Comparative Study of Spectral and Functional Properties of Wild Type and Double Mutant H(L173)L/I(L177)H Reaction Centers of the Purple Bacterium .

Previously, we found that in the reaction center (RC) of the purple bacterium , formation of heterodimeric primary electron donor (P) caused by the substitution of His-L173 by Leu, was compensated by the second mutation Ile-L177 - His. Significant changes in the spectral properties, pigment composition, and redox potential of P observed in the H(L173)L RC, are restored to the corresponding characteristics of the native RC in the RC H(L173)L/I(L177)H, with the difference that the energy of the long-wavelength Q optical transition of P increases significantly (by ~75 meV). In this work, it was shown using light-induced difference FTIR spectroscopy that the homodimeric structure of P is preserved in the RC with double mutation with partially altered electronic properties: electronic coupling in the radical-cation of the P dimer is weakened and localization of the positive charge on one of its halves is increased.

Anomalous Temperature Dependence of the Triplet-Triplet Energy Transfer in I(L177)H Mutant Reaction Centers.

In photosynthetic reaction centers, quenching of the primary donor triplet state by energy transfer to the carotenoid molecule provides efficient suppression of generation of singlet-excited oxygen, potent chemical oxidant. This process in the reaction centers is thermoactivated, and discontinues at temperatures below 40 K. In these reaction centers, substitution of amino acid residue isoleucine at the 177 position of the L-subunit with histidine results in the sharp decrease of activation energy, so that the carotenoid triplets are populated even at 10 K.

Femtosecond Dynamics of the Excited Primary Electron Donor in Reaction Centers of the Purple Bacterium .

Femtosecond transient absorption spectroscopy was used to study the dynamics of the excited primary electron donor in the reaction centers of the purple bacterium . Using global analysis and the interval method, we found a correlation between the vibrational coherence damping of the excited primary electron donor and the lifetime of the charge-separated state PB, indicating the reversibility of electron transfer to the primary electron acceptor, the B molecule. In the reaction centers, the signs of superposition of two electronic states of P were found for a delay time of less than 200 fs.

Role of hydrogen-bond networks on the donor side of photosynthetic reaction centers from purple bacteria.

For the last decades, significant progress has been made in studying the biological functions of H-bond networks in membrane proteins, proton transporters, receptors, and photosynthetic reaction centers. Increasing availability of the X-ray crystal and cryo-electron microscopy structures of photosynthetic complexes resolved with high atomic resolution provides a platform for their comparative analysis. It allows identifying structural factors that are ensuring the high quantum yield of the photochemical reactions and are responsible for the stability of the membrane complexes.

Properties and Crystal Structure of the Photosynthetic Reaction Center with Double Amino Acid Substitution I(L177)H + F(M197)H.

The photosynthetic reaction center of the purple bacterium with two site-directed mutations Ile-L177-His and M197 Phe-His is of double interest. The substitution I(L177)H results in strong binding of a bacteriochlorophyll molecule with L-subunit. The second mutation F(M197)H introduces a new H-bond between the C2-acetyl carbonyl group of the bacteriochlorophyll P and His-M197, which is known to enhance the stability of the complex.

Stabilization of Photosynthetic Reaction Center by the Introduction of Disulfide Bonds.

The photosynthetic reaction center of the purple nonsulfur bacterium is a useful model for the study of mechanisms of photoinduced electron transfer and a promising component for photo-bio-electrocatalytic systems. The basic research and technological applications of this membrane pigment-protein complex require effective approaches to increase its structural stability. In this work, a rational design approach to genetically modify the reaction centers by introducing disulfide bonds is used.

Properties of Mutant Photosynthetic Reaction Centers of Purple Non-Sulfur Bacteria Cereibacter sphaeroides with M206 Ile→Gln Substitution.

In the structure of photosynthetic reaction center (RC) of the purple bacterium Cereibacter sphaeroides the highly conserved amino acid residue Ile-M206 is located near the bacteriochlorophyll dimer P, which is the primary electron donor, and the monomeric bacteriochlorophyll B, which is the nearest electron acceptor. Since Ile-M206 is close to the C2-acetyl group of bacteriochlorophyll P, the hydroxyl group of Tyr-M210, and to the C9-keto group of bacteriochlorophyll B, as well as to the water molecule near the latter group, this site can be used for introducing mutations in order to study mechanisms of primary photochemical processes in the RC. Previously it was shown that the Ile→Glu substitution at the M204 position (analog of M206 in the RC of C.

