A Ya Shkuropatov

Russian Academy of Sciences, Moscow, Moscow, Russia

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Publications (27)57.99 Total impact

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    ABSTRACT: Ultrafast absorption spectroscopy with 20-fs resolution was applied to study primary charge separation in spinach photosystem II (PSII) reaction center (RC) and PSII core complex (RC complex with integral antenna) upon excitation at maximum wavelength 700-710 nm at 278 K. It was found that the initial charge separation between P680* and ChlD1 (Chl-670) takes place with a time constant of ~1 ps with the formation of the primary charge-separated state P680* with an admixture of: P680*((1-δ)) (P680(δ+)ChlD1(δ-)), where δ ~ 0.5. The subsequent electron transfer from P680(δ+)ChlD1(δ-) to pheophytin (Pheo) occurs within 13 ps and is accompanied by a relaxation of the absorption band at 670 nm (ChlD1(δ-)) and bleaching of the PheoD1 bands at 420, 545, and 680 nm with development of the Pheo(-) band at 460 nm. Further electron transfer to QA occurs within 250 ps in accordance with earlier data. The spectra of P680(+) and Pheo(-) formation include a bleaching band at 670 nm; this indicates that Chl-670 is an intermediate between P680 and Pheo. Stimulated emission kinetics at 685 nm demonstrate the existence of two decaying components with time constants of ~1 and ~13 ps due to the formation of P680(δ+)ChlD1(δ-) and P680(+)PheoD1(-), respectively.
    Biochemistry (Moscow) 03/2014; 79(3):197-204. · 1.15 Impact Factor
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    ABSTRACT: 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.
    Biochemistry (Moscow) 04/2013; 78(4):377-384. · 1.15 Impact Factor
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    ABSTRACT: 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.
    Biochemistry (Moscow) 02/2012; 77(2):157-64. · 1.15 Impact Factor
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    ABSTRACT: 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.
    Biochemistry (Moscow) 12/2011; 76(12):1360-6. · 1.15 Impact Factor
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    ABSTRACT: Low temperature (77-90 K) measurements of absorption spectral changes induced by red light illumination in isolated photosystem II (PSII) reaction centers (RCs, D1/D2/Cyt b559 complex) with different external acceptors and in PSII core complexes have shown that two different electron donors can alternatively function in PSII: chlorophyll (Chl) dimer P(680) absorbing at 684 nm and Chl monomer Chl(D1) absorbing at 674 nm. Under physiological conditions (278 K) transient absorption difference spectroscopy with 20-fs resolution was applied to study primary charge separation in spinach PSII core complexes excited at 710 nm. It was shown that the initial electron transfer reaction takes place with a time constant of ~0.9 ps. This kinetics was ascribed to charge separation between P(680)* and Chl(D1) absorbing at 670 nm accompanied by the formation of the primary charge-separated state P(680)(+)Chl(DI)(-), as indicated by 0.9-ps transient bleaching at 670 nm. The subsequent electron transfer from Chl(D1)(-) occurred within 13-14 ps and was accompanied by relaxation of the 670-nm band, bleaching of the Pheo(D1) Q(x) absorption band at 545 nm, and development of the anion-radical band of Pheo(D1)(-) at 450-460 nm, the latter two attributable to formation of the secondary radical pair P(680)(+)Pheo(D1)(-). The 14-ps relaxation of the 670-nm band was previously assigned to the Chl(D1) absorption in isolated PSII RCs [Shelaev, Gostev, Nadtochenko, Shkuropatov, Zabelin, Mamedov, Semenov, Sarkisov and Shuvalov, Photosynth. Res. 98 (2008) 95-103]. We suggest that the longer wavelength position of P(680) (near 680 nm) as a primary electron donor and the shorter wavelength position of Chl(D1) (near 670 nm) as a primary acceptor within the Q(y) transitions in RC allow an effective competition with an energy transfer and stabilization of separated charges. Although an alternative mechanism of charge separation with Chl(D1)* as the primary electron donor and Pheo(D1) as the primary acceptor cannot be ruled out, the 20-fs excitation at the far-red tail of the PSII core complex absorption spectrum at 710 nm appears to induce a transition to a low-energy state P(680)* with charge-transfer character (probably P(D1)(δ+)P(D2)(δ-)) which results in an effective electron transfer from P(680)* (the primary electron donor) to Chl(D1) as the intermediary acceptor.
