Mutant Reaction Centers Research Articles

High-field EPR on the primary electron donor cation radical in single crystals of heterodimer mutant reaction centers of photosynthetic bacteria — first characterization of the G-tensor

First high-field/high-frequency (95 GHz) EPR experiments on the cation radical of the primary electron donor (D) in single crystals of the HL (M202) heterodimer mutant of the photosynthetic bacterium Rhodobacter sphaeroides are reported. The measurements have been performed in order to determine the electronic G-tensor of D+ in the mutant and to compare it to the G-tensor of D+ in wild type (R-26). In the heterodimer mutant of the two bacteriochlorophyll (BCHl) molecules constituting D in R-26 is replaced by a bacteriopheophytin. In D2+ of the mutant the unpaired electron is localized on the BChl, thus, its G-tensor should reflect monomer properties, whereas in D+ of R-26 the unpaired electron is delocalized over the two BChl molecules. The preliminary results reported here show that directions of the principal axes of the G-tensor of D+ in HL (M202) are similar to those of R-26 investigated previously. Possible origins of this result are discussed.

Characterization of bacterial reaction centers having mutations of aromatic residues in the binding site of the bacteriopheophytin intermediary electron carrier.

We report the initial characterization of a series of reaction centers (RCs) from the photosynthetic bacterium Rhodobacter capsulatus having single or double mutations of phenylalanines 97 and 121 on the L polypeptide. Substitution of these aromatic amino acids, which may interact with the photoactive bacteriopheophytin associated with the L polypeptide (BPhL), was carried out to examine their possible roles in electron transfer, charge stabilization, and/or BPhL binding. In some mutant RCs, the wild-type pigment content is obtained while in certain others a bacteriochlorophyll (BChL) replaces BPhL. The mutant RCs with wild-type pigment content are found to have overall photochemistry effectively identical to that of wild-type RCs. This indicates aromatic residues at L97 and L121 are not critical factors in the charge separation process, although an approximate 2-fold increase in the rate of electron transfer from BPhL- to QA is observed in two mutants where residue L121 is leucine. In two double mutants where L121 is histidine and L97 is either valine or cysteine, BPhL is replaced with a BChl (denoted beta). This pigment content is surprising since in the native RC structure amino acid L121 is not in optimum geometry for coordination to the Mg in the center of the pigment macrocycle. Charge separation takes place in the beta-containing mutants with an approximately 70% yield of P+QA- at 285 K compared to approximately 100% for wild-type. The photochemistry of these new beta-type RCs is very similar to that reported previously for the beta RC from Rhodobacter sphaeroides wherein the same pigment change was induced by a mutation in the M polypeptide.

Vibrational coherence and relaxation dynamics in the primary donor state of the mutant reaction center of Rhodobacter capsulatus: Theoretical analysis of pump-probe stimulated emission

The effects of vibrational coherence and vibrational relaxation on the pump-probe stimulated emission are theoretically studied on the basis of the perturbative density operator method and the transient linear susceptibility theory. The time-resolved profiles are simulated for the system with the single vibrational mode at the several temperature at the several probing frequencies. The quantum beats appeared in the profiles are shown to rapidly dephase as the temperature increases. The calculations imply that the time constant of the vibrational relaxation of the intra-dimer mode in the special pair of the D LL mutant od Rhodobacter capsulatus is approximately the order of 300 femtosecond at the low temperatures.

Protonation of Glu L212 following QB- formation in the photosynthetic reaction center of Rhodobacter sphaeroides: evidence from time-resolved infrared spectroscopy.

