Primary Electron Donor Research Articles

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 Cereibacter sphaeroides.

Previously, we found that in the reaction center (RC) of the purple bacterium Cereibacter sphaeroides, 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)LRC, 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 QY optical transition of P increases significantly (by ~75meV). 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. Results of the study of the triple mutant RC, H(L173)L/I(L177)H/F(M197)H, are consistent with the assumption that the observed changes in the P+ electronic structure, as well as considerable blue shift of the QYP absorption band in the RC H(L173)L/I(L177)H, are associated with modification of the spatial position and/or geometry of P. Using femtosecond transient absorption spectroscopy, it was shown that the mutant H(L173)L/I(L177)H RC retains a sequence of reactions P*→P+BA-→P+HA-→P+QA- with electron transfer rates and the quantum yield of the final stateP+QA- close to those observed in the wild-type RC (P*is the singlet-excited state ofP; BA, HA, and QA are molecules of bacteriochlorophyll, bacteriopheophytin, and ubiquinone in the active A-branch of cofactors, respectively). The obtained results, together with the previously published data for the RC with symmetrical double mutation H(M202)L/I(M206)H, demonstrate that by introducing additional point amino acid substitutions, photochemical activity of the isolated RC from C.sphaeroides could be maintained at a high level even in the absence of important structural elements- axial histidine ligands ofthe primary electron donorP.

Kinetics of Electron Transfer between Redox Cofactors in PhotosystemI Measured by High-Frequency EPR Spectroscopy.

The kinetics of the primary electron donor P700+ and the quinone acceptor A1- redox transitions were simultaneously studied for the first time in the time range of 200μs-10ms using high-frequency pulse Q-band EPR spectroscopy at cryogenic temperatures in various complexes of photosystemI(PSI) from the cyanobacterium Synechocystissp. PCC 6803. In the A1-core PSI complexes that lack 4Fe4S clusters, the kinetics of the A1- andP700+ signals disappearance at 100K were similar and had a characteristic time of τ≈500μs, caused by charge recombination in the P700+A1A- ion-radical pair in the A branch of redox cofactors. The kinetics of the backward electron transfer from A1B- to P700+ in the B branch of redox cofactors with τ<100μs could not be resolved due to time limitations of the method. In the native PSI complexes with a full set of redox cofactors and in the FX-core complexes, containing the 4Fe4S cluster FX, the kinetics of the A1- signal was significantly faster than that of the P700+ signal. The disappearance of the A1- signal had a characteristic time of 280-350μs; it was suggested that, in addition to the backward electron transfer from A1A- to P700+ with τ≈500μs, its kinetics also includes the forward electron transfer from A1A- to the 4Fe4S cluster FX, which had slowed down to 150-200μs. In the kinetics of P700+ reduction, it was possible to distinguish components caused by the backward electron transfer from A1-(τ≈500μs) and from 4Fe4S clusters (τ=1ms for the FX-core and τ>5ms for native complexes). These results are in qualitative agreement with the data on the kinetics of P700+ reduction obtained previously using pulse absorption spectrometry at cryogenic temperatures.

Environment-dependent chlorophyll-chlorophyll charge transfer states in Lhca4 pigment-protein complex.

Photosystem I (PSI) light-harvesting antenna complexes LHCI contain spectral forms that absorb and emit photons of lower energy than that of its primary electron donor, P700. The most red-shifted fluorescence is associated with the Lhca4 complex. It has been suggested that this red emission is related to the inter-chlorophyll charge transfer (CT) states. In this work we present a systematic quantum-chemical study of the CT states in Lhca4, accounting for the influence of the protein environment by estimating the electrostatic interactions. We show that significant energy shifts result from these interactions and propose that the emission of the Lhca4 complex is related not only to the previously proposed a603+-a608- state, but also to the a602+-a603- state. We also investigate how different protonation patterns of protein amino acids affect the energetics of the CT states.

Metagenomics and Stable Isotopes Uncover the Augmented Sulfide-Driven Autotrophic Denitrification in a Seasonally Hypoxic, Sulfate-Abundant Reservoir.

