Algorithm for Extracting Weak Bands Kinetics from the Transient Absorption Spectra of the Rhodobacter sphaeroides Reaction Center.

An algorithm to extract kinetics of the ion radical bands from the strong absorption background in the transient absorption spectra of the Rhodobacter sphaeroides reaction centers upon femtosecond excitation of the primary electron donor is suggested. The rising kinetics of the transient absorption band at 1020 nm and the bleaching kinetics of the 545-nm band constructed using the proposed method are adequately fitted by the kinetic equations for sequential electron transfer from the excited primary donor to the BA (monomeric bacteriochlorophyll) molecule, and then to the HA (bacteriopheophytin serving as an electron acceptor) molecule with the rate constants of 3.5 ± 0.2 and 0.8 ± 0.1 ps, respectively. The kinetics of the bacteriochlorophyll absorption band at 600 nm shows both the ultrafast bleaching of the P870 dimer and slower bleaching of the BA monomer due to its transition to the anion radical. The plotted kinetics of the ion radical bands is in agreement with the concentration profiles of the charge-separated states produced by the global target analysis of experimental data using the model of sequential electron transfer in the reaction centers.

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.

Features of Bacteriochlorophylls Axial Ligation in the Photosynthetic Reaction Center of Purple Bacteria.

This review focuses on recent experimental data obtained by site-directed mutagenesis of the reaction center in purple nonsulfur bacteria. The role of axial ligation of (bacterio)chlorophylls in the regulation of spectral and redox properties of these pigments, as well as correlation between the structure of chromophores and nature of their ligands, are discussed. Cofactor ligation in various types of reaction centers is compared, and possible reasons for observed differences are examined in the light of modern ideas on the evolution of photosynthesis.

An Alternative Pathway of Light-Induced Transmembrane Electron Transfer in Photosynthetic Reaction Centers of Rhodobacter sphaeroides.

In the absorption spectrum of Rhodobacter sphaeroides reaction centers, a minor absorption band was found with a maximum at 1053 nm. The amplitude of this band is ~10,000 times less and its half-width is comparable to that of the long-wavelength absorption band of the primary electron donor P870. When the primary electron donor is excited by femtosecond light pulses at 870 nm, the absorption band at 1053 nm is increased manifold during the earliest stages of charge separation. The growth of this absorption band in difference absorption spectra precedes the appearance of stimulated emission at 935 nm and the appearance of the absorption band of anion-radical BA- at 1020 nm, reported earlier by several researchers. When reaction centers are illuminated with 1064 nm light, the absorption spectrum undergoes changes indicating reduction of the primary electron acceptor QA, with the primary electron donor P870 remaining neutral. These photoinduced absorption changes reflect the formation of the long-lived radical state PBAHAQA-.

Charge separation in Rhodobacter sphaeroides mutant reaction centers with increased midpoint potential of the primary electron donor.

Primary charge separation dynamics in four mutant reaction centers (RCs) of the purple bacterium Rhodobacter sphaeroides with increased midpoint potential of the primary electron donor P (M160LH, L131LH, M197FH, and M160LH + L131LH + M197FH) have been studied by femtosecond transient absorption spectroscopy at room temperature. The decay of the excited singlet state in the wild-type and mutant RCs is complex and has two main exponential components, which indicates heterogeneity of electron transfer rates or the presence of reverse electron transfer reactions. The radical anion band of monomeric bacteriochlorophyll B(A) at 1020 nm was first observed in transient absorbance difference spectra of single mutants. This band remains visible, although with somewhat reduced amplitude, even at delays up to tens of picoseconds when stimulated emission is absent and the reaction centers are in the P(+)H(A)(-) state. The presence of this band in this time period indicates the existence of thermodynamic equilibrium between the P(+)B(A)(-)H(A) and P(+)B(A)H(A)(-) states. The data give grounds for assuming that the value of the energy difference between the states P*, P(+)B(A)(-)H(A), and P(+)B(A)H(A)(-) at early times is of the same order of magnitude as the energy kT at room temperature. Besides, monomeric bacteriochlorophyll B(A) is found to be an immediate electron acceptor in the single mutant RCs, where electron transfer is hampered due to increased energy of the P(+)B(A)(-) state with respect to P*.

Primary charge separation within P870* in wild type and heterodimer mutants in femtosecond time domain.

Primary charge separation dynamics in the reaction center (RC) of purple bacterium Rhodobacter sphaeroides and its P870 heterodimer mutants have been studied using femtosecond time-resolved spectroscopy with 20 and 40fs excitation at 870nm at 293K. Absorbance increase in the 1060-1130nm region that is presumably attributed to P(A)(δ+) cation radical molecule as a part of mixed state with a charge transfer character P*(P(A)(δ+)P(B)(δ-)) was found. This state appears at 120-180fs time delay in the wild type RC and even faster in H(L173)L and H(M202)L heterodimer mutants and precedes electron transfer (ET) to B(A) bacteriochlorophyll with absorption band at 1020nm in WT. The formation of the P(A)(δ+)B(A)(δ-) state is a result of the electron transfer from P*(P(A)(δ+)P(B)(δ-)) to the primary electron acceptor B(A) (still mixed with P*) with the apparent time delay of ~1.1ps. Next step of ET is accompanied by the 3-ps appearance of bacteriopheophytin a(-) (H(A)(-)) band at 960nm. The study of the wave packet formation upon 20-fs illumination has shown that the vibration energy of the wave packet promotes reversible overcoming of an energy barrier between two potential energy surfaces P* and P*(P(A)(δ+)B(A)(δ-)) at ~500fs. For longer excitation pulses (40fs) this promotion is absent and tunneling through an energy barrier takes about 3ps. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.