Photosynthesis Research 81: 165180, 2004. 2004 Kluwer Academic Publishers. Printed in the Netherlands.

165

Regular paper

Electron transfer dynamics in Rhodobacter sphaeroides reaction center mutants with a modied ligand for the monomer bacteriochlorophyll on the active side
Evaldas Katilius1,*, Jennie L. Babendure1,2, Su Lin1 & Neal W. Woodbury1
Department of Chemistry and Biochemistry, Arizona Biodesign Institute and the Center for the Study of Early 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
1

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 photosynthetic 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 were created to investigate how the alteration of the ligand residue aects the properties of the BA molecule and changes the dynamics of the primary charge separation in reaction centers. In all of the mutants, the changes in the ligand result in a blue-shift of the BA absorption spectrum, in both visible and near-infrared spectral regions, with the size of the shift varying between mutants. The primary electron transfer time constants in these mutant reaction centers range from 4.5 to 18 ps, being substantially slower than the wildtype value of 3 ps. The decrease in the electron transfer rate is interpreted as being due to an increase in the free 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 quinone molecules 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 photosynthesis occurs in a pigmentprotein complex called the reaction center (RC), where light energy drives electron transfer across the photosynthetic membrane. The reaction center from purple nonsulfur photosynthetic bacteria is probably the beststudied example (for reviews see Parson 1996; Ho and Deisenhofer 1997; van Brederode and

Jones 2000). The RC from Rhodobacter (Rb.) sphaeroides consists of 3 protein subunits L, M and H, which coordinate 10 cofactors: 4 bacteriochlorophyll (BChl), 2 bacteriopheophytin (BPheo), 2 ubiquinone molecules, a carotenoid molecule and a nonheme iron. The BChl, BPheo and quinone cofactors are arranged in two, nearly C2 symmetrical branches. Because of this symmetry, there are two potential pathways for photosynthetic electron transfer (usually labeled A and B),

166 both starting with the initial electron donor, P (a pair of bacteriochlorophylls), and then proceeding through a monomer bacteriochlorophyll (either BA or BB) and a bacteriopheophytin (either HA or HB) to a ubiquinone (either QA or QB). Since the determination of the reaction center crystal structure (Deisenhofer et al. 1984; Allen et al. 1987), the function of the monomer bacteriochlorophylls has been the target of many studies. The earliest kinetic studies revealed electron transfer from the initial electron donor P to the bacteriopheophytin acceptor on the active branch of the cofactors (HA) (Kirmaier et al. 1985a, b). It was proposed initially that the monomer BChl molecule (BA), which is situated between P and HA, acts primarily as a bridge in a superexchange electron transfer process between P and HA. Later, the transient population of the chargeseparated state P+B was detected (Holzapfel et al. 1989; A Arlt et al. 1993, 1990), which led to the conclusion that this state is a real intermediate in the electron transfer process. The crystal structure of the Rb. sphaeroides RC shows that monomer BChl molecules, BA and BB, are ligated by histidines L153 and M182, respectively (Allen et al. 1987; El-Kabbani et al. 1991; Ermler et al. 1994). One of the early site-directed mutagenesis studies of Rb. capsulatus RCs has shown that these histidines can be replaced by leucine (L), serine (S) or threonine (T) and still result in functional reaction centers, while mutation of the histidine M182 to arginine (R) led to a photosynthetically incompetent mutant (Bylina et al. 1990). Later, the histidine to leucine mutation at the position M182 was again created in Rb. capsulatus and Rb. sphaeroides RCs (Gallo 1994; Katilius et al. 1999). It was determined that the BB molecule in this mutant was replaced with a bacteriopheophytin. The exchange of the pigments also caused an alteration of RC photochemical properties, as electron transfer to the normally inactive B-side was observed (Katilius et al. 1999, 2002). Histidine at the position L153 has been mutated to cysteine (C), glutamic acid (E) and leucine (L) in reaction centers from Blastochloris (Bl.) viridis (Arlt et al. 1996). Changing histidine to cysteine did not signicantly alter the RC properties, while mutation to glutamic acid caused a roughly 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 signicant changes in the electron transfer dynamics, as the energetics of the charge separated states were aected by the pigment exchange (Arlt et al. 1996). In this report, we describe a series of mutations of histidine L153 in the Rb. sphaeroides RC. Nine mutations were constructed; histidine (H) was replaced with aspartic acid (D), glutamic acid (E), glutamine (Q), glycine (G), leucine (L), phenylalanine (F), serine (S), tyrosine (Y) and valine (V) (the mutants are referred to as HX(L153), where X is any of the above nine amino acids). The main goal of this study is to characterize how the nature of the ligand aects the spectral and electrochemical properties of the BA molecule in Rb. sphaeroides RCs and, in turn, how these properties aect the electron transfer reactions. The variety of different amino acids inserted at L153 should also provide more information concerning how the energetics of the initial charge separated state P+B aects the electron transfer dynamics in the A reaction centers of purple bacteria.

