ligand deter-mines the spectral and redox properties of the BA

molecule, and at the same time, how the changes inthe BA properties affect the dynamics of the elec-tron transfer in Rb. sphaeroides RCs.

The HF(L153) and HL(L153) mutationsdo not result in the replacement of BA

Mutating histidine to either phenylalanine or toleucine at postion L153 was expected to cause thebacteriochlorophyll in the BA site to be replacedwith bacteriopheophytin, as had been observed

174

previously for analogous mutations of histidineligands of bacteriochlorophylls forming the specialpair P and the monomer bacteriochlorophyll BB aswell as BA in Bl. viridis (Bylina and Youvan 1988;Bylina et al. 1988; Gallo 1994; Arlt et al. 1996;Katilius et al. 1999). However, the absorbancespectra and pigment composition in the HF(L153)and HL(L153) mutants are inconsistent with thereplacement of the pigments. In the case of theHF(L153) mutant, the increase in absorption ofthe BPheo QX band around 530 nm and decreaseof the BChl QX band at 600 nm, as well as thebroadening and shift of the B-band from 804 nm(in WT) to 799 nm and the increase of the H-bandabsorption at 760 nm would support the idea of apigment exchange. However, the mutation alsoresults in a roughly 30% decrease of the P-bandintensity compared to the WT RC spectrum. Thiseffect cannot simply be explained by the replace-ment of BA with BPheo. Similar changes are alsopresent in the spectrum of the HL(L153) mutant.The BChl : BPheo pigment ratio determined forthese mutant RCs by pigment extraction is alsoinconsistent with a BChl to BPheo exchange. Inthe HF(L153) mutant, the pigment ratio is about2. The P-containing fraction of the HL(L153)mutant (see absorption spectrum in Figure 1) hasa pigment ratio of about 1.6, but this fraction isnot very stable and converts to the P-less fractionwith a pigment ratio of 1.4. Due to this, the pureP-containing fraction is very hard to isolate, andits properties are affected by some contaminationwith the P-less fraction. Lower pigment ratio in theHL(L153) mutant might be explained by substoi-chiometric pigment exchange, but it might also bea result of the loss or oxidation of BChl mole-cule(s) (discussed below). In any case, pigmentratios of 1.6 or 2 are not consistent with an ex-change of BChl for BPheo.

P-less mutants

RCs isolated from the mutants HE(L153),HL(L153), HQ(L153) and HY(L153) containfractions without a P-band, as described above.Similar P-less RCs were previously isolated fromthe VR(L174) mutant in which arginine wasintroduced in the vicinity of BA and P (Jacksonet al. 1997). P-less RCs were also generated as aresult of cavity mutations, in which both histidinesligating the BChls of P were replaced with glycine

(Moore and Boxer 1996, 1998). In both cases, thedecrease in the BChl : BPheo pigment ratio wasinterpreted as due to the loss of one or both BChlmolecules forming P. However, the abovemen-tioned studies did not present the complete spectraof the RCs, prohibiting comparison of their resultsto the spectra of the mutants described here.Spectral and pigment analyses of the HE(L153),HL(L153), HQ(L153) and HY(L153) mutant RCscan be interpreted as due to either loss or irre-versible oxidation of BChl molecule(s). Possibleoxidation of the pigments is supported by theobservation of a broad band in the 400–500 nmregion as well as changes in the BChl and BPheoabsorption regions, which are best represented inthe absorption spectrum of the HQ(L153) mutant(see Figures 1 and 2). The spectra of P-less frac-tions isolated from all mutants are very similar tothe RC absorption spectrum when P is oxidized toP+, either by chemical oxidation or light inducedformation of the long lived P+ state (Fajer et al.1975; Hoff and Deisenhofer 1997). This similarityhas also been noted previously in the case of theVR(L174) mutant (Jackson et al. 1997). If theP-less fractions are really the product of P-oxida-tion, then the oxidation must be irreversible, asaddition of reducing agents does not change theirabsorption spectrum. Also, the broad absorptionincrease in 400–500 nm region in the P-less frac-tions cannot be attributed just to P+ alone, how-ever, it might indicate the oxidation of pigments ingeneral.

