Wild-type Reaction Centers Research Articles

Configuration of Spheroidene in the Photosynthetic Reaction Center of Rhodobacter sphaeroides: A Comparison of Wild-Type and Reconstituted R26

We compare the resonance Raman spectra acquired at two excitation wavelengths, 496.5 and 514.5 nm, of spheroidene in the wild-type reaction center of Rhodobacter sphaeroides and reconstituted into the reaction center of the Rhodobacter sphaeroides mutant R26. Our earlier work showed that the reconstituted R26 reaction center holds spheroidene in two configurations: 15,15'-cis and another configuration. Here we show that in the wild-type reaction center only 15,15'-cis spheroidene is present. In the resonance Raman spectra of the reconstituted R26 reaction centers, a transition is identified that arises exclusively from the second configuration. According to density-functional-theory calculations, this transition is specific for the 13,14-cis configuration.

Carotenoid Excited-State Properties in Photosynthetic Purple Bacterial Reaction Centers: Effects of the Protein Environment

Carotenoid excited-state properties are characterized and compared in reaction centers (RCs) of wild-type (WT) Rhodobacter (Rb.) sphaeroides , and a mutant VR(L157), in which the near-infrared absorbance band associated with the primary electron donor, P, is missing. Energy transfer from the carotenoid (spheroidenone) S(2) and relaxed S(1) excited states to an adjacent monomeric-bacteriochlorophyll is unchanged between WT and the mutant RC samples. However, two other excited states, including a vibrationally hot S(1) state and a state referred to as S*, have distinct properties in the two RCs. The lifetime of the hot S(1) state is significantly shortened in the P-less mutant compared to WT RCs (450 fs vs 800 fs, respectively), and there is a nearly 2-fold decrease in the efficiency of energy transfer from the carotenoid to bacteriochlorophyll in the P-less mutant relative to WT RCs. The fact that both the observed hot S(1) excited state lifetime and the energy transfer efficiency decrease in the mutant implies that the intrinsic lifetime of the hot S(1) state in the P-less mutant has decreased. Interestingly, the S* state is observed only in the P-less mutant, and it is not present in the WT. The change in the hot S(1) lifetime between WT and mutant RCs, and the formation of the S* state only in the mutant, suggests that the carotenoid binding pocket in the P-less mutant is substantially altered. The excited-state behavior of spheroidene in WT RCs isolated from anaerobically grown cells was also characterized and compared with previous studies of spheroidene in the light-harvesting complex II (LH2) from Rb. sphaeroides . Differences in the photophysical properties of spheroidene between WT RCs and LH2 parallel those observed for spheroidenone between WT and VR(L157) mutant RCs. On the basis of the structural information available for both RCs and LH2, it appears that the hot S(1) state and the S* state are sensitive to the structural constraints imposed by protein-carotenoid interactions. Finally, in the VR(L157) mutant, it is possible to directly observe the carotenoid triplet state, likely formed via quenching of the bacteriochlorophyll triplet state. This provides direct experimental evidence for triplet energy transfer to the carotenoid, a process that is integral to the photoprotective role of carotenoids in bacterial RCs.

Interquinone Electron Transfer in Photosystem I As Evidenced by Altering the Hydrogen Bond Strength to the Phylloquinone(s)

The kinetics of electron transfer from phyllosemiquinone (PhQ(*-)) to the iron sulfur cluster F(X) in Photosystem I (PS I) are described by lifetimes of approximately 20 and approximately 250 ns. These two rates are attributed to reactions involving the quinones bound primarily by the PsaB (PhQ(B)) and PsaA (PhQ(A)) subunits, respectively. The factors leading to a approximately 10-fold difference between the observed lifetimes are not yet clear. The peptide nitrogen of conserved residues PsaA-Leu722 and PsaB-Leu706 is involved in asymmetric hydrogen-bonding to PhQ(A) and PhQ(B), respectively. Upon mutation of these residues in PS I of the green alga, Chlamydomonas reinhardtii , we observe an acceleration of the oxidation kinetics of the PhQ(*-) interacting with the targeted residue: from approximately 255 to approximately 180 ns in PsaA-L722Y/T and from approximately 24 to approximately 10 ns in PsaB-L706Y. The acceleration of the kinetics in the mutants is consistent with a perturbation of the H-bond, destabilizing the PhQ(*-) state, and increasing the driving force of its oxidation. Surprisingly, the relative amplitudes of the phases reflecting PhQ(A)(*-) and PhQ(B)(*-) oxidation were also affected by these mutations: the apparent PhQ(A)(*-)/PhQ(B)(*-) ratio is shifted from 0.65:0.35 in wild-type reaction centers to 0.5:0.5 in PsaA-L722Y/T and to 0.8:0.2 in PsaB-L706Y. The most consistent account for all these observations involves considering reversibility of oxidation of PhQ(A)(*-) and PhQ(B)(*-) by F(X), and asymmetry in the driving forces for these electron transfer reactions, which in turn leads to F(x)-mediated interquinone electron transfer.

