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. It is shown that the I(L177)Y and I(M206)Y mutations cause a similar shift of the QYP band in the absorption spectra of the mutant RCs and do not affect the distribution of the electron spin density within the photo-oxidized P+ dimer. The differences in the position and amplitude of the QYB band in the I(L177)Y and I(M206)Y RCs were determined. The results indicate the possibility of new pigment-protein interactions in the vicinity of monomeric bacteriochlorophylls in the A and B chains, which might be of interest for future research.

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.

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.

[Therapy of patients with the high risk of arterial hypertension with the use of telmisartan].

AIM: The study assesses the effects of therapy based on the use of telmisartan in patients with arterial hypertension and stable angina on the clinical and functional indicators of the cardiovascularvascular system. MATERIAL AND METHODS: 52 patients with a combination of arterial hypertension (AH) I and II stages and coronary artery disease II with the mean age 63.5 ± 5.4 years (79% males and 21% of females) were enrolled in the trial. The duration of AH and CAD were 17.5±3.8 and 12.5±3.1 accordingly. All patients took a daily 80 mg dose of telmisartan. The efficacy of treatment was estimated in 3,6 and 12 months. RESULTS: The use of telmisartan in complex treatment of high-risk patients with AH has led to decreasing blood pressure to the target level, increasing exercise tolerance (walking the distance from 315.5 m to 410.2 m in 6 minutes). 12 months of therapy based on telmisartan showed significant decrease in left hy­ pertrophy ventricle (LV myocardial mass index on average by 10.4%), a decrease in the total duration of depression ST segment from 9.6±2.9 to 2.7±1.5 mm and a decrease in the depth of depression from 1.5±0.3 mm to 0.3±0.09 mm, with a trend towards decrease in the number of episodes of ST-segment depression, as well as the absence of a significant change in heart rate. After 12 months of en­ dothelium-dependent vasodilation therapy, the condition of 31 (60%) patients improved, and 18 (35%) patients showed the tendency to improvement. The tests with reactive hyperemia conducted after 12 months of treatment revealed decreased linear velocity of blood flow in the brachial artery on average by 17%. CONCLUSION: The use of telmisartan in complex therapy improves the quality of life according to the EQ-VAS questionnaire by 25 points after 12 months of therapy, contributes to lowering blood pressure in 96% of patients, reduc­ ing myocardial hypertrophy, endothelial dysfunction and severity of ischemic manifestations (reducing the need for nitroglycerin intake to an average of 0,5 inhalations, as well as a decrease in the ST segment depression of 40% after 12 months of therapy).

Intestinal colonization with resistant bacteria: a prognostic marker of mortality in decompensated cirrhosis.

Infections due to drug-resistant (DR) bacteria are increasingly recognized as an emerging problem worldwide. Asymptomatically colonized patients may contribute to the reservoir in the hospital setting, causing both horizontal transmission and endogenous infections. We aimed to evaluate the prevalence of intestinal colonization with DR bacteria on subsequent clinical infection development and prognosis in patients with decompensated cirrhosis. One hundred seven patients without infection at baseline were screened and prospectively followed-up for 3 months. Among the patients screened, DR bacteria were isolated in 47 (43.9%), 14 colonized with multidrug- (MDR) and 33 with extensively drug (XDR)-resistant bacteria or a mixture of MDR/XDR bacteria. Severity of liver disease and demographic characteristics were similar among groups. The 20 (42.6%) with DR vs 14 (23.3%) without had hepatic encephalopathy and/or spontaneous bacterial peritonitis episodes over the past 6 months (p = 0.034). One third of both DR and non-DR groups developed infection during follow-up but in only 7 and 5, respectively, the infection was microbiologically documented. In a 3-month-follow-up period, mortality was higher in patients colonized with XDR compared to those without (log rank p = 0.027). In multivariate analysis, colonization with XDR bacteria [HR = 1.074, (CI:1.024-1.126), p = 0.003] and MELD score [HR = 2.579 (1.109-5.996), p = 0.028] were independently associated with low survival. Asymptomatic GI colonization with DR bacteria is a risk factor for increased mortality in decompensated cirrhosis. Frequent hospitalizations for complications of the underlying disease and selective pressure induced by the use of antimicrobials are probably the main determinants.

The complex analysis of risk factors, influencing on the progression of chronic obstructive ulmonary disease.

