Anatoli Ya. Shkuropatov

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 Rhodobacter sphaeroides R-26 with selective replacement of bacteriopheophytin by pheophytin a: I. Characterisation of steady-state absorbance and circular dichroism, and of the P+QA− state

Abstract Bacteriopheophytin (BPheo) a of reaction centers (RCs) of Rhodobacter sphaeroides R-26 has been exchanged with pheophytin (Pheo) a . By varying the incubation temperature of the pigment exchange procedure two types of RCs were obtained, with either only the BPheo in the B-chain (BPheo B ) or both BPheo B and BPheo A replaced by Pheo a . For the two RC types absorption and CD spectra at 6 K as well as P + Q A − difference spectra at 10 K are compared with those of native RCs. The most pronounced differences are observed in the Q Y and Q X regions of the (B)Pheos. The P + Q A − decay halftime is for RCs with Pheo a in both chains 10–15 ms longer than for native RCs and RCs that still have a BPheo in the A-chain, at all temperatures between 10 and 290 K. At low temperatures all three RC types showed biphasic P + Q A − recombination.

Reaction centers of Rhodobacter sphaeroides R-26 with selective replacement of bacteriopheophytin a by pheophytin a: II. Temperature dependence of the quantum yield of P+QA− and 3P formation

Abstract The quantum yield of the formation of the charge-separated state P+QA− in reaction centers (RCs) of Rhodobacter sphaeroides R-26, in which the bacteriopheophytins in both the active (A) and the inactive (B) branch are replaced by pheophytin (Pheo) a (ΦA,B-exchanged RCs), shows a positive temperature dependence: it is 38±5% between 10 and 60 K, increases with temperature to 72±5% at 200 K and shows a minor additional increase above this temperature. The temperature dependence of the quantum yield of P+QA− formation in ΦA,B-exchanged RCs is modelled in the framework of a reaction scheme with the energy level of P+PheoA− placed above P+BA− (Shkuropatov, A.Ya. and Shuvalov, V.A. (1993) FEBS Lett. 322, 168–172), by the introduction of direct electron transfer from BA− to QA, assisted by a superexchange-mechanism via P+PheoA−. The observed triplet formation of ΦA,B-exchanged RCs with pre-reduced QA at cryogenic temperatures (quantum yield≤12%) is attributed to a residual fraction of RCs in which only ΦB was exchanged for Pheo a. The lack of triplet formation in pre-reduced ΦA,B-exchanged RCs is consistent with our kinetic model, since this predicts that at low temperatures the state P+PheoA− is not populated.

The Energy Level of P + B A - in Plant Pheophytin-Exchanged Bacterial Reaction Centers Probed by the Temperature Dependence of Delayed Fluorescence

The role of the accessory bacteriochlorophyll (BChl) BA between the primary electron donor P and the bacteriopheophytin (BPheo) electron acceptor HA in photosynthetic reaction centers (RC) is still subject of debate. The free energy gap between the states P* and P+B A - will have a decisive influence on the mechanism of the first electron transfer steps. We have recently shown [1] that P* and P+B A - accumulates in pheophytin (Pheo) exchanged RCs at 5K and that electron transfer to this state occurs at essentially the same rate as in native RCs. According to these results, the free energy level of P* and P+B A - has to lie below that of P* (Scheme 1.). The measurement of the temperature dependence of the fluorescence quantum yield resulting from thermal repopulation of the state P’ therefore offers a direct access to determine the free energy level of P* and P+B A - relative to that of P*.

WAVE PACKET MOTIONS COUPLED TO ELECTRON TRANSFER IN REACTION CENTERS OF CHLOROFLEXUS AURANTIACUS

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.

Coherent Electron Transfer in Reaction Centers of YM210L and YM210L/HL168L Mutants of Rba. Sphaeroides

A role of tyrosine M210 in the 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 the YM210L and YM210L/HL168L mutant reaction centers (RCs) of Rhodobacter (Rba.) sphaeroides in comparison with those in native RCs of Rba. sphaeroides. Kinetics of P* decay at 940 nm of the both mutants show a significant slowing-down 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 the1020-nm kinetics. 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 − was proposed to explain a rapid stabilization of the P+BA − state in native RCs. A conclusion was done that, probably, the absence of TyrM210 can not be compensated by lowering of the P+BA − free energy that is expected for the double YM210L/HL168L mutant.