Naturally, the question arises, how can onedistinguish between the loss and the oxidation ofpigments? Simple spectrophotometric analysis ofthe pigment extract, as described in (Van der Restand Gingras 1974), is not reliable, as the spectra ofirreversibly oxidized pigments would very likelyalter the intensity of the extracted BChl and/orBPheo pigment bands. The spectrum of theHL(L153) P-less fraction extract contains anadditional band with a peak around 680 nm (datanot shown), which definitely increases the apparentBPheo absorbance value, therefore altering thecalculated pigment ratio. This band could be due toan oxidized BChl; however, more analysis is nec-essary to determine the validity of this assignment.

From the whole series of mutants, only thehistidine to serine mutation did not produce anyP-less fraction (variable amounts of P-less frac-tions were observed in all other mutants). These

175

results suggest that histidine ligand to the BA

molecule is an important structural factor, whichcannot be easily altered to other amino acidswithout affecting other cofactors. It is unclear howa serine side chain could ligate the Mg atom of theBA cofactor, as the distance from the oxygen atomto the Mg would be too long for a direct interac-tion if the protein backbone retained the samestructure in the mutant as in the wild-type. Onepossibility is that in this mutant, as well as in othermutants, a water molecule can be employed as aligand. This has previously been suggested in thecase of a histidine to glycine mutation at M202(Goldsmith et al. 1996), however, the direct proofof that hypothesis can only be obtained from acrystal structure of the mutant RCs.

Effects of mutations on BA absorption

In WT RCs, the absorption spectra of the BA andBB molecules overlap both in the QX and QY re-gions at room temperature. At low temperatures,the QY absorption bands become resolvable, withBA absorbing at around 800 nm and BB at around810 nm (Kirmaier and Holten 1987; Breton 1988).In most of the mutants reported here, a shift of BA

absorption band to the shorter wavelengths can beclearly resolved (see Figures 1 and 2 and Table 1).In most of the mutants, changes in the QX spectralregion around 600 nm are also obvious (especiallyat 77 K), however, they are harder to attribute justto BA molecules, because P contributes to the totalabsorbance intensity in that region as well. Thereis no obvious relationship between the spectralshifts and the chemical nature of the aminoacid, which replaces the histidine ligand. Forexample, in case of the HV(L153) and HY(L153)mutations, the observed band shifts are the same,yet the chemical properties of valine and tyrosineare clearly quite different. The most intriguingspectral changes are observed in the HF(L153) andHG(L153) mutants. The spectrum of theHF(L153) mutant shows that the QY absorbanceband of BA is most likely heterogeneous, with amajor peak at 799 nm and an additional band ataround 790 nm. On the other hand, the spectrumof the HG(L153) reaction centers reveals a 10-nmblue-shift of the BA absorbance to 789 nm, withclear separation of the BA and BB absorbancebands. It is possible only to speculate what exactly

causes these significant spectral changes in themutants of histidine L153. As suggested above,there is a possibility that the actual ligand of theBA molecule in many of the mutat RCs is a watermolecule. However, without obtaining soundstructural evidence, it is difficult to say anythingmore definitive.

Photochemistry in the mutated RCs

In WT RCs it has been determined that electrontransfer from the state P+B�

A to the state P+H�A is

faster than the initial electron transfer from P* toP+B�

A (Holzapfel et al. 1989, 1990; Arlt et al. 1993;Sporlein et al. 2000), making the state P+B�

A veryhard to observe experimentally. In the HF(L153),HG(L153), HS(L153), HV(L153) and HY(L153)mutants, the state P+B�

A was not resolved experi-mentally. The increase in the lifetime of P* decayin these mutants can be interpreted as due toslower primary electron transfer from P* to P+B�

A

compared to WT RCs. It can also be inferred thatthe secondary electron transfer from P+B�

A toP+H�

A does not change in these mutants comparedto WT RCs, or at least remains faster than theinitial electron transfer from P* to P+B�

A.In the HD(L153) and HE(L153) mutants, P*

decay is also much slower than in WT RCs, as itwas in the mutants described above. However, theP* decay is heterogeneous, and at least twoexponential functions are necessary to fit the data.Previously, heterogeneous decay of P* has beendetected in various mutants and even in WT RCs(Kirmaier and Holten 1990, 1991; Chan et al.1991; Du et al. 1992; Muller et al. 1992; Jia et al.1993; Nagarajan et al. 1993; Woodbury et al. 1994;Arlt et al. 1996; Holzwarth and Muller 1996; Linet al. 1996a). Several different explanations havebeen suggested for this heterogeneity. It has beenascribed to the distribution in energy of thecharge-separated states P+B�

A or P+H�A (Kirmaier

and Holten 1990, 1991; Muller et al. 1992), to therelaxation of the radical pair (Nagarajan et al.1993; Woodbury et al. 1994; Lin et al. 1996b), orto the increased kinetic complexity that arises fromconsidering reversible electron transfer reactions(Holzwarth and Muller 1996). The HE(L153)mutant was previously created in Bl. viridis, and inthis mutant P* stimulated emission decay was alsodescribed with two exponential decays. In this

176

case, the slower, heterogeneous decay was attrib-uted to an increase of the free energy of the stateP+B�

A above P* (Arlt et al. 1996).In addition to the possible explanations for

heterogeneity presented above, another possibilityis that the heterogeneous P* decay in theHD(L153) and HE(L153) mutants arises frommultiple subpopulations of RCs, possibly due tothe presence of both protonated and deprotonatedversions of the introduced acidic residues. In thiscase, the two different lifetimes would describe twodistinct RC populations. The first subpopulation,with the shorter lifetime, 9 ps (HE(L153)) or 18 ps(HD(L153)), would show the normal, althoughsomewhat slower electron transfer from P* toP+B�

A. The second subpopulation would show animpaired electron transfer process that must com-pete with the intrinsic P* decay to the ground stateof about 200 ps (Breton et al. 1990), because thespectral properties of the second, 65 ps (HE(L153))or 120 ps (HD(L153)) component show a recoveryof P-ground-state bleaching on this timescale.

Alternatively, the heterogeneous P* decay andrecovery of the P-ground-state bleaching in theHE(L153) and HD(L153) mutants could be dueto an increase in the free energy of the state P+B�

A

in these mutants compared to WT RCs. A changein the free energy of P+B�

A could also result in analtered rate of further charge separation toP+H�

A. In this model, the first component, withthe lifetime of 9 ps in HE(L153) and 18 ps inHD(L153) (Figure 4), would represent the ap-proach to equilibrium of the initial electrontransfer from P* to P+B�

A. The second compo-nent, on the timescale of 60 ps in case ofHE(L153) and 120 ps in case of HD(L153), couldrepresent the decay of the equilibration betweenthe states P* and P+B�

A involving electron trans-fer from P+B�

A to P+H�A in competition with

decay of P* to the ground state, and, possibly,P+B�

A recombination to the ground state. In thismodel, the relative yield of the each of theseprocesses is dependent on the energetics of P* andthe charge-separated states.

Energetics of the first electron transfer stepin the mutants

If the free energy of P+B�A is substantially in-

creased in these mutants relative to P*, it may be

possible to model the decrease in rate constant interms of an increased activation energy for overallelectron transfer in these mutants. Electrontransfer activation energies in the RC can bemodeled using Marcus theory (Bixon et al. 1995;Zinth et al. 1998; Haffa et al. 2002; Huppmannet al. 2002). The rate constant according to thistheory is determined by the free energy differencebetween the initial and product states:

k ¼ 2p�h

V 2ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi4pkkBT

p exp �ðDGþ kÞ2

4kkBT

" #; ð1Þ

where V is the electronic coupling matrix element,DG is the free energy difference between the initialand product states, k is the reorganization energy,kB is Boltzmann constant, and T is temperature(Marcus and Sutin 1985). The free energy differ-ence and the reorganization energy determine theactivation energy Ea for the electron transferprocess. Equation (1) can be rewritten:

k ¼ 2p�h

V 2ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi4pkkBT

p exp � Ea

kBT

� �;

where Ea ¼ðDGþ kÞ2

4k:

ð2Þ

Assuming that the mutations did not alter thereorganization energy and electronic couplingmatrix element, the difference between the activa-tion energies in the various mutants relative to theWT could be calculated using the equation:

Ea;mut � Ea;WT ¼ �kBT lnkmut

kWT

� �: ð3Þ

The activation energy differences were calculatedfor all mutants taking the lifetime of the firstcomponent as the inverse of the initial electrontransfer rate constant. The calculated differencesare presented in Table 3. One can see that thelargest changes in the activation energy are ob-served in the HE(L153) and HD(L153) mutants; inthe latter the increase is almost 50 meV. However,the increase in activation energy does not allowone to estimate the free energy of the state P+B�

A,as the activation energy depends on both thereaction free energy change and the reorganizationenergy (see Equation (2)). Nevertheless, assumingthat the reorganization energy did not change inthe mutants, one can estimate the free energy ofthe state P+B�

A relative to the free energy of P*.