Electronic structure of the primary electron donor of Blastochloris viridis heterodimer mutants: High-field EPR study

High-field electron paramagnetic resonance (HF EPR) has been employed to investigate the primary electron donor electronic structure of Blastochloris viridis heterodimer mutant reaction centers (RCs). In these mutants the amino acid substitution His(M200)Leu or His(L173)Leu eliminates a ligand to the primary electron donor, resulting in the loss of a magnesium in one of the constituent bacteriochlorophylls (BChl). Thus, the native BChl/BChl homodimer primary donor is converted into a BChl/bacteriopheophytin (BPhe) heterodimer. The heterodimer primary donor radical in chemically oxidized RCs exhibits a broadened EPR line indicating a highly asymmetric distribution of the unpaired electron over both dimer constituents. Observed triplet state EPR signals confirm localization of the excitation on the BChl half of the heterodimer primary donor. Theoretical simulation of the triplet EPR lineshapes clearly shows that, in the case of mutants, triplet states are formed by an intersystem crossing mechanism in contrast to the radical pair mechanism in wild type RCs. Photooxidation of the mutant RCs results in formation of a BPhe anion radical within the heterodimer pair. The accumulation of an intradimer BPhe anion is caused by the substantial loss of interaction between constituents of the heterodimer primary donor along with an increase in the reduction potential of the heterodimer primary donor D/D + couple. This allows oxidation of the cytochrome even at cryogenic temperatures and reduction of each constituent of the heterodimer primary donor individually. Despite a low yield of primary donor radicals, the enhancement of the semiquinone–iron pair EPR signals in these mutants indicates the presence of kinetically viable electron donors.

Examination of stability of mutant photosynthetic reaction center of Rhodobacter sphaeroides I(L177)H and determination of location of bacteriochlorophyll covalently bound to the protein

We demonstrated earlier that as a result of the I(L177)H mutation in the photosynthetic reaction center (RC) of the bacterium Rhodobacter sphaeroides, one of the bacteriochlorophylls (BChl) binds with the L-subunit, simultaneously raising coordination stability of the central magnesium atom of the bacteriochlorophyll associated with the protein. In this study, spectral properties of wild type RC and I(L177)H in the presence of urea and SDS as well as at 48 degrees C were examined. It is shown that the I(L177)H mutation decreases the RC stability. Under denaturing conditions, some changes indicating breakdown of oligomeric structure of the complex and loss of interaction between pigments and their protein environment are observed in I(L177)H RC spectra. In addition, pheophytinization of bacteriochlorophylls occurs in both types of RC in the presence of SDS. However, an 811-nm band is observed in the spectrum of the mutant RC under these conditions, which indicates retention of one of the BChl molecules in the protein binding site and stable coordination of its central magnesium atom. It is shown that in both types of RC, monomeric BChl B(B) can be modified by sodium borohydride treatment and then extracted by acetone-methanol mixture. Spectral properties of the BChl covalently bound with the protein in I(L177)H RC do not change. The results demonstrate that BChl P(A) is the molecule of BChl tightly bound with the L-subunit in mutant RC as it was supposed earlier.