The protonation events that occur upon QA-QB-->QAQB- electron transfer in photosynthetic reaction centers from Rhodobacter sphaeroides were investigated by time-resolved infrared spectroscopy using tunable diode lasers as previously described [Mäntele, W., Hienerwadel, R., Lenz, F., Riedel, E. J., Grisar, R., & Tacke, M. (1990) Spectrosc. Int. 2, 29-35; Hienerwadel, R., Thibodeau, D. L., Lenz, F., Nabedryk, E., Breton, J., Kreutz, W., & Mäntele, W. (1992) Biochemistry 31, 5799-5808]. In the mid-infrared region between 1695 and 1780 cm-1, transient signals associated with QA-QB-->QAQB- electron transfer were observed and characterized. The dominant transient absorbance changes are three positive signals at 1732, 1725, and 1706 cm-1 and two negative signals at 1716 and at 1698 cm-1. The 1725 cm-1-signal disappears upon 1H-->2H exchange as expected for an accessible COOH group and is absent in Glu L212 Gln mutant reaction centers. On this basis, we propose an assignment of this signal to the COOH group of Glu L212. The other signals could correspond to intensity changes and/or shifts of other carboxylic residues, although contributions from ester C = O groups of bacteriopheophytins cannot be ruled out. In native reaction centers at pH 7 and at 4 degrees C, biphasic kinetics of the transient components were observed at most frequencies. The major signal at 1725 cm-1 exhibits a fast kinetic component of t 1/2 = 0.18 ms (25% of the total amplitude) and a slow one of t1/2 = 1 ms (75% of the total amplitude). A global fit analysis of the signals between 1695 and 1780 cm-1 revealed that the spectral distributions of the fast and the slow components are different. Biphasic kinetics with comparable half-times were also observed for the Glu L212 to Gln mutant. The simplest model to explain these results is that the fast phase represents electron transfer and the slow phase represents proton transfer and/or conformational changes coupled to electron transfer. The difference spectra of the slow component from native reaction centers show that the 1725 cm-1 band corresponds to an absorbance increase and not to a shift of an existing band. The signal is therefore proposed to arise from the protonation of Glu L212. The amplitude of the 1725 cm-1 signal varies distinctly with pH as expected for protonation of a COO- group. With increasing pH, the amplitude of the slow component increases while that of the fast component decreases slightly.(ABSTRACT TRUNCATED AT 400 WORDS)

ENDOR studies of the primary donor cation radical in mutant reaction centers of Rhodobacter sphaeroides with altered hydrogen-bond interactions.

The electronic structure of the cation radical of the primary electron donor was investigated in genetically modified reaction centers of Rhodobacter sphaeroides. The site-directed mutations were designed to add or remove hydrogen bonds between the conjugated carbonyl groups of the primary donor, a bacteriochlorophyll dimer, and histidine residues of the protein and were introduced at the symmetry-related sites L168 His-->Phe, HF(L168), and M197 Phe-->His, FH(M197), near the 2-acetyl groups of the dimer and at sites M160 Leu-->His, LH(M160), and L131 Leu-->His, LH(L131), in the vicinity of the 9-keto carbonyls of the dimer. The single mutants and a complete set of double mutants were studied using EPR, ENDOR, and TRIPLE resonance spectroscopy. The changes in the hydrogen bond situation of the primary donor were accompanied by changes in the dimer oxidation midpoint potential, ranging from 410 to 710 mV in the investigated mutants [Lin, X., Murchison, H. A., Nagarajan, V., Parson, W. W., Williams, J. C. & Allen, J. P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10265-10269]. It was found that the addition or removal of a hydrogen bond causes large shifts of the spin density between the two halves of the dimer. Measurements on double mutants showed that the unpaired electron can be gradually shifted from a localization on the L-half of the dimer to a localization on the M-half, depending on the hydrogen bond situation. As a control, the effects of the different hydrogen bonds on P.+ in the mutant HL(M202), which contains a BChlL-BPheM heterodimer as the primary donor with localized spin on the BChl aL [Bylina, E. J., & Youvan, D. C. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7226-7230; Schenck, C. C., Gaul, D., Steffen M., Boxer S. G., McDowell L., Kirmaier C., & Holten D. (1990) in Reaction Centers of Photosynthetic Bacteria (Michel-Beyerle M. E., Ed.) pp 229-238, Springer, Berlin] were studied. In this mutant only small local changes of the spin densities (< or = 10%) in the vicinity of the hydrogen bonds were observed. The effects of the introduced hydrogen bonds on the spin density distribution of the dimer in the mutants are discussed in terms of different orbital energies of the two BChl a moieties which are directly influenced by hydrogen bond formation. The observed changes of the spin density distribution for the double mutants are additive with respect to the single mutations.(ABSTRACT TRUNCATED AT 400 WORDS)

13C MAS NMR evidence for a homogeneously ordered environment of tyrosine M210 in reaction centres of Rhodobacter sphaeroides