177

The reorganization energy for the initial electrontransfer reaction has previously been estimated atabout 800 cm)1 (Bixon et al. 1995). Then we cancalculate that DG0(P* ) P+B�

A) for the HD(L153)mutant is about 270 cm)1 or 35 meV above P*.That corresponds to nearly 100 meV increase infree energy compared to wild-type, as the free en-ergy of the state P+B�

A has been determined to bearound 50–70 meV below P* (Arlt et al. 1993;Bixon et al. 1995; Holzwarth and Muller 1996;Nowak et al. 1998; Shuvalov and Yakovlev 1998).A similar magnitude effect was also suggested inthe case of the GD(M203) mutant, in which thenegatively charged aspartic acid residue is placedclose to the ring E of the BA molecule (Heller et al.1995; Heller et al. 1996). This large shift in the freeenergy would explain the significant decrease inthe electron transfer rate and yield in theHE(L153) or HD(L153) mutants. Smaller changesin the free energy of the state P+B�

A may helpexplain the slower initial electron transfer reac-tions as observed in all other mutants. However,we cannot also exclude the possibility that muta-tions of the BA ligand cause changes in the elec-tronic coupling between P and BA, which wassuggested from the temperature dependence studyof primary electron transfer reaction in Bl. viridis(Huppmann et al. 2002).

Acknowledgements

This research was supported by NSF GrantsMCB9817388 and MCB0131766. The transientspectrometer used was funded by the NSF GrantBIR9512970. This is publication no. 582 from theCenter for the Study of Early Events in Photo-synthesis.

References

Allen JP, Feher G, Yeates TO, Komiya H and Rees DC

(1987) Structure of the reaction center from Rhodobacter

sphaeroides R-26: the cofactors. Proc Natl Acad Sci USA 84:

5730–5734

Arlt T, Schmidt S, Kaiser W, Lauterwasser C, Meyer M, Scheer

H and Zinth W (1993) The accessory bacteriochlorophyll: a

real electron carrier in primary photosynthesis. Proc Natl

Acad Sci USA 90: 11757–11761

Arlt T, Dohse B, Schmidt S, Wachtveitl J, Laussermair E, Zinth

W and Oesterhelt D (1996) Electron transfer dynamics of

Rhodopseudomonas viridis reaction centers with a modified

binding site for the accessory bacteriochlorophyll. Biochem-

istry 35: 9235–9244

Bixon M, Jortner J and Michel-Beyerle ME (1995) A kinetic

analysis of the primary charge separation in bacterial

photosynthesis. Energy gaps and static heterogeneity. Chem

Phys 197: 389–404

Breton J (1988) Low temperature linear dichroism study of the

orientation of the pigments in reduced and oxidized reaction

centers of Rps. viridis and Rb. sphaeroides. In: Breton J and

Vermeglio A (eds) The Photosynthetic Bacterial Reaction

Center: Structure and Dynamics, pp 59–69. Plenum Press,

New York

Breton J, Martin J-L, Lambry J-C, Robles SJ and Youvan DC

(1990) Ground state and femtosecond transient absorption

spectroscopy of a mutant of Rhodobacter capsulatus which

lacks the initial electron acceptor bacteriopheophytin. In:

Michel-Beyerle M-E (ed) Reaction Centers of Photosynthetic

Bacteria, pp 293–302. Springer-Verlag, Berlin

Bylina EJ and Youvan DC (1988) Directed mutations affecting

spectroscopic and electron transfer properties of the primary

donor in the photosynthetic reaction center. Proc Natl Acad

Sci USA 85: 7226–7230

Bylina EJ, Kirmaier C, McDowell L, Holten D and Youvan

DC (1988) Influence of an amino-acid residue on the optical

properties and electron transfer dynamics of a photosynthetic

reaction centre complex. Nature 336: 182–184

Bylina EJ, Kolaczkowski SV, Norris JR and Youvan DC

(1990) EPR characterization of genetically modified reaction

centers of Rhodobacter capsulatus. Biochemistry 29: 6203–

6210

Camara-Artigas A, Magee C, Goetsch A and Allen JP (2002)