AIM: The aim of the study is the analysis of risk factors, influencing on the progression of chronic obstructive pulmonary disease. MATERIALS AND METHODS: 259 patients with chronic obstructive pulmonary disease (COPD) I and III stages, mean age 54,3±11,7 and the duration of the disease 10,1±5,7. Patients were divided into 2 groups: I group (n=119) - with III stage of disease, II group (n=140) - COPD with I stage. 30 healthy were involved in control group. The risk factors of progression of COPD were determined with the use of regressive and correlative analyses. RESULTS: The level of markers of systemic inflammation in serum of patients with COPD: TNF-α (RR 3,25); IL-8 (RR 2,1); hs-CRP (RR 3,52) in combination with traditional risk factors (long course of the disease (RR 2,3), age (RR 1,65), smoking (RR 1,65), and also obesity (RR 2,45), early menopause (RR 3,52), hereditary respiratory predisposition (RR 3,05), and also coexisting cardiovascular pathology increase the risk of COPD progression several times. . CONCLUSION: According to the results of this trial, unmodified risk factors such as hereditary predisposition, early menopause, long course of the diseases and coexisting cardiovascular pathology contribute significantly on progression of COPD. The relative risk increases with increasing of concentration of markers of systemic inflammation (TNF-α, IL-8 and hs-CRP), that correlate with clinical manifestations of COPD: cough, shortness of breath on VAS in mm.

[The Analysis of Efficacy of The Application of Interleukin-1 Inhibitor in the Complex Therapy of Secondary Osteoarthritis Taking Into Account the Dynamics of Clinical and Functional Indicators].

BACKGRAUND: Secondary osteoarthritis (OA) adversely affects the underlying disease and can enhance clinical manifestations of articular syndrome. Early detection of comorbid pathology and early treatment with the therapy of underlying condition is of primary importance to preserve adequate functional activity in patients. The Aim is to analyze the efficacy of the application of interleukin-1 inhibitor in the complex treatment of secondary OA in addition to rheumatoid arthritis (RA). MATERIALS AND METHODS: 248 patients with secondary OA and RA were divided in 4 groups: patients of group I (n = 62) took interleukin-1 inhibitor (50 mg x 2 times per day) in combination with laser therapy on the basis of methotrexate (10-20 mg per week), group II (n = 60) received interleukin-1 inhibitor in complex with methotrexate, in group III (n = 62)--laser therapy with methotrexate and in group 4--methotrexate. We estimated the treatment efficacy in 3 and 6 months according to the dynamics of pain on VAS, indexes HAQ and KOOS. RESULTS: according to VAS group 1 showed statistical significant reduction in pain on movement in 3 and 6 months of 28.53 mm (43.6%) and 31.3 mm (48%) respectively, to values of 36.87 ± 1.56* and 33.11 ± 1.11* (p < 0.05). The results showed the statistically significant advantage of HAQ dynamics in groups I and II in comparison with groups III, IV (p = 0.03). The most prominent statistically significant (p = 0.02) results according to the dynamics of index KOOS were noted in groups of patients who took diacerein (I and II) 6 months after the complex treatment. CONCLUSIONS: the inclusion of interleukin-1 inhibitor in the complex treatment of secondary OA in patients with RA contributes to clinical improvement according to VAS (p = 0.03), index KOOS that reflects functional condition of the knee and increases the quality of life by index HAQ (p = 0.03) after 6 months therapy.

[THE COMPARATIVE ANALYSIS OF LEVEL OF OLIGOMERIC MATRIX PROTEIN OF CARTILAGE IN BLOOD SERUM OF PATIENTS WITH DISEASES OF MUSCULO-SKELETAL SYSTEM].

The osteoarthritis and rheumatoid arthritis are considered as the most prevalent diseases in the structure of diseases of musculoskeletal system. The higher social significance of these nosologies dictates necessity of searching reliable cartilage biomarkers having diagnostic validity both in discerning degenerative alterations at early stage of disease of joints and in monitoring of treatment effectiveness. The content of oligomeric matrix protein of cartilage using ELISA was evaluated in blood serum ofpatients with secondary osteoarthritis under rheumatoid arthritis (n=248). The comparison of derived results was carried out using control groups. Within the framework of study relationship was evaluated between level of oligomeric matrix protein of cartilage in patients with secondary osteoarthritis under rheumatoid arthritis with values offunctional KOOS index. The analysis of derived results established trend to increasing of level of oligomeric matrix protein of cartilage in blood serum ofpatients with secondary osteoarthritis under rheumatoid arthritis as compared with control groups. The moderate correlation interdependence between cartilage biomarker and KOOS index.