Materials and methods Mutations were created using the Chameleon or QuikChange mutagenesis kits (Stratagene, La Jolla, California). The mutagenesis reactions were performed on the pUCAH plasmid, which was made up of pUC19 with the Asp718I-HindIII fragment of the L-subunit gene. The mutagenesis reactions were performed according to the kit manuals, and the mutations were veried by DNA sequencing of the whole L-subunit gene fragment. For each mutation, the Asp718I-HindIII fragment of pUCAH was then subcloned into the plasmid pRKSCH7H, which contained the rest of the L-subunit and subunit genes and included seven CAC codons at the end of the M-subunit gene encoding a hepta-histidine tag. Those plasmids were then transferred by conjugation into the Rb. sphaeroides pufLM deletion strain DLM1.1, which lacked the reaction center gene, and cells were grown under semiaerobic conditions (Lin et al. 1994). Cells were harvested by centrifugation at 10,000 g, the pellet was washed and resuspended in 10 mM Tris-HCl (pH 8) buer. Cells were then broken using a french pressure cell at 1800020000 psi, and the lysate was incubated with DNAseI

167 (Sigma, St. Louis, Missouri) and 100 lM Ca2+ and Mg2+ for 15 min to digest chromosomal DNA. This lysate was centrifuged at 16000 g for 10 min to separate cellular debris and unbroken cells from soluble photosynthetic membranes. Chromatophores were incubated at 26 C in the presence of 0.65% LDAO detergent for about 15 20 min. The solubilized RCs were then centrifuged at 32000 g for 30 min to separate them from unsolubilized membranes. To improve the spe-cic binding of the solubilized RCs to the metal-chelating Ni-NTA column (Qiagen, Valencia, California), 150 mM NaCl was added to the solution before loading it on the Ni-NTA column as well as to the washing buer. This improved the specic binding of the poly-His tagged RCs to the column and resulted in faster purication of the RCs. Imidazole was omitted from the washing buer during isolation of the mutant RCs and the RC elution from the Ni-NTA column was performed with a buer containing 50 mM EDTA instead of 50 mM imidazole, because addition of imidazole previously showed variability in preparations of RCs from the HL(M182) mutant (Katilius et al. 2002). Eluted RCs were dialyzed overnight and additionally puried via ion exchange chromatography on Toyopearl DEAE650M (diethylaminoethyl) column (Supelco, St. Louis, Missouri) (Lin et al. 1994). This altered procedure was previously shown to yield samples of the poly-His tagged HL(M182) mutant with the same pigment ratio as measured in the isolated HL(M182) mutant RCs without the poly-His tag (Katilius et al. 2002). However, additional purication using an ion exchange column revealed signicant heterogeneity in some of the mutant RC preparations. For most of the mutants, two reaction center-containing fractions were collected from this column. The rst fraction usually showed very little or no absorbance at around 865 nm; the second reaction center fraction, which eluted o the column a few minutes later, had a P-band absorbance at around 865 nm, although its amplitude was usually reduced compared to the WT RC spectrum. The spectra of the P-containing fractions are presented in Figures 1 and 2 for all mutants, except for HQ(L153), in which a reaction center fraction containing the P band was not isolated. The stability of the P-containing and P-less fractions was assessed by keeping the samples at room temperature for several days and measuring the absorption spectra every several hours. Under these conditions, P-containing fractions showed a decrease in the P absorbance occurring within a few days, which indicated that degradation of the protein was occurring on long time scales. However, no signicant spectral changes were observed after performing transient absorbance measurements, which usually took several hours (spectra of the samples were measured before and after the measurements, results not shown). No signicant degradation of the Pcontaining fractions of the mutant RCs was observed upon exchange of the LDAO detergent for Triton X-100, which was done on an ion exchange column according to a previously published protocol (Peloquin et al. 1994). Investigation of P-less fractions showed that their spectra did not change over the course of several days at room temperature, which would indicate no further degradation of the protein. To test for possible oxidation of P (which could give rise to the apparently P-less fractions and to the decreased intensity of the P-band in some of the P-containing fractions), up to 50 mM of sodium ascorbate was added to the samples, however, no recovery of the P-band was observed. Pigment extractions of the P-containing fractions as well as P-less fractions from HL(L153), HQ(L153) and HY(L153) mutants were performed in 7:2 (v/v) acetone methanol mixture as previously described in (Van der Rest and Gingras 1974). The experimental setup for the transient absorbance measurements has been described previously (Katilius et al. 1999, 2002). For these measurements, RCs were suspended in either 15 mM TrisHCl (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 (reduced samples). The addition of 100 lM ortho-phenanthroline was used to block QA to QB electron transfer in the unreduced samples. In order to block the electron transfer from HA to QA, 1 mM sodium dithionite was used to chemically reduce QA. Measurements of RCs with intact QA were performed in a rotating circular cell with a 2.5 mm optical path containing about 4 mls of sample. The cell rotation allowed complete sample removal from the excitation region between laser pulses. The samples with reduced QA were measured in an airtight, 2-mm pathlength glass cuvette with constant stirring. All measurements were performed at