M234Glu is a component of the proton sponge in the reaction center from photosynthetic bacteria

Bacterial reaction centers use light energy to couple the uptake of protons to the successive semi-reduction of two quinones, namely QA and QB. These molecules are situated symmetrically in regard to a non-heme iron atom. Four histidines and one glutamic acid, M234Glu, constitute the five ligands of this atom. By flash-induced absorption spectroscopy and delayed fluorescence we have studied in the M234EH and M234EL variants the role played by this acidic residue on the energetic balance between the two quinones as well as in proton uptake. Delayed fluorescence from the P+QA− state (P is the primary electron donor) and temperature dependence of the rate of P+QA− charge recombination that are in good agreement show that in the two RC variants, both QA− and QB− are destabilized by about the same free energy amount: respectively ∼100±5 meV and 90±5 meV for the M234EH and M234EL variants, as compared to the WT. Importantly, in the M234EH and M234EL variants we observe a collapse of the high pH band (present in the wild-type reaction center) of the proton uptake amplitudes associated with formation of QA− and QB−. This band has recently been shown to be a signature of a collective behaviour of an extended, multi-entry, proton uptake network. M234Glu seems to play a central role in the proton sponge-like system formed by the RC protein.

Electron transfer in the Rhodobacter sphaeroides reaction center assembled with zinc bacteriochlorophyll

The cofactor composition and electron-transfer kinetics of the reaction center (RC) from a magnesium chelatase (bchD) mutant of Rhodobacter sphaeroides were characterized. In this RC, the special pair (P) and accessory (B) bacteriochlorophyll (BChl) -binding sites contain Zn-BChl rather than BChl a. Spectroscopic measurements reveal that Zn-BChl also occupies the H sites that are normally occupied by bacteriopheophytin in wild type, and at least 1 of these Zn-BChl molecules is involved in electron transfer in intact Zn-RCs with an efficiency of >95% of the wild-type RC. The absorption spectrum of this Zn-containing RC in the near-infrared region associated with P and B is shifted from 865 to 855 nm and from 802 to 794 nm respectively, compared with wild type. The bands of P and B in the visible region are centered at 600 nm, similar to those of wild type, whereas the H-cofactors have a band at 560 nm, which is a spectral signature of monomeric Zn-BChl in organic solvent. The Zn-BChl H-cofactor spectral differences compared with the P and B positions in the visible region are proposed to be due to a difference in the 5th ligand coordinating the Zn. We suggest that this coordination is a key feature of protein-cofactor interactions, which significantly contributes to the redox midpoint potential of H and the formation of the charge-separated state, and provides a unifying explanation for the properties of the primary acceptor in photosystems I (PS1) and II (PS2).

Properties of mutant reaction centers of Rhodobacter sphaeroides with substitutions of histidine L153, the axial Mg2+ ligand of bacteriochlorophyll BA

Mutant reaction centers (RC) from Rhodobacter sphaeroides have been studied in which histidine L153, the axial ligand of the central Mg atom of bacteriochlorophyll B(A) molecule, was substituted by cysteine, methionine, tyrosine, or leucine. None of the mutations resulted in conversion of the bacteriochlorophyll B(A) to a bacteriopheophytin molecule. Isolated H(L153)C and H(L153)M RCs demonstrated spectral properties similar to those of the wild-type RC, indicating the ability of cysteine and methionine to serve as stable axial ligands of the Mg atom of bacteriochlorophyll B(A). Because of instability of mutant H(L153)L and H(L153)Y RCs, their properties were studied without isolation of these complexes from the photosynthetic membranes. The most prominent effect of the mutations was observed with substitution of histidine by tyrosine. According to the spectral data and the results of pigment analysis, the B(A) molecule is missing in the H(L153)Y RC. Nevertheless, being associated with the photosynthetic membrane, this RC can accomplish photochemical charge separation with quantum yield of approximately 7% of that characteristic of the wild-type RC. Possible pathways of the primary electron transport in the H(L153)Y RC in absence of photochemically active chromophore are discussed.