The mechanism governing sulfur cycling in nitrate reduction within sulfate-rich reservoirs during seasonal hypoxic conditions remains poorly understood. This study employs nitrogen and oxygen isotope fractionation in nitrate, along with metagenomic sequencing to elucidate the intricacies of the coupled sulfur oxidation and nitrate reduction process in the water column. In the Aha reservoir, a typical seasonally stratified water body, we observed the coexistence of denitrification, bacterial sulfide oxidation, and bacterial sulfate reduction in hypoxic conditions. This is substantiated by the presence of abundant N/S-related genes (nosZ and aprAB/dsrAB) and fluctuations in N/S species. The lower 15εNO3/18εNO3 ratio (0.60) observed in this study, compared to heterotrophic denitrification, strongly supports the occurrence of sulfur-driven denitrification. Furthermore, we found a robust positive correlation between the metabolic potential of bacterial sulfide oxidation and denitrification (p < 0.05), emphasizing the role of sulfide produced via sulfate reduction in enhancing denitrification. Sulfide-driven denitrification relied on ∑S2- as the primary electron donor preferentially oxidized by denitrification. The pivotal genus, Sulfuritalea, emerged as a central player in both denitrification and sulfide oxidation processes in hypoxic water bodies. Our study provides compelling evidence that sulfides assume a critical role in regulating denitrification in hypoxic water within an ecosystem where their contribution to the overall nitrogen cycle was previously underestimated.

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

Femtosecond transient absorption spectroscopy was used to study the dynamics of the excited primary electron donor in the reaction centers of the purple bacterium Rhodobacter sphaeroides. 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 stateP+BA-, indicating the reversibility of electron transfer to the primary electron acceptor, theBA molecule. In the reaction centers, the signs of superposition of two electronic states of P were found for a delay time of less than 200fs. It is suggested that the admixture value of the charge transfer statePA+PB- with the excited primary electron donorP* is about 24%. The results obtained are discussed in terms of the two-step electron transfer mechanism.

Probing ligands to reaction centers to limit the photocycle in photosynthetic bacterium Rubrivivax gelatinosus

Light-induced electron flow between reaction center and cytochrome bc1 complexes is mediated by quinones and electron donors in purple photosynthetic bacteria. Upon high-intensity excitation, the contribution of the cytochrome bc1 complex is limited kinetically and the electron supply should be provided by the pool of reduced electron donors. The kinetic limitation of electron shuttle between reaction center and cytochrome bc1 complex and its consequences on the photocycle were studied by tracking the redox changes of the primary electron donor (BChl dimer) via absorption change and the opening of the closed reaction center via relaxation of the bacteriochlorophyll fluorescence in intact cells of wild type and pufC mutant strains of Rubrivivax gelatinosus. The results were simulated by a minimum model of reversible binding of different ligands (internal and external electron donors and inhibitors) to donor and acceptor sides of the reaction center. The calculated binding and kinetic parameters revealed that control of the rate of the photocycle is primarily due to 1) the light intensity, 2) the size and redox state of the donor pool, and 3) the unbinding rates of the oxidized donor and inhibitor from the reaction center. The similar kinetics of strains WT and pufC lacking the tetraheme cytochrome subunit attached to the reaction center raise the issue of the physiological importance of this subunit discussed from different points of view. SignificanceA crucial factor for the efficacy of electron donors in photosynthetic photocycle is not just the substantial size of the pool and large binding affinity (small dissociation constant KD = koff/kon) to the RC, but also the mean residence time (koff)−1 in the binding pocket. This is an important parameter that regulates the time of re-activation of the RC during multiple turnovers. The determination of koff has proven challenging and was performed by simulation of widespread experimental data on the kinetics of P+ and relaxation of fluorescence. This work is a step towards better understanding the complex pathways of electron transfer in proteins and simulation-based design of more effective electron transfer components in natural and artificial systems.

Reductive dissolution of As-bearing iron oxides: Mediating mechanism of fulvic acid and dissimilated iron reducing bacteria

Fulvic acid (FA) and iron oxides often play regulating roles in the geochemical behavior and ecological risk of arsenic (As) in terrestrial ecosystems. FA can act as electron shuttles to facilitate the reductive dissolution of As-bearing iron (hydr)oxides. However, the influence of FA from different sources on the sequential conversion of Fe/As in As-bearing iron oxides under biotic and abiotic conditions remains unclear. In this work, we exposed prepared As-bearing iron oxides to FAs derived from lignite (FAL) and plant peat (FAP) under anaerobic conditions, tracked the fate of Fe and As in the aqueous phase, and investigated the reduction transformation of Fe(III)/As(V) with or without the presence of Shewanella oneidensis MR-1. The results showed that the reduction efficiency of Fe(III)/As(V) was increased by MR-1, through its metabolic activity and using FAs as electron shuttles. The reduction of Fe(III)/As(V) was closely associated with goethite being more conducive to Fe/As reduction compared to hematite. It is determined that functional groups such as hydroxy, carboxy, aromatic, aldehyde, ketone and aliphatic groups are the primary electron donors. Their reductive capacities rank in the following sequence: hydroxy> carboxy, aromatic, aldehyde, ketone> aliphatic group. Notably, our findings suggest that in the biotic reduction, Fe significantly reduction precedes As reduction, thereby influencing the latter's reduction process across all incubation systems. This work provides empirical support for understanding iron's role in modulating the geochemical cycling of As and is of significant importance for assessing the release risk of arsenic in natural environments.