The structure of the heterodimer reaction center from

Rhodobacter sphaeroides at 2.55 angstrom resolution. Photo-

synth Res 74: 87–93

Chan C-K, Chen LX-Q, DiMagno TJ, Hanson DK, Nance SL,

Schiffer M, Norris JR and R. FG (1991) Initial electron

Table 3. Results of the free energy calculations for mutated

RCs

Sample sP*, psa DEa, meVb DG(P*)P+B�

A), meVc

HD(L153) 18 45 35

HE(L153) 9 27 5

HF(L153) 6 17 )18

HG(L153) 6.8 20 )11

HS(L153) 5.8 16 )20

HV(L153) 4.5 9.5 )38

HY(L153) 4.5 9.5 )38

WT 3 – )45

aOnly the shortest lifetimes were considered in case of

HD(L153) and HE(L153) mutants.bThe changes in the activation energy compared to WT RCs

were calculated using the Equation (3).cFree energy gaps between the states P* and P+B�

A were

calculated assuming activationless P* to P+B�A electron transfer

in WT RCs. Taking into account the small activation energy,

4–5 meV as can be calculated from the values of reorganization

energy and the free energy gap from (Bixon et al. 1995),

increases the values for DG(P*)P+B�A) by about 5–10 meV.

178

transfer in photosynthetic reaction centers of Rhodobacter

capsulatus mutants. Chem Phys Lett 176: 366–372

Deisenhofer J, Epp O, Miki K, Huber R and Michel H (1984)

X-ray structure analysis of a membrane protein complex.

Electron density map at 3 A resolution and a model of the

chromophores of the photosynthetic reaction center from

Rhodopseudomonas viridis. J Mol Biol 180: 385–398

Du M, Rosenthal SJ, Xie X, DiMagno TJ, Schmidt M,

Hanson DK, Schiffer M, Norris JR and Fleming GR (1992)

Femtosecond spontaneous-emission studies of reaction cen-

ters from photosynthetic bacteria. Proc Natl Acad Sci USA

89: 8517–8521

El-Kabbani O, Chang C-H, Tiede D, Norris J and Schiffer M

(1991) Comparison of reaction centers from Rhodobacter

sphaeroides and Rhodopseudomonas viridis: overall architec-

ture and protein–pigment interactions. Biochemistry 30:

5361–5369

Ermler U, Fritzsch G, Buchanan SK and Michel H (1994)

Structure of the photosynthetic reaction centre from Rho-

dobacter sphaeroides at 2.65 A resolution: cofactors and

protein-cofactor interactions. Structure 2: 925–936

Fajer J, Brune DC, Davis MS, Forman A and Spaulding LD

(1975) Primary charge separation in bacterial photosynthesis:

oxidized chlorophylls and reduced pheophytins. Proc Natl

Acad Sci USA 72: 4956–4960

Gallo Jr DM (1994) Chimeric mutagenesis of the Rb. capsulatus

reaction center: an exploration of the structure/function

relationship. PhD Thesis. Arizona State University, Tempe,

Arizona

Goldsmith JO, King B and Boxer SG (1996) Mg coordination

by amino acid side chains is not required for assembly and

function of the special pair in bacterial photosynthetic

reaction centers. Biochemistry 35: 2421–2428

Haffa ALM, Lin S, Katilius E, Williams JC, Taguchi AKW,

Allen JP and Woodbury NW (2002) The dependence of

the initial electron transfer rate on driving force in Rhodob-

acter sphaeroides reaction centers. J Phys Chem B 106: 7376–

7384

Haffa ALM, Lin S, Williams JC, Taguchi AKW, Allen JP and

Woodbury NW (2003) High yield of long-lived B-side charge

separation at room temperature in mutant bacterial reaction

centers. J Phys Chem B 107: 12503–12510

Haffa ALM, Lin S, Williams JC, Bowen BP, Taguchi AKW,

Allen JP and Woodbury NW (2004) Controlling the pathway

of photosynthetic charge separation in bacterial reaction

centers. J Phys Chem B 108: 4–7

Heller BA, Holten D and Kirmaier C (1995) Control of electron

transfer between the L- and M-sides of photosynthetic

reaction centers. Science 269: 940–945

Heller BA, Holten D and Kirmaier C (1996) Effects of Asp

residues near the L-side pigments in bacterial reaction

centers. Biochemistry 35: 15418–15427

Hoff AJ and Deisenhofer J (1997) Photophysics of photosyn-

thesis. Structure and spectroscopy of reaction centers of

purple bacteria. Phys Rep 287: 1–247

Holzapfel W, Finkele U, Kaiser W, Oesterhelt D, Scheer H,

Stilz HU and Zinth W (1989) Observation of a bacteriochlo-

rophyll anion radical during the primary charge separation in

a reaction center. Chem Phys Lett 160: 1–7

Holzapfel W, Finkele U, Kaiser W, Oesterhelt D, Scheer H,

Stilz HU and Zinth W (1990) Initial electron-transfer in the

reaction center from Rhodobacter sphaeroides. Proc Natl

Acad Sci USA 87: 5168–5172

Holzwarth AR and Muller MG (1996) Energetics and kinetics

of radical pairs in reaction centers from Rhodobacter

sphaeroides. A femtosecond transient absorption study.

Biochemistry 35: 11820–11831

Huppmann P, Arlt T, Penzkofer H, Schmidt S, Bibikova M,

Dohse B, Oesterhelt D, Wachtveitl J and Zinth W (2002)

Kinetics, energetics, and electronic coupling of the primary

electron transfer reactions in mutated reaction centers of

Blastochloris viridis. Biophys J 82:3186–3197

Jackson JA, Lin S, Taguchi AKW, Williams JC, Allen JP and

Woodbury NW (1997) Energy transfer in Rhodobacter

sphaeroides reaction centers with the initial electron donor

oxidized or missing. J Phys Chem 101: 5747–5754

Jia Y, DiMagno TJ, Chan C-K, Wang Z, Du M, Hanson

DK, Schiffer M, Norris JR, Fleming GR and Popov

MS (1993) Primary charge separation in mutant reaction

centers of Rhodobacter capsulatus. J Phys Chem 97: 13180–

13191

Katilius E, Turanchik T, Lin S, Taguchi AKW and Woodbury

NW (1999) B-side electron transfer in a Rhodobacter

sphaeroides reaction center mutant in which the B-side

monomer bacteriochlorophyll is replaced with bacteriopheo-

phytin. J Phys Chem B 103: 7386–7389

Katilius E, Katiliene Z, Lin S, Taguchi AKW and Woodbury

NW (2002) B-side electron transfer in a Rhodobacter

sphaeroides reaction center mutant in which the B-side

monomer bacteriochlorophyll is replaced with bacteriopheo-

phytin: low temperature study and energetics of charge

separated states. J Phys Chem B 106: 1471–1475

Kellogg EC, Kolaczkowski S, Wasielewski MR and Tiede

DM (1989) Measurement of the extent of electron transfer

to the bacteriopheophytin in theM-subunit in reaction centers

of Rhodopseudomonas viridis. Photosynth Res 22: 47–59

Kirmaier C and Holten D (1987) Primary photochemistry of

reaction centers from the photosynthetic purple bacteria.

Photosynth Res 13: 225–260

Kirmaier C and Holten D (1990) Evidence that a distribution of

bacterial reaction centers underlies the temperature and

detection-wavelength dependence of the rates of the primary

electron-transfer reactions. Proc Natl Acad Sci USA 87:

3552–3556

Kirmaier C and Holten D (1991) An assessment of the

mechanism of initial electron transfer in bacterial reaction

centers. Biochemistry 30: 609–613

Kirmaier C, Holten D and Parson WW (1985a) Picosecond-

photodichroism studies of the transient states in Rhodo-

pseudomonas sphaeroides reaction centers at 5 K: effects of

electron transfer on the six bacteriochlorin pigments. Bio-

chim Biophys Acta 810: 49–61

Kirmaier C, Holten D and Parson WW (1985b) Temperature

and detection-wavelength dependence of the picosecond

electron-transfer kinetics measured in Rhodopseudomonas

sphaeroides reaction centers. Resolution of new spectral and

kinetic components in the primary charge-separation process.