Kinetics and Energetics of Electron Transfer Reactions in a Photosynthetic Bacterial Reaction Center Assembled with Zinc Bacteriochlorophylls

Electron transfer processes were studied in the reaction center (RC) of a Rhodobacter sphaeroides magnesium chelatase (bchD) mutant that assembles with six chemically identical chlorin molecules. A previous study [Jaschke & Beatty, Biochemistry, 2007] and this work show the complete absence of bacteriochlorophyll (containing Mg as the metal) and bacteriopheophytin from the bchD mutant RC. Instead, bacteriochlorophylls containing a Zn atom as the metal (Zn-BChl) occupy the binding sites of the special pair (P), accessory bacteriochlorophyll (B), and primary electron acceptor (H). In spite of significant differences in cofactor composition, electron transfer from excited P through B to H proceeds with high efficiency and with rates nearly identical to the wild type RC. The rate of electron transfer from H to QA is also the same as that observed in the wild type RC. Thus, the protein-cofactor interactions, mainly through electron sharing between the metal of the BChl and the protein, play an important role in adjusting the energies of the cofactors to form an efficient electron transfer system. The study also suggests that the overall electron transfer from P to H is more sensitive to the energy change between P and B than B and H, and can tolerate a large variation in the redox energy of H.

Identification of FTIR Bands Due to Internal Water Molecules around the Quinone Binding Sites in the Reaction Center from Rhodobacter sphaeroides

The bacterial reaction center (RC) is a membrane protein complex that performs photosynthetic electron transfer from a bacteriochlorophyll dimer to quinone acceptors Q(A) and Q(B). Q(B) accepts electrons from the primary quinone, Q(A), in two sequential electron transfer reactions coupled to uptake of a proton from solution. It has been suggested that water molecules along the proton uptake pathway are protonated upon quinone reduction on the basis of FTIR difference spectra [Breton, J., and Nabedryk, E. (1998) Photosynth. Res. 55, 301-307]. We examined the possible involvement of water molecules in the photoreaction processes by studying (18)O water isotope effects on FTIR difference spectra resulting from formation of Q(A)(-) and Q(B)(-). Continuum bands in D(2)O due to Q(B)(-) formation in the 2300-1800 cm(-1) region did not show spectral shifts by (18)O water in the wild-type (WT) RC, suggesting that these bands do not originate from (protonated) water. In contrast, the Q(B)(-)/Q(B) spectrum of the EQ-L212 mutant RC showed a spectral shift of a band near 2100 cm(-1) due to (18)O water substitution, consistent with protonation of internal water. FTIR shifts due to (18)O water were also observed following formation of Q(A)(-) and Q(B)(-) in the spectral region of 3700-3500 cm(-1) characteristic of weakly hydrogen bonded water. The water responsible for the Q(B)(-) change was localized near Glu-L212 by spectral shifts in mutant RCs. The weakly hydrogen bonded water perturbed by quinone reduction may play a role in stabilizing the charge-separated state.

Low-Temperature Studies of Electron Transfer to the M Side of YFH Reaction Centers from Rhodobacter capsulatus

There have been extensive experimental and theoretical studies of the temperature dependence of the rates of electron transfer between the cofactors associated primarily with the L polypeptide (or A branch) in the bacterial photosynthetic reaction center (RC). The focus of this paper is to gain further insight into the temperature dependence of rate of initial electron transfer to the parallel cofactor chain associated mainly with the M polypeptide (or B branch), which is inactive in the native RC. To this end, picosecond transient absorption measurements have been carried out on RCs of the YFH mutant of the photosynthetic bacterium Rhodobacter capsulatus at 77 K. In wild-type RCs, symmetry-related residues Phe M208 and Tyr L181 flank the primary electron donor (P) and are key to initial charge separation. In the YFH mutant these residues are swapped, i.e., are Tyr M208 and Phe L181. The third mutation in YFH changes Leu M212 to His and results in replacement of the L-side bacteriopheophytin (H(L)) with a bacteriochlorophyll denoted beta. Studies were carried out at 77 K for RCs in detergent-buffer/glycerol glasses utilizing either the detergent N-lauryl-N,N-dimethylamine N-oxide (LDAO) or Deriphat 160-C. In both media, excitation of P to its lowest singlet excited state (P*) elicits complex kinetic behavior that, in the simplest phenomenological description, involves two P* cofactor-protein populations: one that is capable of charge separation (active) and one that is not (inactive). The amplitudes of the components of the P* stimulated-emission kinetic profiles, and the amplitudes and time course of accompanying P-bleaching recovery, reveal that the two P* populations, active/inactive, are in 60/40 (LDAO) or 40/60 (Deriphat) proportion. In the nonphotoactive fraction, P* decays solely via return to the ground state with its inherent lifetime (i.e., lifetime in the absence of electron transfer) of 170 ps (LDAO) or 350 ps (Deriphat). In the photoactive fraction, P* has a lifetime of 4.5 ps (LDAO) or 13 ps (Deriphat) and decays by parallel electron transfer to H(M) (30%) and beta (70%) on the M and L branches, respectively. The rate constant for P* --> P(+)H(M)(-) electron transfer is (15 ps)(-1) (LDAO) or (43 ps)(-1) (Deriphat) at 77 K. These rate constants are about 2-fold greater than those determined at 295 K in the corresponding detergent/buffer solutions. These results combined with related findings from prior work show that the primary charge-separation events on both sides of the RC are basically activationless processes. In particular, in the functionally active P* population of YFH RCs, there is little or no apparent energy barrier (e.g., involving motions of the cofactors or protein or both) for electron transfer from P* to the either side of the RC. This conclusion holds irrespective of the precise description of the complex kinetic behavior that is observed. In addition to the observations on the temperature-dependent photochemistry, the 77 K transient absorption spectra in the near-infrared resolve a bacteriopheophytin anion band at 955 nm for wild-type RCs and, for the beta-containing mutants L(M212)H and YFH, a bacteriochlorophyll anion band at 1015 nm.