Microbial synergies drive simultaneous biodegradation of ethoxy and alkyl chains of Nonylphenol Ethoxylate in fluidized bed reactors

The widely-used surfactant Nonylphenol Ethoxylate (NPEO) produces endocrine-disrupting compounds during biodegradation, with these byproducts being more harmful than untreated NPEO. This study investigates the effectiveness of a Fluidized Bed Reactor (FBR) in reducing the production of 4-Nonylphenol (4-NP) during the biodegradation of NPEO. Two identical FBR filled with sand were used to assess the NPEO degradation and to enhance the microbial consortia capable of breaking down the complex byproducts, ethanol and fumarate were introduced as co-substrates. Our findings demonstrate the significant potential of the FBR, especially when coupled with fumarate, for enhancing the surfactant degradation. It outperforms the efficiency achieved with ethanol as the primary electron donor, albeit with a higher rate of byproduct production. Microbial community taxonomy and metabolic prediction revealed the high abundance of Geobacter (1.51–31.71%) and Methanobacterium (1.08–13.81%) in non-conductive sand. This may hint a new metabolic interaction and expand our understanding of Direct Interspecies Electron Transfer (DIET) in bioreactors applied to micropollutants degradation. Such an intricate relationship between facultative and anaerobes working together to simultaneously biodegrade the ethoxy and alkyl chains presents a new perspective on NPEO degradation and can potentially be extended to other micropollutants.

APX2 Is an Ascorbate Peroxidase-Related Protein that Regulates the Levels of Plastocyanin in Chlamydomonas.

The function of ascorbate peroxidase-related (APX-R) proteins, present in all green photosynthetic eukaryotes, remains unclear. This study focuses on APX-R from Chlamydomonas reinhardtii, namely, ascorbate peroxidase 2 (APX2). We showed that apx2 mutants exhibited a faster oxidation of the photosystem I primary electron donor, P700, upon sudden light increase and a slower re-reduction rate compared to the wild type, pointing to a limitation of plastocyanin. Spectroscopic, proteomic and immunoblot analyses confirmed that the phenotype was a result of lower levels of plastocyanin in the apx2 mutants. The redox state of P700 did not differ between wild type and apx2 mutants when the loss of function in plastocyanin was nutritionally complemented by growing apx2 mutants under copper deficiency. In this case, cytochrome c6 functionally replaces plastocyanin, confirming that lower levels of plastocyanin were the primary defect caused by the absence of APX2. Overall, the results presented here shed light on an unexpected regulation of plastocyanin level under copper-replete conditions, induced by APX2 in Chlamydomonas.

The role of superoxide anion to Cr(VI) reduction by pine biochar

It has been reported that Cr(VI) can be reduced by biochar because of its redox activity. Considering the anionic form of Cr(VI), we hypothesize that the reduction in aqueous phase is significant. However, the contribution of different reactive oxygen species in the biochar-Cr(VI) reaction system has not been distinguished. Herein, we quantitatively identified Cr(VI) adsorption and reduction in biochar systems. The reduction content of Cr(VI) was 1.5 times higher in untreated conditions than in anaerobic conditions. The disappearance of·O2- under anaerobic conditions illustrated that·O2- may be involved in the reduction of Cr(VI). Quenching of·O2- resulted in a decrease of Cr(VI) reduction by 34%, while 1O2 was negligible, probably due to the stronger electron-donating capacity of·O2-. The degradation of nitrotetrazolium blue chloride (quenching agent of·O2-) confirmed that the reduction process of·O2- mainly occurred in the liquid-phase. Boehm titration and quantification of·O2- further elucidated the significant correlation (P < 0.05) between phenolic groups and the formation of·O2-, which implied that phenolic groups acted as the primary electron donors in generating·O2-. This study highlights the importance of the liquid-phase reduction process in removing Cr(VI), which provides theoretical support for biochar conversion of Cr(VI).