    Journal of photochemistry and photobiology. B, Biology 03/2011; 104(1-2):44-50. · 3.11 Impact Factor
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    ABSTRACT: 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.
    Biochemistry (Moscow) 07/2010; 75(7):832-40. · 1.15 Impact Factor
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    ABSTRACT: Difference absorption spectroscopy with temporal resolution of approximately 20 fsec was used to study the primary phase of charge separation in isolated reaction centers (RCs) of Chloroflexus aurantiacus at 90 K. An ensemble of difference (light-minus-dark) absorption spectra in the 730-795 nm region measured at -0.1 to 4 psec delays relative to the excitation pulse was analyzed. Comparison with analogous data for RCs of HM182L mutant of Rhodobacter sphaeroides having the same pigment composition identified the 785 nm absorption band as the band of bacteriopheophytin Phi(B) in the B-branch. By study the bleaching of this absorption band due to formation of Phi(B)(-), it was found that a coherent electron transfer from P* to the B-branch occurs with a very small delay of 10-20 fsec after excitation of dimer bacteriochlorophyll P. Only at 120 fsec delay electron transfer from P* to the A-branch occurs with the formation of bacteriochlorophyll anion B(A)(-) absorption band at 1028 nm and the appearance of P* stimulated emission at 940 nm, as also occurs in native RCs of Rb. sphaeroides. It is concluded that a nuclear wave packet motion on the potential energy surface of P* after a 20-fsec light pulse excitation leads to the coherent formation of the P(+)Phi(B)(-) and P(+)B(A)(-) states.
    Biochemistry (Moscow) 08/2009; 74(8):846-54. · 1.15 Impact Factor
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    ABSTRACT: 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.
    Photosynthesis Research 11/2008; 98(1-3):95-103. · 3.15 Impact Factor
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    ABSTRACT: Transient absorption difference spectroscopy with approximately 20 femtosecond (fs) resolution was applied to study the time and spectral evolution of low-temperature (90 K) absorbance changes in isolated reaction centers (RCs) of Chloroflexus (C.) aurantiacus. In RCs, the composition of the B-branch chromophores is different with respect to that of purple bacterial RCs by occupying the B(B) binding site of accessory bacteriochlorophyll by bacteriopheophytin molecule (Phi(B)). It was found that the nuclear wave packet motion induced on the potential energy surface of the excited state of the primary electron donor P* by approximately 20 fs excitation leads to a coherent formation of the states P+Phi(B)(-) and P+B(A)(-) (B(A) is a bacteriochlorophyll monomer in the A-branch of cofactors). The processes were studied by measuring coherent oscillations in kinetics of the absorbance changes at 900 nm and 940 nm (P* stimulated emission), at 750 nm and 785 nm (Phi(B) absorption bands), and at 1,020-1028 nm (B(A)(-) absorption band). In RCs, the immediate bleaching of the P band at 880 nm and the appearance of the stimulated wave packet emission at 900 nm were accompanied (with a small delay of 10-20 fs) by electron transfer from P* to the B-branch with bleaching of the Phi(B) absorption band at 785 nm due to Phi(B)(-) formation. These data are consistent with recent measurements for the mutant HM182L Rb. sphaeroides RCs (Yakovlev et al., Biochim Biophys Acta 1757:369-379, 2006). Only at a delay of 120 fs was the electron transfer from P* to the A-branch observed with a development of the B(A)(-) absorption band at 1028 nm. This development was in phase with the appearance of the P* stimulated emission at 940 nm. The data on the A-branch electron transfer in C. aurantiacus RCs are consistent with those observed in native RCs of Rb. sphaeroides. The mechanism of charge separation in RCs with the modified B-branch pigment composition is discussed in terms of coupling between the nuclear wave packet motion and electron transfer from P* to Phi(B) and B(A) primary acceptors in the B-branch and A-branch, respectively.