Mutant reaction centers of Rhodobacter sphaeroides I(L177)H with strongly bound bacteriochlorophyll a: Structural properties and pigment-protein interactions

Methods of photoinduced Fourier transform infrared (FTIR) difference spectroscopy and circular dichroism were employed for studying features of pigment-protein interactions caused by replacement of isoleucine L177 by histidine in the reaction center (RC) of the site-directed mutant I(L177)H of Rhodobacter sphaeroides. A functional state of pigments in the photochemically active cofactor branch was evaluated with the method of photo-accumulation of reduced bacteriopheophytin H(A)(-). The results are compared with those obtained for wild-type RCs. It was shown that the dimeric nature of the radical cation of the primary electron donor P was preserved in the mutant RCs, with an asymmetric charge distribution between the bacteriochlorophylls P(A) and P(B) in the P(+) state. However, the dimers P in the wild-type and mutant RCs are not structurally identical due probably to molecular rearrangements of the P(A) and P(B) macrocycles and/or alterations in their nearest amino acid environment induced by the mutation. Analysis of the electronic absorption and FTIR difference P(+)Q(-)/PQ spectra suggests the 17(3)-ester group of the bacteriochlorophyll P(A) to be involved in covalent interaction with the I(L177)H RC protein. Incorporation of histidine into the L177 position does not modify the interaction between the primary electron acceptor bacteriochlorophyll B(A) and the bacteriopheophytin H(A). Structural changes are observed in the monomer bacteriochlorophyll B(B) binding site in the inactive chromophore branch of the mutant RCs.

Identification of the First Steps in Charge Separation in Bacterial Photosynthetic Reaction Centers of Rhodobacter sphaeroides by Ultrafast Mid-Infrared Spectroscopy: Electron Transfer and Protein Dynamics

Time-resolved visible pump/mid-infrared (mid-IR) probe spectroscopy in the region between 1600 and 1800 cm −1 was used to investigate electron transfer, radical pair relaxation, and protein relaxation at room temperature in the Rhodobacter sphaeroides reaction center (RC). Wild-type RCs both with and without the quinone electron acceptor Q A, were excited at 600 nm (nonselective excitation), 800 nm (direct excitation of the monomeric bacteriochlorophyll (BChl) cofactors), and 860 nm (direct excitation of the dimer of primary donor ( P) BChls ( P L/ P M)). The region between 1600 and 1800 cm −1 encompasses absorption changes associated with carbonyl (C O) stretch vibrational modes of the cofactors and protein. After photoexcitation of the RC the primary electron donor P excited singlet state ( P*) decayed on a timescale of 3.7 ps to the state P + B L − (where B L is the accessory BChl electron acceptor). This is the first report of the mid-IR absorption spectrum of P + B L − ; the difference spectrum indicates that the 9-keto C O stretch of B L is located around 1670–1680 cm −1. After subsequent electron transfer to the bacteriopheophytin H L in ∼1 ps, the state P + H L − was formed. A sequential analysis and simultaneous target analysis of the data showed a relaxation of the P + H L − radical pair on the ∼20 ps timescale, accompanied by a change in the relative ratio of the P L + and P M + bands and by a minor change in the band amplitude at 1640 cm −1 that may be tentatively ascribed to the response of an amide C O to the radical pair formation. We conclude that the drop in free energy associated with the relaxation of P + H L − , is due to an increased localization of the electron hole on the P L half of the dimer and a further consequence is a reduction in the electrical field causing the Stark shift of one or more amide C O oscillators.