The initial electron transfer time in the photosynthetic reaction centre of Rhodobacter sphaeroides is highly sensitive to the replacement of tyrosine M210 by a tryptophan residue. Low-temperature magic angle spinning 13C NMR is used to study Rhodobacter sphaeroides 2.4.1 (M)Y210W mutant reaction centres that are labelled with [4′- 13C]tyrosine. The response of (M)Y210 in R26 is assigned unambiguously to the most upfield narrow signal (linewidth 34 Hz) at σ i = 152.2 ppm, in the region where non-hydrogen bonded tyrosine signals are expected. From the comparison with the signal of labelled R26 it follows that the chemical environment of the (M)Y210 label is unique. The Y(M)210 is in a structurally and electrostatically homogeneous region on the sensitivity scale of the MAS NMR technique. The environment of M210 is structurally stable and the observation of a narrow line shows that the (M)Y210 side chain can be considered static with respect to rotational diffusion on time scales as long as 10 −2s. The narrow signals from the remaining labels in the protein interior for the 2.4.1 (M)Y210W mutant are remarkably similar to those observed for R26. Using a commonly accepted ratio of 150 ppm shift per positive charge equivalent for aromatic carbons, the chemical shift differences between mutant and R26 translate into small variations of the order of 10 −3 electronic equivalents of charge polarization. Also the linewidths are similar, except for a narrow response at σ i = 156.4 ppm that sharpens slightly in the (M)Y210W mutant. Thus, the influence of the (M)Y210W mutation on the global electrostatic properties and structure of the protein, as probed by the tyrosine labels, is minimal. This strongly argues against an explanation of slow and non-exponential electron transfer kinetics in the (M)Y210W RC in terms of loss of structural integrity upon mutation. In contrast, the NMR results strongly support current opinions that (M)Y210 contributes to the fine-tuning of the energy levels of prosthetic groups involved in electron transfer.

Structure-function studies on the interaction of PsaC with the Photosystem 1 heterodimer

The interaction of PsaC with the core heterodimer of Photosystem 1 was studied in wild type Synechocystis sp. PCC 6803 and the site-directed mutant R561E of PsaB. The mutant reaction center was much less stable in urea and the functional reconstitution of the mutant core using PsaC was impaired. However, the extent of reconstitution increased in the presence of divalent cations whereas that of the wild type was inhibited. We conclude that the reaction center in the mutant is unstable, most likely due to the introduction of an unfavorable electrostatic interaction between surface-exposed residues on PsaC and the binding site for the subunit on the core heterodimer in support of the model proposed previously (Rodday et al. (1993) Photosynth Res 36: 1-9).

Flash-induced electrogenic reactions in the SA(L223) reaction center mutant in Rhodobacter sphaeroides chromatophores

The charge transfer events in the SA(L223) reaction center mutant Rhodobacter sphaeroides chromatophores were investigated by direct electrometry. Besides the primary charge separation, the small stigmatellin-sensitive electrogenic reaction due to the electron transfer from the primary to the secondary quinone acceptor in the reaction center complex was observed after the first flash. The second flash-induced electrogenic phase of the secondary quinone protonation and subsequent electrogenic reactions of the cytochrome bc 1 complex were much slower than those in chromatophores of the wild type. It is suggested that replacement of Ser-L223 by Ala impairs both specific proton-conducting pathways leading to the secondary quinone Q B.

Changes in primary donor hydrogen-bonding interactions in mutant reaction centers from Rhodobacter sphaeroides: identification of the vibrational frequencies of all the conjugated carbonyl groups.

Specific changes in the hydrogen-bonding states of the primary donor, P, in reaction centers from Rhodobacter sphaeroides bearing mutations near P were determined using near-infrared excited Fourier transform (FT) Raman spectroscopy. This technique, using 1064-nm excitation, provides the preresonantly enhanced vibrational spectrum of P in its reduced state selectively over the contributions of the other reaction center chromophores and protein and yields structural information concerning P and its hydrogen-bonding interactions. The mutations studied were as follows: Leu M160-->His, Leu L131-->His, the D9 double mutant (Leu M160-->His + Leu L131-->His), Phe M197-->His, and His L168-->Phe. These mutations were designed to introduce new, or to break existing, hydrogen bonds to the C9 and C2 carbonyl groups of P. On the basis of previous assignments [Mattioli, T. A., Hoffmann, A., Robert, B., Schrader, B., & Lutz, M. (1991) Biochemistry 30, 4648-4654], the FT Raman spectra of these mutants show the predicted changes in hydrogen bond interactions of P carbonyl groups with the protein. The results of this study have permitted us to unambiguously identify the C2 and C9 carbonyl vibrators of P in Rb. sphaeroides. The genetically introduced hydrogen bond interactions are discussed in terms of other physicochemical properties of P including the redox potential and electronic asymmetry in the P+ state. It is discussed that changes in protein hydrogen bonding to the conjugated carbonyl groups of P alone are not the sole factor that contributes to the sizeable modifications of the P/P+ redox midpoint potentials, and that the chemical nature of the hydrogen bond donor plays a significant role in this modification.

