Spectral and Photochemical Properties of Rhodobacter sphaeroides R-26 Reaction Center Films in Vacuum.

Using absorption spectroscopy in the visible/near-IR and mid-IR regions, spectral and photochemical properties of isolated reaction centers (RCs) from Rhodobacter sphaeroides R-26 were studied in dried films on the inorganic support surface (quartz or CaF2 plates) under vacuum dehydration conditions (10-2 or 7·10-5 mm Hg). Three detergents, N,N-dimethyldodecylamine N-oxide (LDAO), Triton X-100 (TX100), and n-dodecyl-ß-D-maltoside (DM), were tested for their ability to stabilize the RC-detergent complexes in the vacuum-dried state. It was shown that in the presence of LDAO, RC complexes underwent destruction in vacuum. In contrast, DM provided an environment that minimized irreversible disruptive changes in the RCs in vacuum. The effects of vacuum dehydration on the RC-DM films included a small increase in the content of α-helices in the RC protein, a short-wavelength reversible shift in the optical transitions of pigments, and minor changes in the electronic structure of the P+ dimer. The films retained their photochemical activity upon excitation with high-intensity light (200 mW/cm2). TX100 also helped to maintain spectral and functional properties of the RCs in vacuum; however, in this case, the stabilizing effect was less pronounced than in the presence of DM, especially, at high detergent concentrations. The results are discussed within the framework of a model suggesting that the detergent-protein interactions and the properties of detergent micelles play a dominant role in maintaining the structure of the RCs upon vacuum dehydration of the RC complexes. The obtained data can be useful for developing hybrid photoconverting systems based on bacterial RCs.

Effect of Leucine M196 Substitution by Histidine on Electronic Structure of the Primary Electron Donor and Electron Transfer in Reaction Centers from Rhodobacter sphaeroides.

In our recent X-ray study, we demonstrated that substitution of the natural leucine residue M196 with histidine in the reaction center (RC) from Rhodobacter (Rba.) sphaeroides leads to formation of a close contact between the genetically introduced histidine and the primary electron donor P (bacteriochlorophylls (BChls) PA and PB dimer) creating a novel pigment-protein interaction that is not observed in native RCs. In the present work, the possible nature of this novel interaction and its effects on the electronic properties of P and the photochemical charge separation in isolated mutant RCs L(M196)H are investigated at room temperature using steady-state absorption spectroscopy, light-induced difference FTIR spectroscopy, and femtosecond transient absorption spectroscopy. The results are compared with the data obtained for the RCs from Rba. sphaeroides pseudo-wild type strain. It is shown that the L(M196)H mutation results in a decrease in intensity and broadening of the long-wavelength Qy absorption band of P at ~865 nm. Due to the mutation, there is also weakening of the electronic coupling between BChls in the radical cation P+ and increase in the positive charge localization on the PA molecule. Despite the significant perturbations of the electronic structure of P, the mutant RCs retain high electron transfer rates and quantum yield of the P+QA- state (QA is the primary quinone acceptor), which is close to the one observed in the native RCs. Comparison of our results with the literature data suggests that the imidazole group of histidine M196 forms a π-hydrogen bond with the π-electron system of the PB molecule in the P dimer. It is likely that the specific (T-shaped) spatial organization of the π-hydrogen interaction and its potential heterogeneity in relation to the bonding energy is, at least partially, the reason that this type of interaction between the protein and the pigment and quinone cofactors is not realized in the native RCs.

Temperature Dependence of Light-Induced Absorbance Changes Associated with Chlorophyll Photooxidation in Manganese-Depleted Core Complexes of Photosystem II.

Mid-infrared (4500-1150 cm(-1)) absorbance changes induced by continuous illumination of Mn-depleted core complexes of photosystem II (PSII) from spinach in the presence of exogenous electron acceptors (potassium ferricyanide and silicomolybdate) were studied by FTIR difference spectroscopy in the temperature range 100-265 K. The FTIR difference spectrum for photooxidation of the chlorophyll dimer P680 was determined from the set of signals associated with oxidation of secondary electron donors (ß-carotene, chlorophyll) and reduction of the primary quinone QA. On the basis of analysis of the temperature dependence of the P680(+)/P680 FTIR spectrum, it was concluded that frequencies of 13(1)-keto-C=O stretching modes of neutral chlorophyll molecules PD1 and PD2, which constitute P680, are similar to each other, being located at ~1700 cm(-1). This together with considerable difference between the stretching mode frequencies of keto groups of PD1(+) and PD2(+) cations (1724 and 1709 cm(-1), respectively) is in agreement with a literature model (Okubo et al. (2007) Biochemistry, 46, 4390-4397) suggesting that the positive charge in the P680(+) dimer is mainly localized on one of the two chlorophyll molecules. A partial delocalization of the charge between the PD1 and PD2 molecules in P680(+) is supported by the presence of a characteristic electronic intervalence band at ~3000 cm(-1). It is shown that a bleaching band at 1680 cm(-1) in the P680(+)/P680 FTIR spectrum does not belong to P680. A possible origin of this band is discussed, taking into account the temperature dependence (100-265 K) of light-induced absorbance changes of PSII core complexes in the visible spectral region from 620 to 720 nm.