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. In addition, the yield of RC I(M206)H after its isolation from membranes was significantly lower than the yield of RC I(L177)H. This study shows that replacement of amino acid residues in the M203-M206 positions near BChls PB and BA by symmetry-related residues from the L-subunit near BChls PA and BB leads to further decrease in RC amount in the membranes associated obviously with poor assembly of the complex. Introduction of a new hydrogen bond between BChl PB and its protein environment by means of the F(M197)H mutation stabilized the mutant RC but did not affect its low yield. We suggest that the mutation I(M206)H and substitution of amino acid residues in M203-M205 positions could disturb glycolipid binding on the RC surface near BChl BA that is important for stable assembly of the complex in the membrane.

[Correction of an inflammatory process with an interleukin-1 inhibitor in the combination treatment of secondary osteoarthritis in the presence of comorbid condition].

AIM: To enhance the efficiency of treatment in patients with secondary osteoarthritis (ОА) in the presence of comorbid condition, by using an interleukin-1 inhibitor (IL-1i) and laser therapy (LT). SUBJECTS AND METHODS: A total of 248 patients aged 38 to 65 years with RA and secondary OA who had predominantly Stage II in accordance with the Disease Activity Score 28 (DAS28) were examined. According to the received therapy, the patients were divided into 4 groups: 1) IL-1i + LT + mrthotrexate (MT); 2) IL-1i + MT; 3) LT + MT; 4) MT. The efficiency of treatment was evaluated from changes in the KOOS (Knee injury and Osteoarthritis Outcome Score), DAS 28, IL-1, and cartilage oligomeric matrix protein (COMP) 6 months later. RESULTS: There were statistically significant functional improvements in KOOS and DAS28 in Groups 1 and 2 patients with secondary OA in RA. Clinical efficacy was confirmed by positive changes in serum IL-1 and COMP levels. CONCLUSION: Incorporation of an IL-1i into a therapy regimen for secondary OA in RA patients during basic therapy could not only improve the functional status of patients, but also decrease activity of the underlying disease according to the DAS 28.

[The analysis of dynamics of oxidative modification of proteinsin the blood sera of the patients presenting with secondary osteoarthrosis associated with rheumatoid arthritis and treated by laser therapy].

OBJECTIVE: To evaluate the indicators of oxidative modification of proteins (OMP) in the patients presenting with secondary osteoarthrosis associated with rheumatoid arthritis (RA) and to determine their dynamics under the influence of the combined treatment with the use of low-intensity laser irradiation (LILI). MATERIAL AND METHODS: A total of 50 patients with RA associated with secondary osteoarthritis and 25 healthy subjects were enrolled in this study. The patients of one study sub-group (n = 25) were given combined therapy with the use of LLLI, those in the second sub-group (n = 25) received only drug therapy. We made use of the VAS and DAS 28 scales to estimate dynamics of pain and compared serum OMP in the patients and healthy subjects. RESULTS: The analyses of the data obtained demonstrated the increased OMP in the patients with RA in comparison with the healthy subjects. The patients of sub-group 1 experienced a significant decrease in the clinical parameters of pain based on the VAS and DAS 28 scales accompanied by the marked reduction of OMP. In sub-group 2, the patients also exhibited the statistically significant dynamics of these indicators, but it was less pronounced than in sub-group 2. CONCLUSION: The patients presenting with rheumatoid arthritis are characterized by the elevated level of protein oxidative modification, a marker of oxidative stress. LlLI introduced in the combined treatment of the patients with RA not only increases the anti-inflammatory and analgesic effects but also has the antioxidant properties.

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.

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. The histidine L177 or a water molecule were suggested as putative ligands for P(A) in the RC I(L177)H+H(L173)L although this would imply a reorientation of the His backbone and additional rearrangements in the primary donor environment or even a repositioning of the BChl dimer. The crystal structure of the mutant I(L177)H reaction center determined to a resolution of 2.9Å shows changes at the interface region between the BChl P(A) and the monomeric BChl B(B). Spectral and pigment analysis provided evidence for ß-coordination of the BChl B(B) in the double mutant RC I(L177)H+H(M182)L and for its hexacoordination in the mutant reaction center I(L177)H. Computer modeling suggests involvement of two water molecules in the ß-coordination of the BChl B(B). Possible structural consequences of the L177 mutation affecting the coordination of the two BChls P(A) and B(B) are discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

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.