Protonation and Free Energy Changes Associated with Formation of QBH2 in Native and Glu-L212 .fwdarw. Gln Mutant Reaction Centers from Rhodobacter sphaeroides

Formation of the quinol QBH2 in Glu-L212-->Gln mutant [EQ(L212)] reaction centers (RCs) from Rhodobacter sphaeroides was investigated by measuring the proton uptake (using dyes), UV absorption changes, and free energy changes associated with the two-electron reduction of QB. The advantage of using the EQ(L212) RCs for these studies is that the individual protonation steps can be kinetically resolved and analyzed; conclusions reached regarding the mechanism of formation of QBH2 are expected to apply also to native RCs. The proton uptake by EQ(L212) RCs was strongly biphasic: the fast phase was essentially concomitant with the second electron transfer to QB- (approximately 1 ms at pH 7.5); the slow phase was approximately 2000-fold slower. The rate constant of the slow phase depended on the redox state of the primary quinone QA; for QA- the rate constant was larger (i.e., 8-fold at pH 6.0) than for QA. The electron and proton transfers to QB- in EQ(L212) RCs were modeled with a two-step scheme as follows: (1) fast, QA-QB- + H+(1)-->QA(QBH)-; (2) slow, QA(QBH)- + H+(2)-->QAQBH2, where reaction 1 involves concomitant electron transfer and proton uptake [Paddock, M. L., McPherson, P. H., Feher, G., & Okamura, M. Y. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6803-6807]. The stoichiometry of the fast proton uptake associated with the two-electron reduction of QB varied from 1.1 to 1.4 H+/2e- at pH 6.5-8.5, consistent with the uptake of H+(1) plus an additional fractional proton uptake due to amino acid residues whose pKa values are shifted by interactions with the charge of (QBH)-. The total steady-state proton uptake stoichiometry was 2.0 H+/2e- at pH < or = 7.5, consistent with the formation of the quinol QBH2 (reactions 1 and 2). At pH 8.5, the steady-state proton uptake was 1.6 +/- 0.1 H+/2e-, which is consistent with an apparent pKa for H+(2) of approximately 8.5 [McPherson, P. H., Okamura, M. Y., & Feher, G. (1993) Biochim. Biophys. Acta 1144, 309-324]. The proton uptake kinetics indicate that Glu-L212 is a component of the proton transfer chain for H+(2) that connects reduced QB (buried in the RC protein) to the aqueous solvent as proposed previously [Paddock, M. L., Rongey, S. H., Feher, G., & Okamura, M. Y. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6602-6606].(ABSTRACT TRUNCATED AT 400 WORDS)

Pathway of proton transfer in bacterial reaction centers: role of aspartate-L213 in proton transfers associated with reduction of quinoneto dihydroquinone.

The role of Asp-L213 in proton transfer to reduced quinone QB in the reaction center (RC) from Rhodobacter sphaeroides was studied by site-directed replacement of Asp with residues having different proton donor properties. Reaction centers (RCs) with Asn, Leu, Thr, and Ser at L213 had greatly reduced (approximately 6000-fold) proton-coupled electron transfer [kAB(2)] and proton uptake rates associated with the second electron reduction of QB (QA- QB- + 2H(+)-->QAQBH2) compared to native RCs. RCs containing Glu at L213 showed faster (approximately 90-fold) electron and proton transfer rates than the other mutant RCs but were still reduced (approximately 70-fold) compared with native RCs. These results show that kAB(2) is larger when a carboxylic acid occupies the L213 site, consistent with the proposal that Asp-L213 is a component of a proton transfer chain. The reduced kAB(2) observed with Glu versus Asp at L213 suggests that Asp at L213 is important for proton transfer for some other reason in addition to its proton transfer capabilities. Glu-L213 is estimated to have a higher apparent pKa (pKa > or = 7) than Asp-L213 (pKa < or = 4), as indicated by the slower rate of charge recombination (D+QAQB(-)-->DQAQB) in the mutant RCs. The importance of the pKa and charge of the residue at L213 for proton transfer are discussed. Based on these studies, a model for proton transfer is proposed in which Asp-L213 contributes to proton transfer in native RCs in two ways: (1) it is a component of a proton transfer chain connecting the buried QB molecule with the solvent and/or (2) it provides a negative charge that stabilizes a proton on or near QB.