    Journal of Bioinformatics and Computational Biology 09/2008; 6(4):643-66. · 0.93 Impact Factor
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    ABSTRACT: Femtosecond absorption difference spectroscopy was applied to study the time and spectral evolution of low-temperature (90 K) absorbance changes in isolated reaction centers (RCs) of the HM182L mutant of Rhodobacter (Rb.) sphaeroides. In this mutant, the composition of the B-branch RC cofactors is modified with respect to that of wild-type RCs by replacing the photochemically inactive BB accessory bacteriochlorophyll (BChl) by a photoreducible bacteriopheophytin molecule (referred to as PhiB). We have examined vibrational coherence within the first 400 fs after excitation of the primary electron donor P with 20-fs pulses at 870 nm by studying the kinetics of absorbance changes at 785 nm (PhiB absorption band), 940 nm (P*-stimulated emission), and 1020 nm (BA- absorption band). The results of the femtosecond measurements are compared with those recently reported for native Rb. sphaeroides R-26 RCs containing an intact BB BChl. At delay times longer than approximately 50 fs (maximum at 120 fs), the mutant RCs exhibit a pronounced BChl radical anion (BA-) absorption band at 1020 nm, which is similar to that observed for Rb. sphaeroides R-26 RCs and represents the formation of the intermediate charge-separated state P+ BA-. Femtosecond oscillations are revealed in the kinetics of the absorption development at 1020 nm and of decay of the P*-stimulated emission at 940 nm, with the oscillatory components of both kinetics displaying a generally synchronous behavior. These data are interpreted in terms of coupling of wave packet-like nuclear motions on the potential energy surface of the P* excited state to the primary electron-transfer reaction P*-->P+ BA- in the A-branch of the RC cofactors. At very early delay times (up to 80 fs), the mutant RCs exhibit a weak absorption decrease around 785 nm that is not observed for Rb. sphaeroides R-26 RCs and can be assigned to a transient bleaching of the Qy ground-state absorption band of the PhiB molecule. In the range of 740-795 nm, encompassing the Qy optical transitions of bacteriopheophytins HA, HB, and PhiB, the absorption difference spectra collected for mutant RCs at 30-50 fs resemble the difference spectrum of the P+ PhiB- charge-separated state previously detected for this mutant in the picosecond time domain (E. Katilius, Z. Katiliene, S. Lin, A.K.W. Taguchi, N.W. Woodbury, J. Phys. Chem., B 106 (2002) 1471-1475). The dynamics of bleaching at 785 nm has a non-monotonous character, showing a single peak with a maximum at 40 fs. Based on these observations, the 785-nm bleaching is speculated to reflect reduction of 1% of PhiB in the B-branch within about 40 fs, which is earlier by approximately 80 fs than the reduction process in the A-branch, both being possibly linked to nuclear wave packet motion in the P* state.
    Biochimica et Biophysica Acta 01/2006; 1757(5-6):369-79. · 4.66 Impact Factor
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    ABSTRACT: Coherent components in the dynamics of decay of stimulated emission from the primary electron donor excited state P*, and of population of the product charge-separated states and , were studied in GM203L mutant reaction centers (RCs) of Rhodobacter (Rb.) sphaeroides by measuring oscillations in the kinetics of absorbance changes at 940 nm (P* stimulated emission region), 1020 nm ( absorption region) and 760 nm (HA bleaching region). Absorbance changes were induced by excitation of P (870 nm) with 18 fs pulses at 90 K. In the GM203L mutant, replacement of Gly M203 by Leu results in exclusion of the crystallographically defined water molecule (HOH55) located close to the oxygen of the 131-keto carbonyl group of BA and to His M202, which provides the axial ligand to the Mg of the PB bacteriochlorophyll. The results of femtosecond measurements were compared with those obtained with Rb. sphaeroides R-26 RCs containing an intact water HOH55. The main consequences of the GM203L mutation were found to be as follows: (i) a low-frequency oscillation at 32 cm−1, which is characteristic of the HOH55-containing RCs, disappears from the kinetics of absorbance changes at 1020 and 760 nm in the mutant RC; (ii) electron transfer from P* to BA in the wild type RC was characterized by two time constants of 1.1 ps (80%) and 4.3 ps (20%), but in the GM203L mutant was characterized by a single time constant of 4.3 ps, demonstrating a slowing of primary charge separation. The previously postulated rotation of water HOH55 with a fundamental frequency of 32 cm−1, triggered by electron transfer from P* to BA, was confirmed by observation of an isotopic shift of the 32 cm−1 oscillation in the kinetics of population in deuterated, pheophytin-modified RCs of Rb. sphaeroides R-26, by a factor of 1.6. These data are discussed in terms of the influence of water HOH55 on the energetics of the reaction, and protein dynamic events that occur on the time scale of this reaction.