168 Results Absorption spectra at room temperature Absorption spectra of all mutants at room temperature are shown in Figure 1 (the absorbance maxima of P and the monomer BChls are summarized in Table 1). The spectra of most of the mutants are quite dierent from the WT RC spectrum with the exception of the mutant HS(L153). Despite the replacement of histidine with the much smaller residue serine, the absorption spectrum of HS(L153) mutant RCs is almost identical to the absorption spectrum of WT RCs. In the spectra of other mutants several major changes are observed. First, the amplitude of the P-band at 865 nm signicantly decreased relative to 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 the amplitude of the P-band becomes extreme, as it almost totally disappears. The decrease of the P-band is usually accompanied by a relative increase in the H-band at around 760 nm (normalizing to the peak absorbance near 800 nm), when compared to the WT spectrum. The extent of the P-band decrease is in general not correlated to the nature of the new ligand. The largest decrease is observed in the mutants in which the histidine is replaced with glutamate, glutamine or leucine. While one might conclude from this that the size of the residue is an important factor, mutating histidine to either tyrosine or phenylalanine results in a similar decrease in P-band oscillator strength as is observed in the histidine to glycine or valine mutants. The maximum of the B-band in all of the mutants except HS(L153) shifts from 804 nm in WT to about 799 nm. The shape of this band becomes clearly asymmetric; in the HF(L153), HG(L153), HV(L153) or HY(L153) mutants, a shoulder at around 810 nm is resolved. Shifts and splitting of the QX BChl absorption band are also evident in several mutants. For example, in the HF(L153) mutant, the QX BChl band blue-shifts by about 2 nm. In other mutants the shift is even larger, from 598 nm in WT to 592 nm in HG(L153) and even to 587 nm in the HD(L153) mutant. In the case of the HV(L153) mutant, there is an obvious splitting of the QX BChl band with two bands appearing at about 576 and 598 nm. In general, it

Figure 1. Absorption spectra of reaction centers from the mutants HD(L153), HE(L153), HF(L153), HG(L153), HL(L153), HQ(L153), HS(L153), HV(L153), HY(L153) and WT RCs at room temperature.

room temperature. The OD of the samples was about 1.2 at 800 nm. Measurements for all mutants with the exception of HD(L153) were performed with both unreduced and reduced samples. In the case of the HD(L153) mutant, insucient amounts of the P-containing fraction to perform measurements with unreduced QA were obtained, thus only measurements with reduced QA were performed. The data analysis was performed using a locally written data analysis program ASUFIT based on MATLAB software (Mathworks).

169
Table 1. Positions of the P and monomer BChl QY and QX absorption bands at 295 and 77 K Sample 295 K P, nm HD(L153) HE(L153) HF(L153) HG(L153) HL(L153) HQ(L153) HS(L153) HV(L153) HY(L153) WT ND = not determined. 864 862 866 865 860 853 866 866 863 865 BChl QY,nm 799 799 799 798 799 797 804 800 801 804 BChl QX, nm 587 589 596 592 586 589 597 576, 598 598 598 77 K P, nm 891 887 876 892 ND 870 873 890 891 888 BChl QY, nm 798 800 799 789, 812 ND 799 802 798 799 803 BChl QX, nm 581, 603 583, 602 585, 598 585, 602 ND 588 593 579, 601 580, 601 598

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 eects of the mutations on the QX spectral band of BA are harder to unambiguously determine, because the absorbance of P in the mutants is also signicantly altered and P contributes to the absorbance in the 600 nm region. As mentioned above, the decrease of the P-band observed in most of the mutants is accompanied by an increase in the BPheo absorption intensity at around 760 nm. The increase in the BPheo absorption is also evident in the QX transition region around 530540 nm. The intensity of absorption at 530 nm in most of the mutants increases signicantly compared to the spectra of the HS(L153) mutant or WT RCs. It is not clear whether this increase in absorption intensity at around 530 nm is due to an increased amount of BPheo in the samples, or is associated with the absorption increase in the 400500 nm region, which aects the total absorption intensity in the BPheo QX absorbance region. It should also be noted, that in the HQ(L153) mutant a broad absorption band with a maximum at around 445 nm can be resolved (see Figure 1). The origin of this band will be discussed below.

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

and Table 1). In this case, several general trends are again noticeable. In addition to changes of the P-band amplitude relative to the other two bands in the near infrared, at low temperature, changes in the P-band position are also evident. For example, in the HS(L153) mutant at 77 K the P-band is blue shifted to 873 from 888 nm in WT. A smaller blue-shift is also observed in the HF(L153) mutant, while in most of the other mutants there is a small (23 nm) red-shift of the P-band (see Table 1). It is also notable that in the HQ(L153) mutant, in which the P-band was practically absent at room temperature, at low temperature the P-band is clearly resolved, though it has a decreased oscillator strength relative to wild-type, and its peak position is blue shifted to 870 nm. Signicant changes are also visible in the structure of the QY absorbance band of the monomer BChls in many of the mutants. While the position and width of the band around 800 nm were quite similar in the HS(L153) and WT spectra at room temperature, at low temperature this band is less intense at the peak and somewhat broader in HS(L153) than in WT. Due to the band broadening, the shoulder on the longer wavelength side, which in WT is usually associated with the absorbance of the BB molecule (Kirmaier and Holten 1987; Breton 1988; Ho and Deisenhofer 1997), is not as well resolved in this mutant. In contrast, this shoulder becomes very well resolved in the HV(L153) and HY(L153) mutants, as the BA absorption band peaks at 799 nm. However, the most unusual changes are observed in the HF(L153) and HG(L153) spectra. In the HF(L153)