Chemical modification of photosystem II core complex pigments with sodium borohydride.

The reaction of the irreversible chemical reduction of the 13(1)-keto C=O group of pheophytin a (Pheo a) with sodium borohydride in reaction centers (RCs) of functionally active spinach photosystem II (PS II) core complexes was studied. Stable, chromatographically purified PS II core complex preparations with altered chromophore composition are obtained in which ~25% of Pheo a molecules are modified to 13(1)-deoxo-13(1)-hydroxy-Pheo a. Some of the chlorophyll a molecules in the complexes were also irreversibly reduced with borohydride to 13(1)-deoxo-13(1)-hydroxy-chlorophyll a. Based on the results of comparative study of spectral, biochemical, and photochemical properties of NaBH4-treated and control preparations, it was concluded that: (i) the borohydride treatment did not result in significant dissociation of the PS II core complex protein ensemble; (ii) the modified complexes retained the ability to photoaccumulate the radical anion of the pheophytin electron acceptor in the presence of exogenous electron donor; (iii) only the photochemically inactive pheophytin PheoD2 is subjected to the borohydride treatment; (iv) the Qx optical transition of the PheoD2 molecule in the RC of PS II core complexes is located at 543 nm; (v) in the Qy spectral region, PheoD2 probably absorbs at ~680 nm.

FTIR spectroscopy of the reaction center of Chloroflexus aurantiacus: photooxidation of the primary electron donor.

Photochemical oxidation of the primary electron donor P in reaction centers (RCs) of the filamentous anoxygenic phototrophic bacterium Chloroflexus (C.) aurantiacus was examined by light-induced Fourier transform infrared (FTIR) difference spectroscopy at 95 K in the spectral range of 4000-1200 cm(-1). The light-induced P(+)Q(A)(-)/PQ(A) IR spectrum of C. aurantiacus RCs is compared to the well-characterized FTIR difference spectrum of P photooxidation in the purple bacterium Rhodobacter (R.) sphaeroides R-26 RCs. The presence in the P(+)Q(A)(-)/PQ(A) FTIR spectrum of C. aurantiacus RCs of specific low-energy electronic transitions at ~2650 and ~2200 cm(-1), as well as of associated vibrational (phase-phonon) bands at 1567, 1481, and 1294-1285 cm(-1), indicates that the radical cation P(+) in these RCs has dimeric structure, with the positive charge distributed between the two coupled bacteriochlorophyll a molecules. The intensity of the P(+) absorbance band at ~1250 nm (upon chemical oxidation of P at room temperature) in C. aurantiacus RCs is approximately 1.5 times lower than that in R. sphaeroides R-26 RCs. This fact, together with the decreased intensity of the absorbance band at ~2650 cm(-1), is interpreted in terms of the weaker coupling of bacteriochlorophylls in the P(+) dimer in C. aurantiacus compared to R. sphaeroides R-26. In accordance with the previous (pre)resonance Raman data, FTIR measurements in the carbonyl stretching region show that in C. aurantiacus RCs (i) the 13(1)-keto C=O groups of P(A) and P(B-) molecules constituting the P dimer are not involved in hydrogen bonding in either neutral or photooxidized state of P and (ii) the 3(1)-acetyl C=O group of P(B) forms a hydrogen bond (probably with tyrosine M187) absorbing at 1635 cm(-1). Differential signals at 1757(+)/1749(-) and 1741(+)/1733(-) cm(-1) in the FTIR spectrum of C. aurantiacus RCs are attributed to the 13(3)-ester C=O groups of P in different environments.

Investigation of the redox interaction between Mn-bicarbonate complexes and reaction centers from Rhodobacter sphaeroides R-26, Chromatium minutissimum, and Chloroflexus aurantiacus.