Reinvestigation on primary processes of PSII-dimer from Thermosynechococcus vulcanus by femtosecond pump-probe spectroscopy.

Cyanobacterial photosynthetic apparatus efficiently capture sunlight, and the energy is subsequently transferred to photosystem I (PSI) and II (PSII), to produce electrochemical potentials. PSII is a unique membrane protein complex that photo-catalyzes oxidation of water and majorly contains photosynthetic pigments of chlorophyll a and carotenoids. In the present study, the ultrafast energy transfer and charge separation dynamics of PSII from a thermophilic cyanobacterium Thermosynechococcus vulcanus were reinvestigated by femtosecond pump-probe spectroscopic measurements under low temperature and weak intensity excitation condition. The results imply the two possible models of the energy transfers and subsequent charge separation in PSII. One is the previously suggested "transfer-to-trapped limit" model. Another model suggests that the energy transfers from core CP43 and CP47 antennas to the primary electron donor ChlD1 with time-constants of 0.71ps and 3.28ps at 140K (0.17 and 1.33ps at 296K), respectively and that the pheophytin anion (PheoD1-) is generated with the time-constant of 43.0ps at 140K (14.8ps at 296K) upon excitation into the Qy band of chlorophyll a at 670nm. The secondary electron transfer to quinone QA: PheoD1-QA → PheoD1QA- is observed with the time-constant of 650ps only at 296K. On the other hand, an inefficient β-carotene → chlorophyll a energy transfer (33%) occurred after excitation to the S2 state of β-carotene at 500nm. Instead, the carotenoid triplet state appeared in an ultrafast timescale after excitation at 500nm.

A three-stage process of Mn(VII)-Fe(III)/PDS system for enhancing sludge dewaterability: Effective driving of Fe(II)/Fe(III) cycle and adequate assurance of ROS

Sludge dewatering helps to reduce the cost of sewage treatment plants and environmental impact. Hence, we used a type of permanganate (Mn(IV)) activated ferric chloride (Fe(III))/ peroxydisulfate (PDS) system to enhance sludge dewaterability. At optimal dose, the simultaneous addition of Mn(IV), Fe(III) and PDS (MFP treatment) reduced the value of specific resistance to filtration (SRF) from 6.76 × 108 s2/g to 2.01 × 108 s2/g. There is a three-stage process during MFP conditioning: oxidation/flocculation, adsorption/oxidation, and reflocculation. X-ray photoelectron spectroscopy (XPS) and Electron spin resonance (ESR) analyses were employed to investigate the oxidation mechanism. Meanwhile, the analysis of Mn valence was improved by considering metal leaching rates. The cycles of Mn(II)/Mn(III)/Mn(IV) were initiated under the reaction between Mn(VII) and extracellular polymeric substances (EPS)/PDS. This process sustained the cycle of Fe(II)/Fe(III) to promote activation of PDS, which continuously promoted to generate OH•, SO4-•, •O2–, 1O2 and Fe(IV). Additionally, sludge EPS was proven to be the primary electron donor of Fe(II)/Fe(III) cycle, resulting in the increasing of Fe(II) proportion. Notably, the hydrophilicity and water-holding capacity of EPS declined significantly following excellent oxidation, which promoted the conversion of bound water. The effective destruction of hydrogen bonds and protein secondary structures explained the outstanding effects. Meanwhile, XDLVO calculation revealed that sludge coagulation performance enhanced through Fe(III) flocculation, MnO2 adsorption and hydrophobic flocculation. Raw and converted free water would be effectively removed through the more hydrophobic surface of large and tough drain pores formed after the reflocculation process. The efficient drainage of sludge water created strong sludge dewaterability. This study verified the practicality of MFP treatment by exploring the oxidation and coagulation mechanisms to clarify the dewatering mechanism.

Rumen Lachnospiraceae isolate NK3A20 exhibits metabolic flexibility in response to substrate and coculture with a methanogen.

