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.

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.

Primary light-energy conversion in tetrameric chlorophyll structure of photosystem II and bacterial reaction centers: II. Femto- and picosecond charge separation in PSII D1/D2/Cyt b559 complex.

In Part I of the article, a review of recent data on electron-transfer reactions in photosystem II (PSII) and bacterial reaction center (RC) has been presented. In Part II, transient absorption difference spectroscopy with 20-fs resolution was applied to study the primary charge separation in PSII RC (DI/DII/Cyt b 559 complex) excited at 700 nm at 278 K. It was shown that the initial electron-transfer reaction occurs within 0.9 ps with the formation of the charge-separated state P680(+)Chl(D1)(-), which relaxed within 14 ps as indicated by reversible bleaching of 670-nm band that was tentatively assigned to the Chl(D1) absorption. The subsequent electron transfer from Chl(D1)(-) within 14 ps was accompanied by a development of the radical anion band of Pheo(D1) at 445 nm, attributable to the formation of the secondary radical pair P680(+)Pheo(D1)(-). The key point of this model is that the most blue Q(y) transition of Chl(D1) in RC is allowing an effective stabilization of separated charges. Although an alternative mechanism of charge separation with Chl(D1)* as a primary electron donor and Pheo(D1) as a primary acceptor can not be ruled out, it is less consistent with the kinetics and spectra of absorbance changes induced in the PSII RC preparation by femtosecond excitation at 700 nm.