The change in the dark reduction rate of photooxidized reaction centers (RC) of type II from three anoxygenic bacteria (Rhodobacter sphaeroides R-26, Chromatium minutissimum, and Chloroflexus aurantiacus) having different redox potentials of the P(+)/P pair and availability of RC for exogenous electron donors was investigated upon the addition of Mn(2+) and HCO(3)(-). It was found that the dark reduction of P(870)(+) from Rb. sphaeroides R-26 is considerably accelerated upon the combined addition of 0.5 mM MnCl(2) and 30-75 mM NaHCO(3) (as a result of formation of "low-potential" complexes [Mn(HCO(3))(2)]), while MnCl(2) and NaHCO(3) added separately had no such effect. The effect is not observed either in RC from Cf. aurantiacus (probably due to the low oxidation potential of the primary electron donor, P(865), which results in thermodynamic difficulties of the redox interaction between P(865)(+) and Mn(2+)) or in RC from Ch. minutissimum (apparently due to the presence of the RC-bound cytochrome preventing the direct interaction between P(870)(+) and Mn(2+)). The absence of acceleration of the dark reduction of P(870)(+) in the RC of Rb. sphaeroides R-26 when Mn(2+) and HCO(3)(-) were replaced by Mg(2+) or Ca(2+) and by formate, oxalate, or acetate, respectively, reveals the specificity of the Mn2+-bicarbonate complexes for the redox interaction with P(+). The results of this work might be considered as experimental evidence for the hypothesis of the participation of Mn(2+) complexes in the evolutionary origin of the inorganic core of the water oxidizing complex of photosystem II.

Primary electron transfer in reaction centers of YM210L and YM210L/HL168L mutants of Rhodobacter sphaeroides.

The role of tyrosine M210 in charge separation and stabilization of separated charges was studied by analyzing of the femtosecond oscillations in the kinetics of decay of stimulated emission from P* and of a population of the primary charge separated state P(+)B(A)(-) in YM210L and YM210L/HL168L mutant reaction centers (RCs) of Rhodobacter sphaeroides in comparison with those in native Rba. sphaeroides RCs. In the mutant RCs, TyrM210 was replaced by Leu. The HL168L mutation placed the redox potential of the P(+)/P pair 123 mV below that of native RCs, thus creating a theoretical possibility of P(+)B(A)(-) stabilization. Kinetics of P* decay at 940 nm of both mutants show a significant slowing of the primary charge separation reaction in comparison with native RCs. Distinct damped oscillations in these kinetics with main frequency bands in the range of 90-150 cm(-1) reflect mostly nuclear motions inside the dimer P. Formation of a very small absorption band of B(A)(-) at 1020 nm is registered in RCs of both mutants. The formation of the B(A)(-) band is accompanied by damped oscillations with main frequencies from ~10 to ~150 cm(-1). Only a partial stabilization of the P(+)B(A)(-) state is seen in the YM210L/HL168L mutant in the form of a small non-oscillating background of the 1020-nm kinetics. A similar charge stabilization is absent in the YM210L mutant. A model of oscillatory reorientation of the OH-group of TyrM210 in the electric fields of P(+) and B(A)(-) is proposed to explain rapid stabilization of the P(+)B(A)(-) state in native RCs. Small oscillatory components at ~330-380 cm(-1) in the 1020-nm kinetics of native RCs are assumed to reflect this reorientation. We conclude that the absence of TyrM210 probably cannot be compensated by lowering of the P(+)B(A)(-) free energy that is expected for the double YM210L/HL168L mutant. An oscillatory motion of the HOH55 water molecule under the influence of P(+) and B(A)(-) is assumed to be another potential contributor to the mechanism of P(+)B(A)(-) stabilization.

Femtosecond stage of electron transfer in reaction centers of the triple mutant SL178K/GM203D/LM214H of Rhodobacter sphaeroides.

Coherent processes in an initial phase of charge transfer in reaction centers (RCs) of the triple mutant S(L178)K/G(M203)D/L(M214)H of Rhodobacter sphaeroides were investigated by difference (light - dark) absorption spectroscopy with 18 fsec time resolution. Electron transfer in the B cofactor branch is activated in this mutant, while the A-branch electron transfer is slowed in comparison with native RCs of Rba. sphaeroides. A bulk of absorption difference spectra was analyzed in the 940-1060 nm range (stimulated emission of excited bacteriochlorophyll dimer P* and absorption of bacteriochlorophyll anions B(A)(-) and beta(-), where beta is a bacteriochlorophyll substituting the native bacteriopheophytin H(A)) and in the 735-775 nm range (bleaching of the absorption band of the bacteriopheophytin H(B) in the B-branch) in the -0.1 to 4 psec range of delays with respect to the moment of photoexcitation of P at 870 nm. Spectra were measured at 293 and 90 K. The kinetics of P* stimulated emission at 940 nm shows its decay with a time constant of approximately 14 psec at 90 K and approximately 18 psec at 293 K, which is accompanied by oscillations with a frequency of approximately 150 cm(-1). A weak absorption band is found at 1018 nm that is formed approximately 100 fsec after excitation of P and reflects the electron transfer from P* to beta and/or B(A) with accumulation of the P(+)beta(-) and/or P(+)B(A)(-) states. The kinetics of DeltaA at 1018 nm contains the oscillations at approximately 150 cm(-1) and distinct low-frequency oscillations at 20-100 cm(-1); also, the amplitude of the oscillations at 150 cm(-1) is much smaller at 293 than at 90 K. The oscillations in the kinetics of the 1018 nm band do not contain a 32 cm(-1) mode that is characteristic for native Rba. sphaeroides RCs having water molecule HOH55 in their structure. The DeltaA kinetics at 751 nm reflects the electron transfer to H(B) with formation of the P(+)H(B)(-) state. The oscillatory part of this kinetics has the form of a single peak with a maximum at ~50 fsec completely decaying at ~200 fsec, which might reflect a reversible electron transfer to the B-branch. The results are analyzed in terms of coherent nuclear wave packet motion induced in the P* excited state by femtosecond light pulses, of an influence of the incorporated mutations on the mutual position of the energy levels of charge separated states, and of the role of water HOH55 in the dynamics of the initial electron transfer.