Hydrogen (H2) is the primary electron donor for methane formation in ruminants, but the H2-producing organisms involved are largely uncharacterized. This work integrated studies of microbial physiology and genomics to characterize rumen bacterial isolate NK3A20 of the family Lachnospiraceae. Isolate NK3A20 was the first recognized isolate of the NK3A20 group, which is among the ten most abundant bacterial genera in 16S rRNA gene surveys of rumen microbiota. NK3A20 produced acetate, butyrate, H2, and formate from glucose. The end product ratios varied when grown with different substrates and at different H2 partial pressures. NK3A20 produced butyrate as a major product using glucose or under high H2 partial pressures and switched to mainly acetate in the presence of galacturonic acid (an oxidized sugar) or in coculture with a methanogen. Growth with galacturonic acid was faster at elevated H2 concentrations, while elevated H2 slowed growth with glucose. Genome analyses revealed the presence of multiple hydrogenases including a membrane-bound Ech hydrogenase, an electron bifurcating butyryl-CoA dehydrogenase (Bcd-Etf), and an Rnf complex that may be involved in modulating the observed metabolic pathway changes, providing insight into H2 formation in the rumen. IMPORTANCE The genus-level NK3A20 group is one of the ten most abundant genera of rumen bacteria. Like most of the rumen bacteria that produce the hydrogen that is converted to methane in the rumen, it is understudied, without any previously characterized isolates. We investigated isolate NK3A20, a cultured member of this genus, and showed that it modulates hydrogen production in response to its growth substrates and the hydrogen concentration in its environment. Low-hydrogen concentrations stimulated hydrogen formation, while high concentrations inhibited its formation and shifted the fermentation to more reduced organic acid products. We found that growth on uronic acids, components of certain plant polymers, resulted in low hydrogen yields compared to glucose, which could aid in the selection of low-methane feeds. A better understanding of the major genera that produce hydrogen in the rumen is part of developing strategies to mitigate biogenic methane emitted by livestock agriculture.

Tuning of the ChlD1 and ChlD2 properties in photosystem II by site-directed mutagenesis of neighbouring amino acids

Photosystem II is the water/plastoquinone photo-oxidoreductase of photosynthesis. The photochemistry and catalysis occur in a quasi-symmetrical heterodimer, D1D2, that evolved from a homodimeric ancestor. Here, we studied site-directed mutants in PSII from the thermophilic cyanobacterium Thermosynechoccocus elongatus, focusing on the primary electron donor chlorophyll a in D1, ChlD1, and on its symmetrical counterpart in D2, ChlD2, which does not play a direct photochemical role. The main conserved amino acid specific to ChlD1 is D1/T179, which H-bonds the water ligand to its Mg2+, while its counterpart near ChlD2 is the non-H-bonding D2/I178. The symmetrical-swapped mutants, D1/T179I and D2/I178T, and a second ChlD2 mutant, D2/I178H, were studied. The D1 mutations affected the 686 nm absorption attributed to ChlD1, while the D2 mutations affected a 663 nm feature, tentatively attributed to ChlD2. The mutations had little effect on enzyme activity and forward electron transfer, reflecting the robustness of the overall enzyme function. In contrast, the mutations significantly affected photodamage and protective mechanisms, reflecting the importance of redox tuning in these processes. In D1/T179I, the radical pair recombination triplet on ChlD1 was shared onto a pheophytin, presumably PheD1 and the detection of 3PheD1 supports the proposed mechanism for the anomalously short lifetime of 3ChlD1; e.g. electron transfer quenching by QA− of 3PheD1 after triplet transfer from 3ChlD1. In D2/I178T, a charge separation could occur between ChlD2 and PheD2, a reaction that is thought to occur in ancestral precursors of PSII. These mutants help understand the evolution of asymmetry in PSII.

Effect of cationic antiseptics on fluorescent characteristics and electron transfer in cyanobacterial photosystem I complexes.

In this study, the effects of cationic antiseptics such as chlorhexidine, picloxidine, miramistin, and octenidine at concentrations up to 150µM on fluorescence spectra and its lifetimes, as well as on light-induced electron transfer in protein-pigment complexes of photosystem I (PSI) isolated from cyanobacterium Synechocystis sp. PCC 6803 have been studied. In doing so, octenidine turned out to be the most "effective" in terms of its influence on the spectral and functional characteristics of PSI complexes. It has been shown that the rate of energy migration from short-wavelength forms of light-harvesting chlorophyll to long-wavelength ones slows down upon addition of octenidine to the PSI suspension. After photo-separation of charges between the primary electron donor P700 and the terminal iron-sulfur center(s) FA/FB, the rate of forward electron transfer from (FA/FB)- to the external medium slows down while the rate of charge recombination between reduced FA/FB- and photooxidized P700+ increases. The paper considers the possible causes of the observed action of the antiseptic.