Primary charge separation in mutant reaction centers of Rhodobacter capsulatus

Excited-state decays of the special pair of Rhodobacter capsulatus and 10 mutants as a function of temperature are presented. The mutations are at the symmetry-related sites L181 and M208. All the decays are nonexponential, although the degree of nonexponentially depends strongly on the mutant and the temperature. We correlated the changes in decay time with mutation-induced changes in the redox potential of the ground state of the special pair. The qualitative form of the decays led us to explore a model based on a small Gaussian distribution of free energy gaps for the electron-transfer process. The model is used to estimate the reorganization energy arising from low-frequency protein and intramolecular modes coupled to the electron transfer. Our fitting procedure gives values of [le]250 and 500 cm[sup [minus]1] at 292 and 22 K, respectively. A consideration of possible errors leads us to conclude that the room temperature value could be no larger than twice the fitted value (i.e., [le]500 cm[sup [minus]1]). The implications of our results for the mechanism of primary charge separation are discussed. 89 refs., 9 figs., 2 tabs.

Fourier transform infrared study of the primary electron donor in chromatophores of Rhodobacter sphaeroides with reaction centers genetically modified at residues M160 and L131.

Structural changes in chromatophores of Rhodobacter sphaeroides reaction center mutants associated with the substitution of amino acid residues near the primary electron donor P have been investigated by light-induced FTIR difference spectroscopy. The single-site mutations Leu-L131 to His and Leu-M160 to His and the corresponding double mutation were designed to introduce a proton-donating residue that could form a hydrogen bond with the keto carbonyl of ring V of each bacteriochlorophyll (PL and PM) of the dimer. The presence of large positive bands at approximately 1550, 1480, and 1295 cm-1, as well as at 2600-2800 cm-1 in the light-induced P+QA-/PQA FTIR difference spectra, corresponding to the photooxidation of P and the photoreduction of the primary quinone QA, demonstrates that the BChl dimer state of P+ is preserved in the LH(L131), LH(M160), and LH(M160)+LH(L131) mutants, although frequency shifts and amplitude changes can be observed, notably for LH(M160). Compared to wild type, these changes are thought to reflect a different charge repartition over the two BChls in P+. Large frequency downshifts in the 9-keto C=O stretching region of the P+QA-/PQA FTIR difference spectra of chromatophores are observed in the mutant samples relative to wild type. For the LH(M160) mutant, a large differential signal at 1678/1664 cm-1 is assigned to a shift, upon photooxidation, of the 9-keto C=O of PM hydrogen-bonded to His-M160, while that at 1718/1696 cm-1 corresponds to the free 9-keto C=O of PL.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics and free energy gaps of electron-transfer reactions in Rhodobacter sphaeroides reaction centers.

The rates of the light-driven, electron-transfer reactions in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides are examined in mutant strains in which tyrosine (M)210 is replaced by phenylalanine, isoleucine, or tryptophan. The spectra of the absorbance changes between 700 and 975 nm, following excitation by 0.6-ps pulses at 605 nm, are analyzed globally by singular value decomposition. The spectra measured at room temperature are interpreted in terms of a model in which the excited bacteriochlorophyll dimer (P*) transfers an electron to a bacteriopheophytin (HL) with time constants of 3.5 +/- 0.3, 10.5 +/- 1.0, 16 +/- 2, and 41 +/- 4 ps in wild-type RCs and the Phe, Ile, and Trp mutants, respectively, and an electron then moves from HL- to a quinone (QA) with a time constant of 0.16 ns in wild-type RCs, 0.24 ns in the Phe mutant, and 0.20 ns in the Ile and Trp mutants. The first step speeds up with decreasing temperature in wild-type RCs, remains virtually unchanged in the Phe mutant, and slows down in the Ile and Trp mutants. At 80 K, the signals in the 850-975-nm region include an apparent shift of the stimulated emission or absorption spectrum of P*, with a time constant of 5 ps in the Ile mutant and 13 pcs in the Trp mutant. Most of the electron transfer to HL occurs with time constants of 55 and 155 ps in the Ile and Trp mutants, respectively, and probably occurs from the relaxed form of P*. Electron transfer from the initial state cannot be ruled out, however. Relaxations of P* are not resolved in wild-type RCs or the Phe mutant. The midpoint potential (Em) of the P/P+ redox couple is measured by an electrochemical technique; the Em values are 500 +/- 5, 530 +/- 6, 533 +/- 3, and 552 +/- 10 mV for the wild-type and the Phe, Ile, and Trp mutant RCs, respectively. These values are corroborated by chemical titrations. The free energy change (delta G degrees) associated with formation of the P+HL-radical pair from P* also is determined by measuring the amplitude of fluorescence on the nanosecond time scale after blocking electron transfer from HL- to QA. The free energy of P+HL- is elevated by an amount comparable to that calculated from the increase in the Em of P in the Ile mutant and by about 16 meV more than this in the Phe and Trp mutants.(ABSTRACT TRUNCATED AT 400 WORDS)