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.

[Mechanism of charge separation and their stabilization in bacterial reaction centers]. / Mekhanizm razdeleniia zariadov i ikh stabilizatsii v bakterial'nykh reaktsionnykh tsentrakh.

The nuclear wavepacket formed by 20-fs excitation on the P* potential energy surface in native and mutant (YM210W and YM210L) reaction centers of Rhodobacter (Rb.) sphaeroides and Chloroflexus (C.) aurantiacus RCs was found to be reversibly transferred to the P+BA- surface at 120, 380, and 640-fs delays (monitored by measurements of BA- absorption at 1020-1028 nm). The reaction centers of YM210W(L) mutant show the most simple pattern of fs oscillations with a period of 230 fs in stimulated emission from P* and in the product P+BA-. The mechanisms of the electron transfer pathway between P* and BA and of the stabilization of the state P+BA- in bacterial reaction centers are discussed.

Reaction centers of photosystem II with a chemically-modified pigment composition: exchange of pheophytins with 13(1)-deoxo-13(1)-hydroxy-pheophytin a.

Isolated reaction centers of photosystem II with an altered pigment content were obtained by chemical exchange of the native pheophytin a molecules with externally added 13(1)-deoxo-13(1)-hydroxy-pheophytin a. Judged from a comparison of the absorption spectra and photochemical activities of exchanged and control reaction centers, 70-80% of the pheophytin molecules active in charge separation are replaced by 13(1)-deoxo-13(1)-hydroxy-pheophytin a after double application of the exchange procedure. The new molecule at the active branch was not active photochemically. This appears to be the first stable preparation in which a redox active chromophore of the reaction center of photosystem II was modified by chemical substitution. The data are compatible with the presence of an active and inactive branch of cofactors, as in bacterial reaction centers. Possible applications of the 13(1)-deoxo-13(1)-hydroxy-pheophytin a-exchanged preparation to the spectral and functional analysis of native reaction centers of photosystem II are discussed.

Spectral and photochemical properties of borohydride-treated D1-D2-cytochrome b-559 complex of photosystem II.

The D1-D2-cytochrome b-559 reaction center complex of photosystem II with an altered pigment composition was prepared from the original complex by treatment with sodium borohydride (BH4-). The absorption spectra of the modified and original complexes were compared to each other and to the spectra of purified chlorophyll a and pheophytin a (Pheo a) treated with BH4- in methanolic solution. The results of these comparisons are consistent with the presence in the modified complex of an irreversibly reduced Pheo a molecule, most likely 13(1)-deoxo-13(1)-hydroxy-Pheo a, replacing one of the two native Pheo a molecules present in the original complex. Similar to the original preparation, the modified complex was capable of a steady-state photoaccumulation of Pheo- and P680+. It is concluded that the pheophytin a molecule which undergoes borohydride reduction is not involved in the primary charge separation and seems to represent a previously postulated photochemically inactive Pheo a molecule. The Qy and Qx transitions of this molecule were determined to be located at 5 degrees C at 679.5-680 nm and 542 nm, respectively.

Formation of charge separated state P+OA- and triplet state 3P at low temperature in Rhodobacter sphaeroides (R-26) reaction centers in which bacteriopheophytin a is replaced by plant pheophytin a.

Low temperature optical and photochemical properties of Rhodobacter sphaeroides (R-26) reaction centers, in which bacteriopheophytin a has been replaced by plant pheophytin a, are reported. Modified reaction centers preserve the ability for photoinduced electron transfer from the primary electron donor P to the primary quinone acceptor QA at 80K. The triplet state ESR signal of modified reaction centers with prereduced QA at 10K shows an electron spin polarization pattern and ZFS parameters analogous to those for the triplet state 3P in non-treated reaction centers. It was found that at low temperature both P+QA- and 3P states are formed via a precursor radical pair P+I- in which I is the introduced plant pheophytin molecule. This shows that acceptor systems of bacterial and plant (photosystem II) reaction centers are mutually replacable in structural and functional aspects.