170 In the HF(L153) and HG(L153) mutants the BChl QX transitions are clearly split into two bands at around 585 and 600 nm. In the HV(L153) and HY(L153) mutants the two bands are even more separated, with their maxima at around 580 and 600 nm. The splitting is, however, not observed in the HQ(L153) mutant, instead there is a broadening and blue-shift of the band to about 588 nm. BChl:BPheo pigment ratio analysis The results of the pigment extractions are summarized in Table 2. In most of the mutants the BChl to BPheo pigment ratio remained the same as in WT RCs (1.9 0.1). It is quite surprising that in the HF(L153) mutant the pigment ratio is the same as in WT, while an analogous mutation of either of the histidine ligands to the special pair induces the replacement of BChl with BPheo, resulting in 1:1 pigment ratio (Bylina and Youvan 1988; Kirmaier et al. 1988; CamaraArtigas et al. 2002). On the other hand, the pigment ratio in the HL(L153) mutant is lower than in WT (see Table 2), but not 1, as would be expected if the BA pigment had been replaced with a BPheo. A reduced BChl : BPheo pigment ratio is also found in the mutants in which the histidine at L153 has been mutated to glutamic acid or glutamine. Interestingly, the same reduced pigment ratio of about 1.51.6 is found for the P-less fractions isolated from several mutants (for example HL(L153) and HY(L153)). In each case (HL(L153), HE(L153), HQ(L153) and the P-less reaction center fractions of other mutants), the oscillator strength of P is greatly reduced. Possible explanations for the dierences in the pigment ratios in various mutants as well as in P-less fractions of the samples will be discussed below. Transient absorption results Photochemical properties of all mutants were characterized using transient absorption spectroscopy. Mutants which still have the P-band were excited at 860 nm, while the P-less fractions from the mutants HL(L153) and HQ(L153) were investigated by exciting them at 795 nm. The results of kinetic analyses are summarized in Table 2 and Figures 35. P* stimulated emission decay time in all mutants is slower than in WT RCs,

Figure 2. Absorption spectra of reaction centers from the mutants HD(L153), HE(L153), HF(L153), HG(L153), HQ(L153), HS(L153), HV(L153), HY(L153) and WT RCs at 77 K.

mutant, the maximum of the monomer BChl band in the QY region is at 799 nm, but in addition to the shoulder at around 809 nm, another shoulder at 790 nm can be resolved (see Figure 2). On the other hand, in the spectrum of the HG(L153) mutant, the BA and BB absorption bands at 789 and 812 nm, respectively, are clearly resolved. At 77 K, the BChl QX band is split in practically all mutants. The smallest shift is observed in the HS(L153) spectrum, where at least two transitions can be resolved peaking at around 592 and 600 nm.

171
Table 2. Properties of L153 mutants reaction centers Sample HD(L153) HE(L153) HF(L153) HG(L153) HL(L153) HL(L153) P-less fraction HQ(L153) P-less fraction HS(L153) HV(L153) HY(L153) HY(L153) P-less fraction WT ND = not determined. Pigment ratio 1.9 0.1 1.6 0.1 2 0.1 1.9 0.1 1.6 0.1 1.4 0.1 1.6 0.1 1.9 0.1 1.8 0.1 1.8 0.1 1.6 0.1 1.9 0.1 P* decay, ps 18 1; 120 5 9 1; 65 5 6 0.5 6.8 0.2 ND 5.8 0.2 4.5 0.2 4.5 0.2 3.1 0.2 P+H decay, ps A ND ND 190 10 200 10 ND 180 10 180 10 190 10 200 20

and it ranges from about 4.5 ps in the HV(L153) and HY(L153) mutants to about 18 ps in HD(L153). In the HE(L153) and HD(L153) mutants, P* decay becomes heterogeneous, and at least two exponential functions are necessary to t the kinetics (see Table 2 for lifetimes). The kinetics of secondary electron transfer from P+H to A P+Q (see Table 2) do not signicantly change in A most of the mutants compared to that in WT RCs (the rate constant of secondary electron transfer has not been determined in the HE(L153) and HD(L153) mutants). These results suggest that the mutations primarily aect the properties of P* and/or P+B and do not dramatically aect the A properties of the charge-separated states P+H or A P+Q as will be discussed further below. A Transient absorbance kinetics of the samples with neutral QA were analyzed using the sequential decay model: P* P H P Q ! A A ! Using a sequential model instead of the simple sum of exponential functions to t the data directly provides interpretation of the data according to the assumed physical model. The tting results in this case contain the rate constants (lifetimes) for electron transfer from one state to another and the species-associated decay spectra, describing the spectral properties of the dierent states. In the analysis of the data we did not include the state P+B , which is believed to be the A
k1 k2