Temperature Dependence of Electron Transfer to the M-Side Bacteriopheophytin in Rhodobacter capsulatus Reaction Centers

Subpicosecond time-resolved absorption measurements at 77 K on two reaction center (RC) mutants of Rhodobacter capsulatus are reported. In the D(LL) mutant the D helix of the M subunit has been substituted with the D helix from the L subunit, and in the D(LL)-FY(L)F(M) mutant, three additional mutations are incorporated that facilitate electron transfer to the M side of the RC. In both cases the helix swap has been shown to yield isolated RCs that are devoid of the native bacteriopheophytin electron carrier HL (Chuang, J. I.; Boxer, S. G.; Holten, D.; Kirmaier, C. Biochemistry 2006, 45, 3845-3851). For D(LL), depending whether the detergent Deriphat 160-C or N-lauryl-N,N-dimethylamine-N-oxide (LDAO) is used to suspend the RCs, the excited state of the primary electron donor (P*) decays to the ground state with an average lifetime at 77 K of 330 or 170 ps, respectively; however, in both cases the time constant obtained from single-exponential fits varies markedly as a function of the probe wavelength. These findings on the D(LL) RC are most easily explained in terms of a heterogeneous population of RCs. Similarly, the complex results for D(LL)-FY(L)F(M) in Deriphat-glycerol glass at 77 K are most simply explained using a model that involves (minimally) two distinct populations of RCs with very different photochemistry. Within this framework, in 50% of the D(LL)-FY(L)F(M) RCs in Deriphat-glycerol glass at 77 K, P* deactivates to the ground state with a time constant of approximately 400 ps, similar to the deactivation of P* in the D(LL) mutant at 77 K. In the other 50% of D(LL)-FY(L)F(M) RCs, P* has a 35 ps lifetime and decays via electron transfer to the M branch, giving P+HM- in high yield (> or =80%). This result indicates that P* --> P(+)H(M)(-) is roughly a factor of 2 faster at 77 K than at 295 K. In alternative homogeneous models the rate of this M-side electron-transfer process is the same or up to 2-fold slower at low temperature. A 2-fold increase in rate with a reduction in temperature is the same behavior found for the overall L-side process P* --> P(+)H(L)(-) in wild-type RCs. Our results suggest that, as for electron transfer on the L side, the M-side electron-transfer reaction P* --> P(+)H(M)(-) is an activationless process.

Proton and electron transfer in wild-type and mutant reaction centers from Rhodobacter sphaeroides followed by rapid-scan FTIR spectroscopy

Rapid-scan FTIR difference spectroscopy was used to investigate proton and electron transfer reactions in photosynthetic reaction centers from Rhodobacter sphaeroides. Experiments at different temperatures and in the presence of D 2O have provided strong indication that a transient band at 1707 cm −1 previously identified after both the 1st and the 2nd flashes [A. Mezzetti, W. Leibl, Eur. Biophys. J. 34 (2005) 921] is given by a transient protonation of the side chain of a Asp or Glu residue situated on the proton transfer pathway from the cytoplasm to the Q B site. Experiments in D 2O on a Asp-M17 → Asn mutant reaction center, where the proton and electron transfer reactions are slowed down compared to the wild-type, showed that the kinetic isotope effect induced by H/D exchange slows down the electron transfer reaction after the 1st flash, confirming the strong coupling between proton and electron transfer. Rapid-scan FTIR experiments on Cd 2+-treated reaction centers, in agreement with previous UV–vis measurements [P. Adelroth, M.L. Paddock, L.B. Sagle, G. Feher, M.Y. Okamura, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 13086], showed that upon addition of Cd 2+, which inhibits proton uptake, the Q A −Q B → Q AQ B − reaction is slowed. Interestingly, the transient 1707 cm −1 band is not visible in the first spectrum recorded early after the flash. This strongly suggests its identification with a residue situated on the proton transfer pathway, which is perturbed upon metal cation binding.