    Chemical Physics 01/2005; · 1.96 Impact Factor
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    ABSTRACT: Energy and electron transfer in Photosystem II reaction centers in which the photochemically inactive pheophytin had been replaced by 13(1)-deoxo-13(1)-hydroxy pheophytin were studied by femtosecond transient absorption-difference spectroscopy at 77 K and compared to the dynamics in untreated reaction center preparations. Spectral changes induced by 683-nm excitation were recorded both in the Q(Y) and in the Q(X) absorption regions. The data could be described by a biphasic charge separation. In untreated reaction centers the major component had a time constant of 3.1 ps and the minor component 33 ps. After exchange, time constants of 0.8 and 22 ps were observed. The acceleration of the fast phase is attributed in part to the redistribution of electronic transitions of the six central chlorin pigments induced by replacement of the inactive pheophytin. In the modified reaction centers, excitation of the lowest energy Q(Y) transition produces an excited state that appears to be localized mainly on the accessory chlorophyll in the active branch (B(A) in bacterial terms) and partially on the active pheophytin H(A). This state equilibrates in 0.8 ps with the radical pair. B(A) is proposed to act as the primary electron donor also in untreated reaction centers. The 22-ps (pheophytin-exchanged) or 33-ps (untreated) component may be due to equilibration with the secondary radical pair. Its acceleration by H(B) exchange is attributed to a faster reverse electron transfer from B(A) to. After exchange both and are nearly isoenergetic with the excited state.
    Biophysical Journal 04/2004; 86(3):1664-72. · 3.67 Impact Factor
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    ABSTRACT: The photosynthetic reaction centers (RCs) of Rhodobacter sphaeroides are membrane-bound pigment-protein complexes. Detailed three-dimensional structure of the Rb. sphaeroides RC has been resolved by X-ray diffraction [1]. It was found that native RC contained three protein subunits (L, M, and H); four molecules of bacteriochlorophyll (BChl) a , with two of them forming a special pair that serves as the primary electron donor (P); two bacteriopheophytin molecules (Bpheo); two ubiquinone molecules (Q A and Q B ); one atom of nonheme iron (Fe 2+ ); and one carotenoid molecule. According to current concepts, the efficiency of the photochemical reaction in RC is determined by the structure of the electron-transfer cofactors and their interaction with each other and with the protein moiety of the RC [2]. The methods of chemical modification of RC pigments [3] and site-directed modification of RC polypeptides using the methods of genetic engineering [4] provide a promising opportunity for studying the mechanisms of this process. The goal of this work was to find mutations in the vicinity of the primary electron donor that would modify spectral, photochemical, and structural characteristics of RC bacteriochlorophyll molecules constituting P. As a result of screening for these mutations, new mutant reaction centers were obtained, in which isoleucine-206 in the M-subunit of RC was substituted by histidine. Methods of mutagenesis and conditions of cultivation of bacterial cells were described in [5]. Reaction centers were isolated from chromatophores treated with the detergent lauryl dimethylamine oxide (LDAO), as described in [6], and purified by ion-exchange chromatography on DEAE-cellulose. The extent of purification of RC preparations was estimated by the ratio of absorption bands of protein and BChl monomer. Absorption spectra were measured using a Shimadzu UV-1601PC spectrophotometer. Photoinduced absorption changes ( Δ A ) with millisecond time resolution were recorded using a single-beam differential spectrophotometer equipped with a phosphoroscope (effectivelight wavelength, 875 nm). All measurements were performed at room temperature. The three-dimensional structure of the Rb. sphaeroides RC was taken from the Protein Data Bank (http://www.rcsb.org/pdb/#1AIJ). The procedure of the protein matrix energy minimization was implemented using the Swiss-PDB-viewer software (www.genebee.msu.su, Moscow State University).