initial charge separation state. In WT RCs it has been shown that the P+B state decays faster than A it is formed (Holzapfel et al. 1989, 1990; Arlt et al. 1993; Sporlein et al. 2000), thus the population of this state at any given time is small compared to the populations of P* or P+H . In all our invesA tigated mutants we observe that P* decay is longer than in WT RCs. At the same time no clear spectral evidence for P+B formation is observed, A indicating that the electron transfer from BA to HA must be as fast as in WT RCs, or at least much faster than the determined P* decay. The transient absorbance kinetics are well described by tting the data according to the above sequential model which leaves out an explicit P+B state. A Species-associated decay spectra (SADS) obtained from the ts of the transient absorbance kinetics of mutant and WT RCs are presented in Figure 3. In all of the mutants, the spectra corresponding to the P* state are essentially the same. In the HS(L153) mutant, SADS corresponding to the dierent charge-separated states are basically the same as in WT (data not shown, spectra are available in supplementary material). The spectral features in the SADS of the other mutants show some dierences from the WT spectra. In the case of the HF(L153) and HG(L153) mutants, there is an obvious additional bleaching of the band around 785 nm in the charge-separated states, while in the HV(L153) and HY(L153) mutants this bleaching is redshifted towards 795 nm (the SADS of HV(L153) and HY(L153) are nearly identical,

172

(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 tting 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 are presented in Table 2. The state P+Q is nondecaying on the time scale of A A measurements, its lifetime was xed at 10 ns during tting.

thus, only the SADS for the HV(L153) are shown; the spectra for HY(L153) are available in supplementary material). This bleaching obviously affects the spectra corresponding to the P+H and A P+Q states, as the positive band present in WT at A around 780 nm is reduced or contains a small dip in case of the HF(L153) mutant. The spectral feature at around 800 nm in the P+H and P+Q A A dierence spectra of WT is usually associated with the electrochromic shift of the monomer BChl ground state absorbance due to the formation of the charge-separated state P+H and, later, A P+Q . The changes in the spectral shape of this A electrochromic shift may be due to the BA ground

state absorbance changes, as observed in the ground state absorption spectra both at 77 K and at room temperature. As already mentioned above, in the HE(L153) and HD(L153) mutants, at least two exponential functions are necessary to describe the decay of P*. This indicates that there is some kind of the heterogeneity in the sample. Thus, transient absorbance kinetics in these two mutants were analyzed using a simple sum of the exponential functions, the decay-associated spectra corresponding to the dierent components are presented in the Figure 4. The kinetics of the HE(L153) mutant RCs were measured with neutral QA. P* decay in this mutant

173

(a)

(b)

Figure 4. Decay-associated spectra of HE(L153) and HD(L153) mutant RCs. The spectra were obtained from global tting of transient absorbance data measured over 1-ns time-scale at room temperature. The lifetime of the longest component was xed at 10 ns during tting.

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.

can be described by two exponential components with the lifetimes of 9 ps and 60 ps. The decayassociated spectrum of the 9-ps component contains the prominent spectral features corresponding to P* stimulated emission decay centered at about 900 nm, and very little ground state recovery of P. However, the spectrum of the 60-ps component shows both P* stimulated emission decay as well as a signicant recovery of the

P-ground-state bleaching on this time scale (see Figure 4a). The spectrum of the longest component (non-decaying on the time scale of our measurements) corresponds well to the spectrum of P+Q , A as observed in WT RCs (Figure 3). From the ratio of the initial bleaching at 865 nm and the longest component amplitude at this wavelength, it can be concluded that P+Q is formed with about 65% A yield in this mutant.

174 Primary photochemistry of the HD(L153) mutant was characterized only in the QA prereduced sample (the measurements were not performed with the intact QA due to limited amounts of sample). P* decay lifetimes in this mutant are 18 and 120 ps. The decay-associated spectrum of the 18-ps component is similar to the spectrum of the 9-ps component in the HE(L153) mutant, while the decay-associated spectrum of the second, 120ps component again shows that there is a signicant recovery of the P-ground-state bleaching on the 120-ps timescale. The spectrum of the longlived component does not correspond very well to the spectrum of the state P+H in WT RC. The A spectrum of this component, however, correlates well to the SADS of the P+H state from the A HF(L153) or HG(L153) mutant RCs. The changes in the spectrum of the long-lived component in HD(L153) might be also due to the BA ground state absorption band-shift, as observed in the ground state absorption spectrum. From the decrease of the P-bleaching intensity, it can be concluded that the state P+H in the HD(L153) A mutant is formed with approximately 60% yield. The possible reasons for the observed heterogeneous decay of P* in these two mutants will be discussed below. As mentioned above, a P-less fraction of the samples can be isolated in the mutants HQ(L153), HL(L153), HY(L153) and in smaller amounts from other mutants. Upon excitation of the P-less reaction centers isolated from the HQ(L153) mutant at 795 nm, the bleaching of the band at 799 nm decays non-exponentially, which can be best described by a sum of exponential functions with the lifetimes of 5, 200 ps and several ns. Surprisingly, the transient spectra in the visible region of the spectrum show that in addition to the bleaching of BChl QX band around 585 nm, there is also bleaching of BPheo QX band at around 530 nm, as well as a broad absorbance band at around 630640 nm (see Figure 5). The bleaching of the BPheo band is unexpected, as excitation of BChl molecules in the RC should not result in the energy transfer to the BPheo. At the same time, the band around 630640 nm was previously associated with the formation of a BPheo anion, in particular H (Kellogg et al. 1989; Heller et al. B 1995). Thus, these spectral changes imply that there is formation of a charge-separated state involving the bacteriopheophytin HB and bacteriochlorophyll. Previously, transient formation of the state with the same spectral characteristics was observed when WT RCs were excited with blue (390 nm) light (Lin et al. 2001) or in ND(L170)/ ND(M199) mutant RCs upon excitation at 800 nm (Haa et al. 2003). In that case, the spectral evolution was interpreted as formation of the charge separated state B H ; thus, we can also B B speculate that the state formed in the P-less sample upon excitation at 795 nm could be B H . The B B putative charge separated state B H apparently B B lives at least several hundred picoseconds (see Figure 5), which is similar to a long lived state observed in ND(L170)/ND(M199) or HE(L168)/ ND(L170) mutant RCs where the stabilization of the charge separated state B H was attributed to B B introduction of potentially negatively charged amino acids in the vicinity of P and the monomer BChls (Haa et al. 2003, 2004). Of course, the possibility cannot be excluded that the spectral properties of BA and HA and their respective cation/anion states become altered in the P-less fractions such that the spectral properties of the anion band associated with HA shifts, mimicking the normal position of H . However, it is interesting B to note that the same transient spectral changes were also detected in the P-less fraction isolated from the HL(L153) mutant. This indicates that the transient charge separation between monomer BChl and BPheo may be a general characteristic of P-less samples.