Sensitively probing the cofactor redox species and photo-induced electron transfer of wild-type and pheophytin-replaced photosynthetic proteins reconstituted in self-assembled monolayers

An ultrathin, ordered, and packed protein film, consisting of the 2-mercaptoacetic acid (MAA), polydimethyldiallylammonium chloride (PDDA), and wild-type (WT) photosynthetic reaction center (RC; termed as WT-RC) or its pheophytin (Phe)-replaced counterpart (termed as Phe-RC), was fabricated by self-assembling technique onto gold electrode for facilitating the electron transfer (ET) between RC and the electrode surface. Near-infrared (NIR)-visible (Vis) absorption and fluorescence (FL) emission spectra revealed the influence of pigment substitution on the cofactors arrangement and excitation relaxation of the proteins, respectively. Square wave voltammetry (SWV) and photoelectric tests were employed to systematically address the differences between the WT-RC films and mutant ones on the direct and photo-induced ET. The electrochemical results demonstrated that ET initiated by the oxidation of the primary donor (P) was obviously slowed down, and the formed P+ had more population as well as more positive redox potential in the Phe-RC films compared with those in the WT ones. The photoelectrochemical results displayed the dramatically enhanced photoelectric performances of the mutant ones, further suggesting the slow-down formation of final charge-separated state in Phe-RC. The functionalized protein films introduced in this paper provided an efficient approach to sensitively probe the redox cofactors and ET differences resulting from only minor changes in pigment arrangement in the pigment–protein complex. The favored ET process observed for the membrane proteins RC was potentially valuable for a deep understanding of the multi-step biological ET process and development of versatile bioelectronic devices.

High-field EPR, ENDOR and ELDOR on bacterial photosynthetic reaction centers

We report on recent 95 and 360 GHz high-field electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR) and pulsed electron-electron double resonance (PELDOR) studies of wild-type and mutant reaction centers (RCs) from the photosynthetic bacteriumRhodobacter sphaeroides. Taking advantage of the excellent spectral and temporal resolution of EPR at 95 and 360 GHz, the electron-transfer (ET) cofactors radical ions and spin-correlated radical pairs were characterized by theirg- and hyperfine-tensor components, their anisotropicT2 relaxation as well as by the dipolar interaction between P865•+QA•− radical pairs. The goal of these studies is to better understand the dominant factors determining the specificity and directionality of transmembrane ET processes in photosynthetic RC proteins. In particular, our multifrequency experiments elucidate the subtle cofactor-protein interactions, which are essential for fine-tuning the ET characteristics, e.g., the unidirectionality of the light-induced ET pathways along the A branch of the RC protein. By our high-field techniques, frozen-solution RCs of novel site-specific single and double mutants ofR. sphaeroides were studied to modulate the ET characteristics, e.g., even to the extent that dominant B branch ET prevails. The presented multifrequency EPR work culminates in first 360 GHz ENDOR results from organic nitroxide radicals as well as in first 95 GHz high-field PELDOR results from orientationally selected spin-polarized radical pairs P865•+QA•−, which allow to determine the full geometrical structure of the pairs even in frozen-solution RCs.

Proton Uptake in the Reaction Center Mutant L210DN from Rhodobacter sphaeroides via Protonated Water Molecules,

The reaction center (RC) of Rhodobacter sphaeroides uses light energy to reduce and protonate a quinone molecule, QB (the secondary quinone electron acceptor), to form quinol, QBH2. Asp210 in the L-subunit has been shown to be a catalytic residue in this process. Mutation of Asp210 to Asn leads to a deceleration of reoxidation of QA- in the QA-QB --> QAQB- transition. Here we determined the structure of the Asp210 to Asn mutant to 2.5 A and show that there are no major structural differences as compared to the wild-type protein. We found QB in the distal position and a chain of water molecules between Asn210 and QB. Using time-resolved Fourier transform infrared (trFTIR) spectroscopy, we characterized the molecular reaction mechanism of this mutant. We found that QB- formation precedes QA- oxidation even more pronounced than in the wild-type reaction center. Continuum absorbance changes indicate deprotonation of a protonated water cluster, most likely of the water chain between Asn210 and QB. A detailed analysis of wild-type structures revealed a highly conserved water chain between Asp210 or Glu210 and QB in Rb. sphaeroides and Rhodopseudomonas viridis, respectively.