Tyrosine 162 of the photosynthetic reaction center L-subunit plays a critical role in the cytochrome c2 mediated rereduction of the photooxidized bacteriochlorophyll dimer in Rhodobacter sphaeroides. 1. Site-directed mutagenesis and initial characterization.

Five site-directed mutants were engineered to substitute phenylalanine, serine, leucine, methionine, and glycine for tyrosine residue 162 of the pufL gene in Rhodobacter (R.) sphaeroides. Each of the mutations and the wild-type (WT) genes was expressed in the R. sphaeroides puf deletion strain PUF delta LMX21/3. Initial characterization revealed that all of the mutants were photoheterotrophically competent but that L162G and L162S were impaired. The amounts of mutant reaction centers expressed, the spectral characteristics, and the rates of intraprotein electron transfer and turnover were similar to the values obtained for WT. Kinetic measurements of photooxidized special pair rereduction mediated by the physiological donor cytochrome c2 in intact chemoheterotrophically grown cells revealed that the fast phase was abolished in all mutants and that the overall kinetics of rereduction was drastically slowed. It is concluded that L162Y plays a vital role in facilitating the rapid rereduction of the photooxidized bacteriochlorophyll dimer in R. sphaeroides.

Triplet state EPR of reaction centers from the HisL173?LeuL173 mutant of Rhodobacter sphaeroides which contains a heterodimer primary donor

Electron paramagnetic resonance (EPR) spectroscopy has been used to examine the triplet states in reaction centers of Rhodobacter sphaeroides which have undergone a genetic modification affecting the primary donor. Reaction centers containing the His(L173)→Leu(L173) substitution in the amino acid sequence have a primary donor which consists of a BChl-BPh heterodimer. The triplets formed in this heterodimer reaction center were compared with those formed in the wild-type reaction center which contains the BChl-BChl homodimer. Both reaction centers transfer triplet energy to the carotenoid under illumination at liquid nitrogen temperatures (∼90 K). However, the intensity of the carotenoid triplet signal is significantly decreased in the Leu(L173) mutant compared with the wild-type reaction center. At 12 K, in wild-type reaction centers only the primary donor triplet is observed. The Leu(L173) mutant exhibits a signal similar to that observed by Bylina et al. (1990) in His(M200)→Leu(M200) mutant reaction centers from Rb. capsulatus. The values of the zero-field splitting parameters of this triplet are discussed within the context of various models for the primary donor triplet state. No alteration in the ability of the carotenoid to quench the primary donor triplet state results from mutations at these sites.

Insights into the factors controlling the rates of the deactivation processes that compete with charge separation in photosynthetic reaction centers

This article addresses the factors that underlie the unity quantum yield of charge separation in the bacterial photosynthetic reaction center (RC). Of particular interest are the factors that suppress charge recombination and other deactivation processes which could compete effectively with charge separation. Studies on mutant RCs with enhanced charge recombination show that rates of decay processes can be increased tenfold or more upon a relatively small fractional increase in the free energy gap to the ground state. This behavior is opposite to that expected on the basis of Franck-Condon effects, where an increase in Δ G should decrease the deactivation rate. In the mutants, it is proposed that the enhanced deactivation rates of the intermediate charge-separated states result from (i) increased quantum mechanical mixing with electronic states at slightly higher energy that have inherently strong deactivation properties, and (ii) increased thermal repopulation of such strongly deactivated states. The thermal repopulation mechanism is proposed to play an important role in the heterodimer mutants studied here. This mechanism requires that the free energy gaps between the charge-separated and strongly deactivated states must decrease with temperature to be consistent with the weakly temperature-dependent deactivation behavior. Potential mediating states include the excited dimeric primary electron donor and a state involving the oxidized dimer and the reduced accessory bacteriochlorophyll molecule. It is concluded that increasing the free energy of charge-separated intermediates in order to minimize the Franck-Condon factors can enhance the deactivation rates and thus reduce the quantum yield of charge separation.

