Valentina A. Shkuropatova

Photoreactions of bacteriopheophytins and bacteriochlorophylls in reaction centers of Rhodopseudomonas sphaeroides and Chloroflexus aurantiacus

Abstract A comparison of spectral properties of reaction centers from Chloroflexus aurantiacus and Rhodopseudomonas sphaeroides (R-26) is reported. Treatment of reaction centers from Rps. sphaeroides with NaBH4 leads to a decrease of the dipole strength of the 800-nm band by factor of approx. 1.75-1.95 and to the formation of new bacteriopheophytin, BPh-715, which is almost completely removed during the purification of reaction centers. The modification of the reaction centers does not change the quantum yield of P photooxidation and the spectrum of BPh-545 (H1) photoreduction which includes the changing of the 800-nm band. This implies the preservation of the photoactive chain P-B1-H1-QA (where B1 is the bacteriochlorophyll (BChl)-800 molecule situated between P and H1) and the modification of the second BChl-800 (B2). The preparation of modified reaction centers is a mixture of at least three types of reaction centers with different contents of B2 and of the second BPh (H2). Some of the reaction centers (5-25%) contain the original B2 and H2 molecules (type I). In the CD spectrum of modified reaction centers a decrease of the 800-nm band and the appearance of a positive band at 765 nm is observed. This spectrum is similar to the CD spectrum of Chloroflexus reaction centers containing 3 BPhs and 3 BChls. This implies that in some (approx. 40%) of the modified Rps. sphaeroides reaction centers (type II) B2 has been replaced by BPh a which interacts with H2. Probably some of the modified reaction centers (approx. 40%) have lost both B2 and H2 (type III). The modification of reaction centers leads to a considerable decrease of the CD bands at 800 (+) nm and 810 (−) nm and to a decrease of the absorbance changes near 800 nm in the difference absorption spectrum of the oxidation of P. The data are interpreted in terms of the interaction between P and B1 molecules which gives two transitions at 790-800 and 810 nm with different orientations in modified Rps. sphaeroides as well as in Chloroflexus reaction centers. Similar transitions are observed for the interaction between P and B2. The spectral analysis shows the existence of two chains P-B1-H1, and P-B2-H2 in which the distances between the centers of molecules are 1.3 nm or less.

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 (BH− 4). 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 BH− 4 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 131‐deoxo‐131‐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°C at 679.5–680 nm and 542 nm, respectively.

Reaction centers of photosystem II with a chemically-modified pigment composition: exchange of pheophytins with 131-deoxo-131-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 131‐deoxo‐131‐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 131‐deoxo‐131‐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 131‐deoxo‐131‐hydroxy‐pheophytin a‐exchanged preparation to the spectral and functional analysis of native reaction centers of photosystem II are discussed.

Free-radical and correlated radical-pair spin-polarized signals in Rhodobacter sphaeroides R-26 reaction centers

Abstract Two g = 2.0 spin-polarized ESR signals with different properties are detected in native and QA-substituted reaction centers from R. sphaeroides R-26. The signal from substituted RCs is attributed to the primary donor cation radical. Native RCs demonstrate a signal with strong temperature dependence which most likely arises from spin-correlated radical pair P+−Q−AFe2+.

FTIR spectroscopy of the reaction center of Chloroflexus aurantiacus: photoreduction of the bacteriopheophytin electron acceptor.

Mid-infrared spectral changes associated with the photoreduction of the bacteriopheophytin electron acceptor H(A) in reaction centers (RCs) of the filamentous anoxygenic phototrophic bacterium Chloroflexus (Cfl.) aurantiacus are examined by light-induced Fourier transform infrared (FTIR) spectroscopy. The light-induced H(A)(-)/H(A) FTIR (1800-1200cm(-1)) difference spectrum of Cfl. aurantiacus RCs is compared to that of the previously well characterized purple bacterium Rhodobacter (Rba.) sphaeroides RCs. The most notable feature is that the large negative IR band at 1674cm(-1) in Rba. sphaeroides R-26, attributable to the loss of the absorption of the 13(1)-keto carbonyl of H(A) upon the radical anion H(A)(-) formation, exhibits only a very minor upshift to 1675cm(-1) in Cfl. aurantiacus. In contrast, the absorption band of the 13¹-keto C=O of H(A)(-) is strongly upshifted in the spectrum of Cfl. aurantiacus compared to that of Rba. sphaeroides (from 1588 to 1623cm(-1)). The data are discussed in terms of: (i) replacing the glutamic acid at L104 in Rba. sphaeroides R-26 RCs by a weaker hydrogen bond donor, a glutamine, at the equivalent position L143 in Cfl. aurantiacus RCs; (ii) a strengthening of the hydrogen-bonding interaction of the 13¹-keto C=O of H(A) with Glu L104 and Gln L143 upon H(A)(-) formation and (iii) a possible influence of the protein dielectric environment on the 13¹-keto C=O stretching frequency of neutral H(A). A conformational heterogeneity of the 13³-ester C=O group of H(A) is detected for Cfl. aurantiacus RCs similar to what has been previously described for purple bacterial RCs.

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+BA− 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+BA− 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 BA− at 1020 nm is registered in RCs of both mutants. The formation of the BA− band is accompanied by damped oscillations with main frequencies from ∼10 to ∼150 cm−1. Only a partial stabilization of the P+BA− 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 BA− is proposed to explain rapid stabilization of the P+BA− 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+BA− 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 BA− is assumed to be another potential contributor to the mechanism of P+BA− stabilization.

Formation of charge separated state P+Q− A 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+Q− A 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.

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 BA− and β−, where β is a bacteriochlorophyll substituting the native bacteriopheophytin HA) and in the 735–775 nm range (bleaching of the absorption band of the bacteriopheophytin HB 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 ∼14 psec at 90 K and ∼18 psec at 293 K, which is accompanied by oscillations with a frequency of ∼150 cm−1. A weak absorption band is found at 1018 nm that is formed ∼100 fsec after excitation of P and reflects the electron transfer from P* to β and/or BA with accumulation of the P+β− and/or P+BA− states. The kinetics of ΔA at 1018 nm contains the oscillations at ∼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 ΔA kinetics at 751 nm reflects the electron transfer to HB with formation of the P+HB− 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 HA−. 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 PA and PB 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 PA and PB 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 173-ester group of the bacteriochlorophyll PA 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 BA and the bacteriopheophytin HA. Structural changes are observed in the monomer bacteriochlorophyll BB binding site in the inactive chromophore branch of the mutant RCs.

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 Mn2+ and HCO3−. It was found that the dark reduction of P870+ from Rb. sphaeroides R-26 is considerably accelerated upon the combined addition of 0.5 mM MnCl2 and 30–75 mM NaHCO3 (as a result of formation of “low-potential” complexes [Mn(HCO3)2]), while MnCl2 and NaHCO3 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, P865, which results in thermodynamic difficulties of the redox interaction between P865+ and Mn2+) or in RC from Ch. minutissimum (apparently due to the presence of the RC-bound cytochrome preventing the direct interaction between P870+ and Mn2+). The absence of acceleration of the dark reduction of P870+ in the RC of Rb. sphaeroides R-26 when Mn2+ and HCO3− were replaced by Mg2+ or Ca2+ 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 Mn2+ complexes in the evolutionary origin of the inorganic core of the water oxidizing complex of photosystem II.