    Doklady Biochemistry and Biophysics 01/2004; 394:26-9. · 0.32 Impact Factor
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    ABSTRACT: The nuclear wave packet formed by 20 fs excitation on the P* potential energy surface in native and mutant (YM210W and YM210L) reaction centers (RCs) of Rhodobacter (Rb.) sphaeroides and in Chloroflexus (C.) aurantiacus RCs was found to be reversibly transferred to the P+BA- surface at 120, 380, etc. fs delays (monitored by measurements of BA- absorption at 1020−1028 nm). The YM210W(L) mutant RCs show the most simple pattern of femtosecond oscillations with a period of 230 fs in stimulated emission from P* and with the initial amplitude comparable to that in plant pheophytin a (Pheo)-modified Rb. sphaeroides R-26 RCs. Similar reversible oscillations are observed in the 1020 nm band of the mutants, the initial amplitude of which is smaller by a factor of 10 with respect to Pheo-modified Rb. sphaeroides R-26 RCs. In contrast to native and Pheo-modified Rb. sphaeroides R-26 RCs, irreversible quasi-exponential stabilization of P+BA- is considerably suppressed in the mutant RCs in the picosecond time domain. The water rotational mode with a frequency of 32 cm-1 and its overtones, described earlier (Yakovlev; et al. Biochemistry 2002, 41, 2667−2674), are decreased in the YM210W(L) mutants and strongly suppressed in dry films of the mutant RCs. In the dry film of both YM210W and YM210L RCs neither reversible nor irreversible P+BA- formation monitored at 1020 nm is observed despite the preservation of fs oscillations with a frequency of 144 cm-1 in the 935 nm kinetics of stimulated emission from P*. Furthermore, the 1020 nm band is not formed inside of P*. In C. aurantiacus RCs, containing leucine instead of tyrosine at the M208 position, the P* decay is slowed to 5 ps at 90 K (1.5 ps in Rb. sphaeroides RCs) and characterized by fs oscillations with the amplitude comparable to that measured in native Rb. sphaeroides R-26 RCs. The BA- absorption band development at 1028 nm is observed at 90 K with fs oscillations similar to those described for native Rb. sphaeroides R-26 RCs at 293 K but with the amplitude being smaller by a factor of 6. The kinetics of absorbance changes in the 1028 nm band in C. aurantiacus RCs includes the stabilization of P+BA- within 5 ps with subsequent decay due to electron transfer to HA within 1 ps. The mechanisms of the electron-transfer between P* and BA and of the stabilization of the state P+BA- in bacterial RCs are discussed.
    The Journal of Physical Chemistry A 09/2003; 107(40). · 2.77 Impact Factor
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    ABSTRACT: We present an electric field modulated absorption spectroscopy (Stark effect) study of isolated photosystem II reaction center complexes, including a preparation in which the inactive pheophytin H(B) was exchanged for 13(1)-deoxo-13(1)-hydroxy-pheophytin. The results reveal that the Stark spectrum of the Q(x) and Q(y) transitions of the pheophytins has a second-derivative line shape, indicating that the Stark effect is dominated by differences in the dipole moment between the ground and the electronically excited states of these transitions (Delta mu). The Delta mu values for the Q(x) and Q(y) transitions of H(B) are small (Delta mu = 0.6-1.0 D f(-1)), whereas that of the Q(x) transition of the active pheophytin H(A) is remarkably large (Delta mu = 3 D f(-1)). The Stark spectrum of the red-most absorbing pigments also shows a second-derivative line shape, but this spectrum is considerably red-shifted as compared to the second derivative of the absorption spectrum. This situation is unusual but has been observed before in heterodimer special pair mutants of purple bacterial reaction centers [Moore, L. J., Zhou, H., and Boxer, S. G. (1999) Biochemistry 38, 11949-11960]. The red-shifted Stark spectra can be explained by a mixing of exciton states with a charge-transfer state of about equal energy. We conclude that the charge transfer state involves H(A) and its immediate chlorophyll neighbor (B(A)), and we suggest that this (B(A)(delta+)H(A)(delta-)) charge transfer state plays a crucial role in the primary charge separation reaction in photosystem II. In contrast to most other carotenes, the two beta-carotene molecules of the photosystem II reaction center display a very small Delta mu, which can most easily be explained by excitonic coupling of both molecules. These results favor a model that locates both beta-carotene molecules at the same side of the complex.