Discussion A series of mutants was created by replacing the histidine ligand of the reaction center BA molecule with a number of other amino acids. The mutants were created to investigate how the ligand determines the spectral and redox properties of the BA molecule, and at the same time, how the changes in the BA properties aect the dynamics of the electron transfer in Rb. sphaeroides RCs. The HF(L153) and HL(L153) mutations do not result in the replacement of BA Mutating histidine to either phenylalanine or to leucine at postion L153 was expected to cause the bacteriochlorophyll in the BA site to be replaced with bacteriopheophytin, as had been observed

175 previously for analogous mutations of histidine ligands of bacteriochlorophylls forming the special pair P and the monomer bacteriochlorophyll BB as well 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 absorbance spectra and pigment composition in the HF(L153) and HL(L153) mutants are inconsistent with the replacement of the pigments. In the case of the HF(L153) mutant, the increase in absorption of the BPheo QX band around 530 nm and decrease of the BChl QX band at 600 nm, as well as the broadening and shift of the B-band from 804 nm (in WT) to 799 nm and the increase of the H-band absorption at 760 nm would support the idea of a pigment exchange. However, the mutation also results in a roughly 30% decrease of the P-band intensity compared to the WT RC spectrum. This eect cannot simply be explained by the replacement of BA with BPheo. Similar changes are also present in the spectrum of the HL(L153) mutant. The BChl : BPheo pigment ratio determined for these mutant RCs by pigment extraction is also inconsistent with a BChl to BPheo exchange. In the HF(L153) mutant, the pigment ratio is about 2. The P-containing fraction of the HL(L153) mutant (see absorption spectrum in Figure 1) has a pigment ratio of about 1.6, but this fraction is not very stable and converts to the P-less fraction with a pigment ratio of 1.4. Due to this, the pure P-containing fraction is very hard to isolate, and its properties are aected by some contamination with the P-less fraction. Lower pigment ratio in the HL(L153) mutant might be explained by substoichiometric pigment exchange, but it might also be a result of the loss or oxidation of BChl molecule(s) (discussed below). In any case, pigment ratios of 1.6 or 2 are not consistent with an exchange of BChl for BPheo. P-less mutants RCs isolated from the mutants HE(L153), HL(L153), HQ(L153) and HY(L153) contain fractions without a P-band, as described above. Similar P-less RCs were previously isolated from the VR(L174) mutant in which arginine was introduced in the vicinity of BA and P (Jackson et al. 1997). P-less RCs were also generated as a result of cavity mutations, in which both histidines ligating the BChls of P were replaced with glycine (Moore and Boxer 1996, 1998). In both cases, the decrease in the BChl : BPheo pigment ratio was interpreted as due to the loss of one or both BChl molecules forming P. However, the abovementioned studies did not present the complete spectra of the RCs, prohibiting comparison of their results to the spectra of the mutants described here. Spectral and pigment analyses of the HE(L153), HL(L153), HQ(L153) and HY(L153) mutant RCs can be interpreted as due to either loss or irreversible oxidation of BChl molecule(s). Possible oxidation of the pigments is supported by the observation of a broad band in the 400500 nm region as well as changes in the BChl and BPheo absorption regions, which are best represented in the absorption spectrum of the HQ(L153) mutant (see Figures 1 and 2). The spectra of P-less fractions isolated from all mutants are very similar to the RC absorption spectrum when P is oxidized to P+, either by chemical oxidation or light induced formation of the long lived P+ state (Fajer et al. 1975; Ho and Deisenhofer 1997). This similarity has also been noted previously in the case of the VR(L174) mutant (Jackson et al. 1997). If the P-less fractions are really the product of P-oxidation, then the oxidation must be irreversible, as addition of reducing agents does not change their absorption spectrum. Also, the broad absorption increase in 400500 nm region in the P-less fractions cannot be attributed just to P+ alone, however, it might indicate the oxidation of pigments in general. Naturally, the question arises, how can one distinguish between the loss and the oxidation of pigments? Simple spectrophotometric analysis of the pigment extract, as described in (Van der Rest and Gingras 1974), is not reliable, as the spectra of irreversibly oxidized pigments would very likely alter the intensity of the extracted BChl and/or BPheo pigment bands. The spectrum of the HL(L153) P-less fraction extract contains an additional band with a peak around 680 nm (data not shown), which denitely increases the apparent BPheo absorbance value, therefore altering the calculated pigment ratio. This band could be due to an oxidized BChl; however, more analysis is necessary to determine the validity of this assignment. From the whole series of mutants, only the histidine to serine mutation did not produce any P-less fraction (variable amounts of P-less fractions were observed in all other mutants). These