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*.

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. The H-bond networks are known to be responsible for proton transport associated with electron transfer from the primary to the secondary quinone as well as in the processes of water oxidation in photosystem II. Participation of such networks in reactions proceeding on the periplasmic side of bacterial photosynthetic reaction centers is less studied. This review summarizes the current understanding of the role of H-bond networks on the donor side of photosynthetic reaction centers from purple bacteria. It is discussed that the networks may be involved in providing close association with mobile electron carriers, in light-induced proton transport, in regulation of the redox properties of bacteriochlorophyll cofactors, and in stabilization of the membrane protein structure at the interface of membrane and soluble phases.

Coherent phenomena of charge separation in reaction centers of LL131H and LL131H/LM160H/FM197H mutants of Rhodobacter sphaeroides.

Primary stage of charge separation and transfer of charges was studied in reaction centers (RCs) of point mutants LL131H and LL131H/LM160H/FM197H of the purple bacterium Rhodobacter sphaeroides by differential absorption spectroscopy with temporal resolution of 18 fsec at 90 K. Difference absorption spectra measured at 0-4 psec delays after excitation of dimer P at 870 nm with 30 fsec step were obtained in the spectral range of 935-1060 nm. It was found that a decay of P* due to charge separation is considerably slower in the mutant RCs in comparison with native RCs of Rba. sphaeroides. Coherent oscillations were found in the kinetics of stimulated emission of the P* state at 940 nm. Fourier analysis of the oscillations revealed a set of characteristic bands in the frequency range of 20-500 cm(-1). The most intense band has the frequency of ~130 cm(-1) in RCs of mutant LL131H and in native RCs and the frequency of ~100 cm(-1) in RCs of the triple mutant. It was found that an absorption band of bacteriochlorophyll anion B(A)(-) which is registered in the difference absorption spectra of native RCs at 1020 nm is absent in the analogous spectra of the mutants. The results are analyzed in terms of the participation of the B(A) molecule in the primary electron transfer in the presence of a nuclear wave packet moving along the inharmonic surface of P* potential energy.

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. (1999) J. Phys. Chem. B, 103, 7386-7389). We have shown that in the presence of the additional mutation I(L177)H the coordination of the BChl B(B) Mg atom in the double mutant I(L177)H+H(M182)L RC still remains. Changes in the double mutant RC absorption spectrum attributed to BChl absorption suggest that BChl B(B) Mg atom axial ligation might be realized not from the usual α-side of the BChl macrocycle, but from the opposite, ß-side. Weaker coordination of BChl B(B) Mg atom compared to the other mutant RC BChl molecules suggests that not an amino acid residue but a water molecule might be a possible ligand. The results are discussed in the light of the structural changes that occurred in the RC upon Ile/His substitution in the L177 position.

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. Therefore they represent a useful model system both for manipulating of the electron transfer parameters to study detailed mechanisms of its separate steps as well as to investigate the common principles of the photosynthetic RC structure, function, and evolution. This review is focused on the research papers devoted to chemical and genetic modifications of the RCs of purple bacteria in order to study principles and mechanisms of their functioning. Investigations of the last two decades show that the maximal rates of the electron transfer reactions in the RC depend on a number of parameters. Chemical structure of the cofactors, distances between them, their relative orientation, and interactions to each other are of great importance for this process. By means of genetic and spectral methods, it was demonstrated that RC protein is also an essential factor affecting the efficiency of the photochemical charge separation. Finally, some of conservative water molecules found in RC not only contribute to stability of the protein structure, but are directly involved in the functioning of the complex.

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. Under denaturing conditions, some changes indicating breakdown of oligomeric structure of the complex and loss of interaction between pigments and their protein environment are observed in I(L177)H RC spectra. In addition, pheophytinization of bacteriochlorophylls occurs in both types of RC in the presence of SDS. However, an 811-nm band is observed in the spectrum of the mutant RC under these conditions, which indicates retention of one of the BChl molecules in the protein binding site and stable coordination of its central magnesium atom. It is shown that in both types of RC, monomeric BChl B(B) can be modified by sodium borohydride treatment and then extracted by acetone-methanol mixture. Spectral properties of the BChl covalently bound with the protein in I(L177)H RC do not change. The results demonstrate that BChl P(A) is the molecule of BChl tightly bound with the L-subunit in mutant RC as it was supposed earlier.