    Biochemistry 09/2003; 42(30):9205-13. · 3.38 Impact Factor
  • A G Yakovlev, A Ya Shkuropatov, V A Shuvalov
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    ABSTRACT: Results are presented of a study of primary processes of formation of the charge separated states P(+)B(A)(-) and P(+)H(A)(-) (where P is the primary electron donor, B(A) and H(A) the primary and secondary electron acceptors) in native and pheophytin-modified reaction centers (RCs) of Rhodobacter sphaeroides R-26 by methods of femtosecond spectroscopy of absorption changes at low temperature. Coherent oscillations were studied in the kinetics at 935 nm (P* stimulated emission band), at 1020 nm (B(A)(-) absorption band), and at 760 nm (H(A) absorption band). It was found that when the wavepacket created under femtosecond light excitation approaches the intersection between P* and P(+)B(A)(-) potential surfaces at 120- and 380-fsec delays, the formation of two electron states emitting light at 935 nm (P*) and absorbing light at 1020 nm (P(+)B(A)(-)) takes place. At the later time the wavepacket motion has a frequency of 32 cm(-1) and is accompanied by electron transfer from P* to B(A) in pheophytin-modified and native RCs and further to H(A) in native RCs. It was shown that electron transfer processes monitored by the 1020-nm absorption band development as well as by bleaching of 760-nm absorption band have the enhanced 32 cm(-1) mode in the Fourier transform spectra.
    Biochemistry (Moscow) 06/2003; 68(5):541-50. · 1.15 Impact Factor
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    ABSTRACT: The bacteriopheophytin a molecules at the H(A) and H(B) binding sites of reaction centers (RCs) of the Y(M210)W mutant of Rhodobacter sphaeroides were chemically exchanged with plant pheophytin a. The Y(M210)W mutation slows down the formation of H(A)(-), presumably by raising the free energy level of the P(+)B(A)(-) state above that of P* due to increasing the oxidation potential of the primary electron donor P and lowering the reduction potential of the accessory bacteriochlorophyll B(A). Exchange of the bacteriopheophytins with pheophytin a on the contrary lowers the redox potential of H(A), inhibiting its reduction. A combination of the mutation and pigment exchange was therefore expected to make the A-side of the RC incapable of electron transfer and cause the excited state P* to deactivate directly to the ground state or through the B-side, or both. Time-resolved absorption difference spectroscopy at 10 K on the RCs that were modified in this way showed a lifetime of P* lengthened to about 500 ps as compared to about 200 ps measured in the original Y(M210)W RCs. We show that the decay of P* in the pheophytin-exchanged preparations is accompanied by both return to the ground state and formation of a new charge-separated state, the absorption difference spectrum of which is characterized by bleachings at 811 and 890 nm. This latter state was formed with a time constant of ca. 1.7 ns and a yield of about 30%, and lasted a few nanoseconds. On the basis of spectroscopic observations these bands at 811 and 890 nm are tentatively attributed to the presence of the P(+)B(B)(-) state, where B(B) is the accessory bacteriochlorophyll in the "inactive" B-branch of the cofactors. The B(B) molecules in Y(M210)W RCs are suggested to be spectrally heterogeneous, absorbing in the Q(y) region at 813 or 806 nm. The results are discussed in terms of perturbation of the free energy level of the P(+)B(B)(-) state and absorption properties of the B(B) bacteriochlorophyll in the mutant RCs due to a long-range effect of the Y(M210)W mutation on the protein environment of the B(B) binding pocket.
    Biochimica et Biophysica Acta 04/2003; 1557(1-3):1-12. · 4.66 Impact Factor
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    ABSTRACT: Time-resolved cw EPR measurements of the Rhodobacter (Rb) sphaeroides R-26 reaction center primary donor triplet state excited with plane-polarised light are reported. The pigment composition of the reaction center was chemically modified, so that the bacteriopheophytin molecule in the cofactor branch which is inactive towards electron transfer was replaced by plant pheophytin a. This enabled selective excitation of the bacteriopheophytin and pheophytin molecules, and provided conditions for a high-quality magnetophotoselection study. For the first time, orientation of the Qy optical transition dipole moment relative to the molecular frame of the bacteriopheophytin in the active cofactor branch is determined. Of the four orientations allowed by magnetophotoselection, one was chosen as the most plausible. The corresponding Qy vector is tilted from the bacteriopheophytin tetrapyrrole plane by 15°, and projects onto this plane almost on the y-molecular axis. It is suggested that the deviation of the vector from the molecular plane results from an interaction of bacteriopheophytin with the neighbouring molecule of accessory bacteriochlorophyll.