Iron as a Bound Secondary Electron Donor in Modified Bacterial Reaction Centers

The binding and oxidation of ferrous iron were studied in wild-type reaction centers and in mutants that have been modified to be both highly oxidizing and able to bind manganese [Thielges et al. (2005) Biochemistry 44, 7389-7394]. After illumination of wild-type reaction centers, steady-state optical spectroscopy showed that the oxidized bacteriochlorophyll dimer, P+, could oxidize iron but only as a second-order reaction at iron concentrations above 100 microM. In the modified reaction centers, P+ was reduced by iron in the presence of sodium bicarbonate with dissociation constants of approximately 1 microM for two mutants with different metal-binding sites. Transient optical spectroscopy showed that P+ was rapidly reduced with first-order rates of 170 and 275 s-1 for the two mutants. The dependence of the amplitude of this rate on the iron concentration yielded a dissociation constant of approximately 1 microM for both mutants, in agreement with the steady-state determination. The oxidation of bound iron by P+ was confirmed by the observation of a light-induced EPR signal centered at g values of 2.2 and 4.3 and attributed to high-spin Fe3+. Bicarbonate was required at pH 7 for low dissociation constants for both iron and manganese binding. The similarity between iron and manganese binding in these mutants provides insight into general properties of metal-binding sites in proteins.

Mechanism of Carotenoid Singlet Excited State Energy Transfer in Modified Bacterial Reaction Centers

Ultrafast transient laser spectroscopy has been used to investigate carotenoid singlet excited state energy transfer in various Rhodobacter (Rb.) sphaeroides reaction centers (RCs) modified either genetically or chemically. The pathway and efficiency of energy transfer were examined as a function of the structures and energies of the donor and acceptor molecules. On the donor side, carotenoids with various extents of pi-electron conjugation were examined. RCs studied include those from the anaerobically grown wild-type strain containing the carotenoid spheroidene, which has 10 conjugated carbon-carbon double bonds; the GA strain containing neurosporene, which has nine conjugated double bonds; and aerobically grown wild-type cells, as well as aerobically grown H(M182)L mutant, both containing the carbonyl-containing carotenoid spheroidenone, which has 11 conjugated double bonds. By varying the structure of the carotenoid, we observed the effect of altering the energies of the carotenoid excited states on the rate of energy transfer. Both S(1)- and S(2)-mediated carotenoid-to-bacteriochlorophyll energy transfer processes were observed. The highest transfer efficiency, from both the S(1) and S(2) states, was observed using the carotenoid with the shortest chain. The S(1)-mediated carotenoid-to- bacteriochlorophyll energy transfer efficiencies were determined to be 96%, 84%, and 73% for neurosporene, spheroidene, and spheroidenone, respectively. The S(2)-mediated energy transfer efficiencies follow the same trend but could not be determined quantitatively because of limitations in the time resolution of the instrumentation. The dependence of the energy transfer rate on the energetics of the energy transfer acceptor was verified by performing measurements with RCs from the H(M182)L mutant. In this mutant, the bacteriochlorophyll (denoted B(B)) located between the carotenoid and the RC special pair (P) is replaced by a bacteriopheophytin (denoted phi(B)), where the Q(X) and Q(Y) bands of phi(B) are 1830 and 1290 cm(-1), respectively, higher in energy than those of B(B). These band shifts associated with phi(B) in the H(M182)L mutant significantly alter the spectral overlap between the carotenoid and phi(B), resulting in a significant decrease of the transfer efficiency from the carotenoid S(1) state to phi(B). This leaves energy transfer from the carotenoid S(2) state to phi(B) as the dominant channel. Largely because of this change in mechanism, the overall efficiency of energy transfer from the carotenoid to P decreases to less than 50% in this mutant. Because the spectral signature of phi(B) is different from that of B(A) in this mutant, we were able to demonstrate clearly that the carotenoid-to-P energy transfer is via phi(B). This finding supports the concept that, in wild-type RCs, the carotenoid-to-P energy transfer occurs through the cofactor located at the B(B) position.