Flash-induced electric potential generation in wild type and L212EQ mutant chromatophores of Rhodobacter sphaeroides: Q BH 2 is not released from L212EQ mutant reaction centers

The nature of the protonation reactions altered in the L212EQ mutant of photosynthetic purple bacterium Rhodobacter sphaeroides, in which glutamate at position 212 of the L-subunit (Glu L212) is replaced by glutamine, was examined through the flash-induced electric potential generation in chromatophores, measured at various pH values. The second flash-induced electrogenic phase of secondary acceptor quinone (Q B) protonation in mutant reaction centers was 2–3-times smaller than that in reaction centers of wild type. Furthermore, the electrogenic reactions of the cytochrome bc 1 complex were absent in the mutant at pH 7.6, but appeared at lower pH, with normal sensitivity to antimycin and myxothiazol. Hence, in L212EQ chromatophores ubiquinol generated in the reaction center fails to take up one of its two protons and remains bound to protein at neutral pH. At acid pH, the second proton is taken up and ubiquinol is released from the reaction center.

Mutations designed to modify the environment of the primary electron donor of the reaction center from Rhodobacter sphaeroides: phenylalanine to leucine at L167 and histidine to phenylalanine at L168.

Two mutations, L168 His to Phe and L167 Phe to Leu, were made in residues near the primary electron donor, a bacteriochlorophyll dimer, of the reaction center from Rhodobacter sphaeroides. Blue shifts of 10-15 nm in the 865-nm band of the donor were observed in the optical absorption spectra of both of the mutant reaction centers. The rate of initial electron transfer was determined by measurement of the kinetics of the decay of the excited state of the donor, and the rate of charge recombination was determined by measurement of the recovery of the bleaching of the donor. The initial electron transfer time constant and the charge recombination time constant were determined to be 3.6 ps and 220 ms, respectively, in the L168 His to Phe mutant and 5.0 ps and 85 ms in the L167 Phe to Leu mutant, compared to 3.8 ps and 100 ms measured for the wild type. The oxidation potential of the donor measured by oxidation-reduction titrations was found to decrease by 80 mV in the L168 His to Phe mutant and increase by 25 mV in the L167 Phe to Leu mutant. Time-resolved fluorescence decay measurements indicated that the change in the oxidation potential of the donor in the L168 His to Phe mutant resulted in a change in the energies of the charge-separated states. The results show that an increase in the driving force does not increase the rate of the initial electron transfer reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of mutations near the bacteriochlorophylls in reaction centers from Rhodobacter sphaeroides.

Mutations were made in four residues near the bacteriochlorophyll cofactors of the photosynthetic reaction center from Rhodobacter sphaeroides. These mutations, L131 Leu to His and M160 Leu to His, near the dimer bacteriochlorophylls, and M203 Gly to Asp and L177 Ile to Asp, near the monomer bacteriochlorophylls, were designed to result in the placement of a hydrogen bond donor group near the ring V keto carbonyl of each bacteriochlorophyll. Perturbations of the electronic structures of the bacteriochlorophylls in the mutants are indicated by additional resolved transitions in the bacteriochlorophyll absorption bands in steady-state low-temperature and time-resolved room temperature spectra in three of the resulting mutant reaction centers. The major effect of the two mutations near the dimer was an increase up to 80 mV in the donor oxidation-reduction midpoint potential. Correspondingly, the calculated free energy difference between the excited state of the primary donor and the initial charge separated state decreased by up to 55 mV, the initial forward electron-transfer rate was up to 4 times slower, and the rate of charge recombination between the primary quinone and the donor was approximately 30% faster in these two mutants compared to the wild type. The two mutations near the monomer bacteriochlorophylls had minor changes of 25 mV or less in the donor oxidation-reduction potential, but the mutation close to the monomer bacteriochlorophyll on the active branch resulted in a roughly 3-fold decrease in the rate of the initial electron transfer.