    Chemical Physics 01/2003; 294(3):451-458. · 1.96 Impact Factor
  • Andrei G Yakovlev, Anatoli Ya Shkuropatov, Vladimir A Shuvalov
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    ABSTRACT: In Rhodobacter sphaeroides R-26 reaction centers (RCs) the nuclear wave packet induced by 25 fs excitation at 90 K moves on the primary electron donor P* potential energy hypersurface with initial frequency at approximately 130 cm(-1) (monitored by stimulated emission measurement). At the long-wavelength side of P* stimulated emission at 935 nm the wave packet is transferred to the surface with P(+)B(A)(-) character at 120, 380, 1.2 fs, etc. delays (monitored by measurement of the primary electron acceptor B(A)(-) band at 1020 nm). However, only beginning from 380 fs delay and later the relative stabilization of the state P(+)B(A)(-) is observed. This is accompanied by the electron transfer to bacteriopheophytin H(A) (monitored by H(A) band measurement at 760 nm). The most active mode of 32 cm(-1) in the electron transfer and its overtones up to the seventh were found in the Fourier transform spectrum of the oscillatory part of the kinetics of the P* stimulated emission and of the P(+)B(A)(-) and P(+)H(A)(-) formation. This mode and its overtones are apparently populated via the 130 cm(-1) vibrational mode. The deuteration of the sample shifts the fundamental frequency (32 cm(-1)) and all overtones by the same factor of approximately 1.3. This mode and its overtones are suppressed by a factor of approximately 4.7 in the dry film of RCs. The results obtained indicate that the 32 cm(-1) mode might be related to a rotation of hydrogen-containing groups (possibly the water molecule) participating in the modulation of the primary electron transfer from P* to B(A)(-) in at least 35% of RCs. The Brookhaven Protein Data Bank (1PRC) displays the water molecule located at the position HOH302 between His M200 (axial ligand for P(B)) and the oxygen of ring V of B(A) which might be a part (approximately 35%) of the molecular pathway for electron transfer from P* to B(A).
    Biochemistry 12/2002; 41(47):14019-27. · 3.38 Impact Factor
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    ABSTRACT: Soret-excited resonance Raman spectra of two types of pheophytin-exchanged photosystem II RCs are reported. The cofactor composition of the reaction centers was modified by exchanging pheophytin a for 13(1)-deoxo-13(1)-hydroxypheophytin a, yielding one preparation with selective replacement of the photochemically inactive pheophytin (H(B)) and a second one exhibiting total replacement of H(B) and 40% replacement of H(A), the primary electron acceptor. Resonance Raman spectra indicate that the other bound cofactors present are not significantly perturbed by Pheo substitution. The resonance Raman contributions from H(A) and H(B) in the carbonyl stretching region are identified at 1679 and 1675 cm(-)(1), respectively, indicating that both pheophytin molecules in the photosystem II reaction center have hydrogen-bonded keto-carbonyl groups. This conclusion differs from what is observed in the functionally related RCs of purple non-sulfur bacteria, where the keto-carbonyl group of H(B) is not hydrogen bonded, but confirms predictions from models based on protein sequence alignments.
    Biochemistry 10/2002; 41(38):11449-55. · 3.38 Impact Factor

Publication Stats

215 Citations
57.99 Total Impact Points

Institutions

  • 1997–2012
    • Russian Academy of Sciences
      • • Puschino Institute of Basic Biological Problems
      • • N. N. Semenov Institute of Chemical Physics
      • • Institute of Soil Science and Photosynthesis
      Moscow, Moscow, Russia
    • Leiden University
      Leyden, South Holland, Netherlands
  • 2003–2010
    • Lomonosov Moscow State University
      • Department of Biophysics
      Moscow, Moscow, Russia
  • 2002–2008
    • Moscow State Textile University
      Moskva, Moscow, Russia
  • 2005
    • University of Bristol
      • School of Biochemistry
      Bristol, ENG, United Kingdom