X-ray structure of the reaction center with an M197 Phe→His substitution clarifies the properties of the mutant complex.

The first steps of the global process of photosynthesis take place in specialized membrane pigment-protein complexes called photosynthetic reaction centers (RCs). The RC of the photosynthetic purple bacterium , a relatively simple analog of the more complexly organized photosystem II in plants, algae and cyanobacteria, serves as a convenient model for studying pigment-protein interactions that affect photochemical processes. In bacterial RCs the bacteriochlorophyll (BChl) dimer P serves as the primary electron donor, and its redox potential is a critical factor in the efficient functioning of the RC.

Effect of Detergents and Osmolytes on Thermal Stability of Native and Mutant Rhodobacter sphaeroides Reaction Centers.

Photosynthetic reaction center (RC) of the purple bacterium Rhodobacter sphaeroides is one of the most well-studied transmembrane pigment-protein complexes. It is a relatively stable protein with established conditions for its isolation from membranes, purification, and storage. However, it has been shown that some amino acid substitutions can affect stability of the RC, which results in a decrease of the RCs yield during its isolation and purification, disturbs spectral properties of the RCs during storage, and can lead to sample heterogeneity.

Mutation H(M202)L does not lead to the formation of a heterodimer of the primary electron donor in reaction centers of Rhodobacter sphaeroides when combined with mutation I(M206)H.

In photosynthetic reaction centers (RCs) of purple bacteria, conserved histidine residues [His L173 and His M202 in Rhodobacter (Rba.) sphaeroides] are known to serve as fifth axial ligands to the central Mg atom of the bacteriochlorophyll (BChl) molecules (P and P, respectively) that constitute the homodimer (BChl/BChl) primary electron donor P. In a number of previous studies, it has been found that replacing these residues with leucine, which cannot serve as a ligand to the Mg ion of BChl, leads to the assembly of heterodimer RCs with P represented by the BChl/BPheo pair.

Properties of Rhodobacter sphaeroides Reaction Centers with the Ile→Tyr Substitution at Positions L177 and M206.

Studying pigment-protein interactions in the photosynthetic reaction centers (RCs) is important for the understanding of detailed mechanisms of the photochemical process. This paper describes spectral and photochemical characteristics, pigment composition, and stability of the Rhodobacter sphaeroides RCs with the I(L177)Y and I(M206)Y amino acid substitutions. The obtained data are compared with the properties of I(L177)H, I(L177)D, and I(M206)H RCs reported previously.

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) P and P 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.

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.

Controlling Photosynthetic Excitons by Selective Pigment Photooxidation.

As a basis of photosynthesis, photoinduced oxidation of (bacterio)chlorophyll molecules in the special reaction center complexes has been a subject of extensive research. In contrast, the generally harmful photooxidation of antenna chromoproteins has received much less attention. Here, we have established the permanent structural changes in the LH2 antenna bacteriochlorophyll-protein complex from a sulfur photosynthetic purple bacterium Ectothiorhodospira haloalkaliphila taking place at physiological conditions upon intense optical irradiation.

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 P. 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.

Different effects of identical symmetry-related mutations near the bacteriochlorophyll dimer in the photosynthetic reaction center of Rhodobacter sphaeroides.

In the bacterial photosynthetic reaction center (RC), asymmetric protein environment of the bacteriochlorophyll (BChl) dimer largely determines the photophysical and photochemical properties of the primary electron donor. Previously, we noticed significant differences in properties of Rhodobacter sphaeroides RCs with identical mutations in symmetry-related positions - I(M206)H and I(L177)H. The substitution I(L177)H resulted in covalent binding of BChl PA with the L-subunit, as well as in 6-coordination of BChl BB, whereas in RC I(M206)H no such changes of pigment-protein interactions were found.

The L(M196)H mutation in Rhodobacter sphaeroides reaction center results in new electrostatic interactions.