176 results suggest that histidine ligand to the BA molecule is an important structural factor, which cannot be easily altered to other amino acids without aecting other cofactors. It is unclear how a serine side chain could ligate the Mg atom of the BA cofactor, as the distance from the oxygen atom to the Mg would be too long for a direct interaction if the protein backbone retained the same structure in the mutant as in the wild-type. One possibility is that in this mutant, as well as in other mutants, a water molecule can be employed as a ligand. This has previously been suggested in the case of a histidine to glycine mutation at M202 (Goldsmith et al. 1996), however, the direct proof of that hypothesis can only be obtained from a crystal structure of the mutant RCs. causes these signicant spectral changes in the mutants of histidine L153. As suggested above, there is a possibility that the actual ligand of the BA molecule in many of the mutat RCs is a water molecule. However, without obtaining sound structural evidence, it is dicult to say anything more denitive.

Photochemistry in the mutated RCs In WT RCs it has been determined that electron transfer from the state P+B to the state P+H is A A faster than the initial electron transfer from P* to P+B (Holzapfel et al. 1989, 1990; Arlt et al. 1993; A Sporlein et al. 2000), making the state P+B very A hard to observe experimentally. In the HF(L153), HG(L153), HS(L153), HV(L153) and HY(L153) mutants, the state P+B was not resolved experiA mentally. The increase in the lifetime of P* decay in these mutants can be interpreted as due to slower primary electron transfer from P* to P+B A compared to WT RCs. It can also be inferred that the secondary electron transfer from P+B to A P+H does not change in these mutants compared A to WT RCs, or at least remains faster than the initial 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 it was in the mutants described above. However, the P* decay is heterogeneous, and at least two exponential functions are necessary to t the data. Previously, heterogeneous decay of P* has been detected 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; Lin et al. 1996a). Several dierent explanations have been suggested for this heterogeneity. It has been ascribed to the distribution in energy of the charge-separated states P+B or P+H (Kirmaier A A and Holten 1990, 1991; Muller et al. 1992), to the relaxation of the radical pair (Nagarajan et al. 1993; Woodbury et al. 1994; Lin et al. 1996b), or to the increased kinetic complexity that arises from considering reversible electron transfer reactions (Holzwarth and Muller 1996). The HE(L153) mutant was previously created in Bl. viridis, and in this mutant P* stimulated emission decay was also described with two exponential decays. In this

Eects of mutations on BA absorption In WT RCs, the absorption spectra of the BA and BB molecules overlap both in the QX and QY regions at room temperature. At low temperatures, the QY absorption bands become resolvable, with BA absorbing at around 800 nm and BB at around 810 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 be clearly resolved (see Figures 1 and 2 and Table 1). In most of the mutants, changes in the QX spectral region around 600 nm are also obvious (especially at 77 K), however, they are harder to attribute just to BA molecules, because P contributes to the total absorbance intensity in that region as well. There is no obvious relationship between the spectral shifts and the chemical nature of the amino acid, which replaces the histidine ligand. For example, in case of the HV(L153) and HY(L153) mutations, the observed band shifts are the same, yet the chemical properties of valine and tyrosine are clearly quite dierent. The most intriguing spectral changes are observed in the HF(L153) and HG(L153) mutants. The spectrum of the HF(L153) mutant shows that the QY absorbance band of BA is most likely heterogeneous, with a major peak at 799 nm and an additional band at around 790 nm. On the other hand, the spectrum of the HG(L153) reaction centers reveals a 10-nm blue-shift of the BA absorbance to 789 nm, with clear separation of the BA and BB absorbance bands. It is possible only to speculate what exactly