Biochim Biophys Acta 810: 33–48

Kirmaier C, Holten D, Bylina EJ and Youvan DC (1988)

Electron transfer in a genetically modified bacterial reaction

center containing a heterodimer. Proc Natl Acad Sci USA 85:

7562–7566

179

Lin S, Lin X, Williams JC, Taguchi AKW, Allen JP and

Woodbury NW (1996a) Reaction center heterogeneity

probed by multipulse photoselection experiments with pico-

second time resolution. In: Michel-Beyerle ME (ed) The

Reaction Center of Photosynthetic Bacteria. Structure and

Dynamics, pp 217–223. Springer-Verlag, Berlin

Lin S, Taguchi AKW and Woodbury NW (1996b) Excitation

wavelength dependence of energy transfer and charge sepa-

ration in reaction centers from Rhodobacter sphaeroides:

evidence for adiabatic electron transfer. J Phys Chem 100:

17067–17078

Lin S, Katilius E, Haffa ALM, Taguchi AKW and Woodbury

NW (2001) Blue light drives B-side electron transfer in

bacterial photosynthetic reaction centers. Biochemistry 40:

13767–13773

Lin X, Williams JC, Allen JP and Mathis P (1994) Relationship

between rate and free energy difference for electron transfer

from cytochrome c2 to the reaction center in Rhodobacter

sphaeroides. Biochemistry 33: 13517–13523

Marcus RA and Sutin N (1985) Electron transfers in chemistry

and biology. Biochim Biophys Acta 811: 265–322

Moore LJ and Boxer SG (1996) Cavity mutants involving

residue L168 near the special pair dimer in reaction centers of

Rb. sphaeroides. Biophys J 70: A142

Moore LJ and Boxer SG (1998) Inter-chromophore interactions

in pigment-modified and dimer-less bacterial photosynthetic

reaction centers. Photosynth Res 55: 173–180

Muller MG, Griebenow K and Holzwarth AR (1992) Primary

processes in isolated bacterial reaction centers from Rhodob-

acter sphaeroides studied by picosecond fluorescence kinetics.

Chem Phys Lett 199: 465–469

Nagarajan V, Parson WW, Davis D and Schenck CC (1993)

Kinetics and free energy gaps of electron-transfer reactions in

Rhodobacter sphaeroides reaction centers. Biochemistry 32:

12324–12336

Nowak FR, Kennis JTM, Franken EM, Shkurupatov AY,

Yakovlev A, Gast P, Hoff AJ, Aartsma TJ and Shuvalov VA

(1998) The energy level of P+B�A in plant pheophytin-

exchanged bacterial reaction centers probed by the temper-

ature dependence of delayed fluorescence. In: Garab G (ed)

Photosynthesis: Mechanisms and Effects, pp 783–786. Klu-

wer Academic Publishers, Dordrecht, The Netherlands

Parson WW (1996) Photosynthetic bacterial reaction centres.

In: Bendall S. D. (ed) Protein Electron Transfer, pp 125–160.

BIOS Scientific Publishers, Oxford

Peloquin JM, Williams JC, Lin X, Alden RG, Murchison HA,

Taguchi AKW, Allen JP and Woodbury NW (1994) Time-

dependent thermodynamics during early electron transfer in

reaction centers from Rhodobacter sphaeroides. Biochemistry

33: 8089–8100

Shuvalov VA and Yakovlev AG (1998) Energy level of P+B)

with respect to P* found from recombination fluorescence

measurements in pheophytin-modified reaction centres.

Membr Cell Biol 12: 563–569

Sporlein S, Zinth W, Meyer M, Scheer H and Wachtveitl J

(2000) Primary electron transfer in modified bacterial reac-

tion centers: optimization of the first events in photosynthe-

sis. Chem Phys Lett 322: 454–464

van Brederode ME and Jones MR (2000) Reaction centres of

purple bacteria. Subcell Biochem 35: 621–676

Van der Rest M and Gingras G (1974) The pigment comple-

ment of the photosynthetic reaction center isolated from

Rhodospirillum rubrum. J Biol Chem 249: 6446–6453

Woodbury NW, Peloquin JM, Alden RG, Lin X, Lin S,

Taguchi AKW, Williams JC and Allen JP (1994) Rela-

tionship between thermodynamics and mechanism dur-

ing photoinduced charge separation in reaction

centers from Rhodobacter sphaeroides. Biochemistry 33:

8101–8112

Zinth W, Huppmann P, Arlt T and Wachtveitl J (1998)

Ultrafast spectroscopy of the electron transfer in photosyn-

thetic reaction centres: towards a better understanding of

electron transfer in biological systems. Phil Trans R Soc

London A 356: 465–476

180