New histidine residue was introduced in M196 position in the reaction center of Rhodobacter sphaeroides in order to alter polarity of the BChl dimer's protein environment and to study how it affects properties and structure of the primary electron donor P. It was shown that in the absorption spectrum of the mutant RC the 6 nm red shift of the Q Y P band was observed together with considerable decrease of its amplitude. The mid-point potential of P/P (+) in the mutant RC was increased by +65 (±15) mV as compared to the E m P/P (+) value in the wild-type RC suggesting that the mutation resulted in new pigment-protein interactions.

Expression, purification, crystallization and preliminary X-ray structure analysis of wild-type and L(M196)H-mutant Rhodobacter sphaeroides reaction centres.

The electron and proton transport mediated by protein-bound cofactors in photosynthesis have been investigated by various methods in order to determine the energetics, the dynamics and the pathway of this process. In purple bacteria, primary photosynthetic charge separation and the build-up of a proton gradient across the periplasmic membrane are catalyzed by the photosynthetic reaction centre (RC). Here, the purification, crystallization and preliminary X-ray analysis of wild-type and L(M196)H-mutant RCs of Rhodobacter sphaeroides are presented, enabling study of the influence of the protein environment of the primary electron donor on the spectral properties and photochemical activity of the RC.

The site-directed mutation I(L177)H in Rhodobacter sphaeroides reaction center affects coordination of P(A) and B(B) bacteriochlorophylls.

To explore the influence of the I(L177)H single mutation on the properties of the nearest bacteriochlorophylls (BChls), three reaction centers (RCs) bearing double mutations were constructed in the photosynthetic purple bacterium Rhodobacter sphaeroides, and their properties and pigment content were compared with those of the correspondent single mutant RCs. Each pair of the mutations comprised the amino acid substitution I(L177)H and another mutation altering histidine ligand of BChl P(A) or BChl B(B). Contrary to expectations, the double mutation I(L177)H+H(L173)L does not bring about a heterodimer RC but causes a 46nm blue shift of the long-wavelength P absorbance band.

Structure-function investigations of bacterial photosynthetic reaction centers.

During photosynthesis light energy is converted into energy of chemical bonds through a series of electron and proton transfer reactions. Over the first ultrafast steps of photosynthesis that take place in the reaction center (RC) the quantum efficiency of the light energy transduction is nearly 100%. Compared to the plant and cyanobacterial photosystems, bacterial RCs are well studied and have relatively simple structure.

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.

[Triplet state of the primary donor in reaction centers of the phototrophic bacterium Rhodobacter sphaeroides R26 with active photoinduced electron transfer].

EPR characteristics of transient paramagnetic states photoinduced in isolated reaction centers of Rhodobacter sphaeroides R26 with intact electron transfer have been studied. It was demonstrated that the detected weak triplet state EPR signal belongs to the primary donor molecule and is populated via the conventional mechanism of radical pair S-T0 mixing. The distortion of the spectral shape of this signal is explained by the triplet quantum yield anisotropy brought about by the short lifetime of precursor radical pairs.

Properties of Rhodobacter sphaeroides photosynthetic reaction center with double amino acid substitution I(L177)H+H(M182)L.

Histidine M182 in the reaction center (RC) of Rhodobacter sphaeroides serves as the fifth ligand of the bacteriochlorophyll (BChl) B(B) Mg atom. When this His is substituted by an amino acid that is not able to coordinate Mg, bacteriopheophytin appears in the B(B) binding site instead of BChl (Katilius, E., et al.

Examination of stability of mutant photosynthetic reaction center of Rhodobacter sphaeroides I(L177)H and determination of location of bacteriochlorophyll covalently bound to the protein.

We demonstrated earlier that as a result of the I(L177)H mutation in the photosynthetic reaction center (RC) of the bacterium Rhodobacter sphaeroides, one of the bacteriochlorophylls (BChl) binds with the L-subunit, simultaneously raising coordination stability of the central magnesium atom of the bacteriochlorophyll associated with the protein. In this study, spectral properties of wild type RC and I(L177)H in the presence of urea and SDS as well as at 48 degrees C were examined. It is shown that the I(L177)H mutation decreases the RC stability.