177 case, the slower, heterogeneous decay was attributed to an increase of the free energy of the state P+B above P* (Arlt et al. 1996). A In addition to the possible explanations for heterogeneity presented above, another possibility is that the heterogeneous P* decay in the HD(L153) and HE(L153) mutants arises from multiple subpopulations of RCs, possibly due to the presence of both protonated and deprotonated versions of the introduced acidic residues. In this case, the two dierent lifetimes would describe two distinct RC populations. The rst subpopulation, with the shorter lifetime, 9 ps (HE(L153)) or 18 ps (HD(L153)), would show the normal, although somewhat slower electron transfer from P* to P+B . The second subpopulation would show an A impaired electron transfer process that must compete with the intrinsic P* decay to the ground state of about 200 ps (Breton et al. 1990), because the spectral properties of the second, 65 ps (HE(L153)) or 120 ps (HD(L153)) component show a recovery of P-ground-state bleaching on this timescale. Alternatively, the heterogeneous P* decay and recovery of the P-ground-state bleaching in the HE(L153) and HD(L153) mutants could be due to an increase in the free energy of the state P+B A in these mutants compared to WT RCs. A change in the free energy of P+B could also result in an A altered rate of further charge separation to P+H . In this model, the rst component, with A the lifetime of 9 ps in HE(L153) and 18 ps in HD(L153) (Figure 4), would represent the approach to equilibrium of the initial electron transfer from P* to P+B . The second compoA nent, on the timescale of 60 ps in case of HE(L153) and 120 ps in case of HD(L153), could represent the decay of the equilibration between the states P* and P+B involving electron transA fer from P+B to P+H in competition with A A decay of P* to the ground state, and, possibly, P+B recombination to the ground state. In this A model, the relative yield of the each of these processes is dependent on the energetics of P* and the charge-separated states. possible to model the decrease in rate constant in terms of an increased activation energy for overall electron transfer in these mutants. Electron transfer activation energies in the RC can be modeled using Marcus theory (Bixon et al. 1995; Zinth et al. 1998; Haa et al. 2002; Huppmann et al. 2002). The rate constant according to this theory is determined by the free energy dierence between the initial and product states: " # 2p V2 DG k2 p exp ; 1 k h  4pkkB T 4kkB T where V is the electronic coupling matrix element, DG is the free energy dierence between the initial and product states, k is the reorganization energy, kB is Boltzmann constant, and T is temperature (Marcus and Sutin 1985). The free energy dierence and the reorganization energy determine the activation energy Ea for the electron transfer process. Equation (1) can be rewritten:   2p V2 Ea p exp k ; h  4pkkB T kB T 2 DG k2 : where Ea 4k Assuming that the mutations did not alter the reorganization energy and electronic coupling matrix element, the dierence between the activation energies in the various mutants relative to the WT could be calculated using the equation:   kmut Ea;mut Ea;WT kB T ln : 3 kWT The activation energy dierences were calculated for all mutants taking the lifetime of the rst component as the inverse of the initial electron transfer rate constant. The calculated dierences are presented in Table 3. One can see that the largest changes in the activation energy are observed in the HE(L153) and HD(L153) mutants; in the latter the increase is almost 50 meV. However, the increase in activation energy does not allow one to estimate the free energy of the state P+B , A as the activation energy depends on both the reaction free energy change and the reorganization energy (see Equation (2)). Nevertheless, assuming that the reorganization energy did not change in the mutants, one can estimate the free energy of the state P+B relative to the free energy of P*. A

Energetics of the rst electron transfer step in the mutants If the free energy of P+B is substantially inA creased in these mutants relative to P*, it may be

178
Table 3. Results of the free energy calculations for mutated RCs Sample HD(L153) HE(L153) HF(L153) HG(L153) HS(L153) HV(L153) HY(L153) WT
a

Acknowledgements This research was supported by NSF Grants MCB9817388 and MCB0131766. The transient spectrometer used was funded by the NSF Grant BIR9512970. This is publication no. 582 from the Center for the Study of Early Events in Photosynthesis. References
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sP*, psa 18 9 6 6.8 5.8 4.5 4.5 3

DEa, meVb DG(P*)P+B ), meVc A 45 27 17 20 16 9.5 9.5 35 5 )18 )11 )20 )38 )38 )45

Only the shortest lifetimes were considered in case of HD(L153) and HE(L153) mutants. b The changes in the activation energy compared to WT RCs were calculated using the Equation (3). c Free energy gaps between the states P* and P+B were A calculated assuming activationless P* to P+B electron transfer A in WT RCs. Taking into account the small activation energy, 45 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 ) by about 510 meV. A

The reorganization energy for the initial electron transfer reaction has previously been estimated at about 800 cm)1 (Bixon et al. 1995). Then we can calculate that DG0 (P* ) P+B ) for the HD(L153) A mutant is about 270 cm)1 or 35 meV above P*. That corresponds to nearly 100 meV increase in free energy compared to wild-type, as the free energy of the state P+B has been determined to be A around 5070 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 eect was also suggested in the case of the GD(M203) mutant, in which the negatively charged aspartic acid residue is placed close to the ring E of the BA molecule (Heller et al. 1995; Heller et al. 1996). This large shift in the free energy would explain the signicant decrease in the electron transfer rate and yield in the HE(L153) or HD(L153) mutants. Smaller changes in the free energy of the state P+B may help A explain the slower initial electron transfer reactions as observed in all other mutants. However, we cannot also exclude the possibility that mutations of the BA ligand cause changes in the electronic coupling between P and BA, which was suggested from the temperature dependence study of primary electron transfer reaction in Bl. viridis (Huppmann et al. 2002).

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