The impact of water table fluctuation and salinity on LNAPL distribution and geochemical properties in the smear zone under completely anaerobic conditions

Climate and groundwater are always in a state of dynamic equilibrium. Subsurface systems contaminated by light non-aqueous phase liquid (LNAPL) present a challenge to understand the overall impact of water table dynamics, due to various interacting mechanisms, including volatilization, and LNAPL mobilization/dissolution along the groundwater flow direction and oscillating redox conditions. We investigated the impact of water table fluctuations on LNAPL natural attenuation and soil geochemical characteristics in semi-arid coastal areas under saline conditions. Four soil columns operated for 151 days under anoxic conditions where a layer of benzene and toluene were subjected to a stable and fluctuating water table associated with low and high salinity conditions. The bottom of stable and fluctuating columns reached an anaerobic state after 40 days, while the middle of stable column took 60 days. pH values of the fluctuating columns covered a wide range, and at the end shifted towards alkaline conditions, unlike the stable columns. In fluctuating columns, pore water sulfate decreased in the middle, but in stable columns, it decreased in the first 40 days, which suggested that sulfate was the primary electron donor and sulfate-reducing bacteria were present. At the source zone, benzene and toluene reached their maximum concentration after 30 and 10 days for the stable and the fluctuating columns, respectively. Significant decrease in benzene and toluene concentrations occurred under the fluctuating water table. Salinity did not affect benzene and toluene concentrations in the aqueous phase, although water table fluctuations have the most effect. Soil solid-phase analysis shows fluctuating columns have less toluene than stable columns. Solid-phase analysis showed the fluctuating columns have less benzene and toluene concentrations as compared to the stable columns.

Charge separation in the photosystem II reaction center resolved by multispectral two-dimensional electronic spectroscopy

The photosystem II reaction center (PSII RC) performs the primary energy conversion steps of oxygenic photosynthesis. While the PSII RC has been studied extensively, the similar time scales of energy transfer and charge separation and the severely overlapping pigment transitions in the Qy region have led to multiple models of its charge separation mechanism and excitonic structure. Here, we combine two-dimensional electronic spectroscopy (2DES) with a continuum probe and two-dimensional electronic vibrational spectroscopy (2DEV) to study the cyt b559-D1D2 PSII RC at 77 K.This multispectral combination correlates the overlapping Qy excitons with distinct anion and pigment-specific Qx and mid-infrared transitions to resolve the charge separation mechanism and excitonic structure. Through extensive simultaneous analysis of the multispectral 2D data, we find that charge separation proceeds on multiple time scales from a delocalized excited state via a single pathway in which PheoD1 is the primary electron acceptor, while ChlD1 and PD1 act in concert as the primary electron donor.

Primary charge separation in native and plant pheophytin a-modified reaction centers of Chloroflexus aurantiacus: Ultrafast transient absorption measurements at low temperature

Ultrafast transient absorption (TA) spectroscopy was used to study electron transfer (ET) at 100 K in native (as isolated) reaction centers (RCs) of the green filamentous photosynthetic bacterium Chloroflexus (Cfl.) aurantiacus. The rise and decay of the 1028 nm anion absorption band of the monomeric bacteriochlorophyll a molecule at the BA binding site were monitored as indicators of the formation and decay of the P+BA− state, respectively (P is the primary electron donor, a dimer of bacteriochlorophyll a molecules). Global analysis of the TA data indicated the presence of at least two populations of the P⁎ excited state, which decay by distinct means, forming the state P+HA− (HA is a photochemically active bacteriopheophytin a molecule). In one population (~65 %), P⁎ decays in ~2 ps with the formation of P+HA− via a short-lived P+BA− intermediate in a two-step ET process P⁎ → P+BA−→ P+HA−. In another population (~35 %), P⁎ decays in ~20 ps to form P+HA− via a superexchange mechanism without producing measurable amounts of P+BA−. Similar TA measurements performed on chemically modified RCs of Cfl. aurantiacus containing plant pheophytin a at the HA binding site also showed the presence of two P⁎ populations (~2 and ~20 ps), with P⁎ decaying through P+BA− only in the ~2 ps population. At 100 K, the quantum yield of primary charge separation in native RCs is determined to be close to unity. The results are discussed in terms of involving a one-step P⁎ → P+HA− superexchange process as an alternative highly efficient ET pathway in Cfl. aurantiacus RCs.

The Photoactive Photosynthetic Reaction Center of a Rhodobacter sphaeroides Mutant Lacking 3-Vinyl (Bacterio)Chlorophyllide a Hydratase Contains 3-Vinyl Bacteriochlorophyll a.

Rhodobacter sphaeroides mutant BF-lacking 3-vinyl (bacterio)chlorophyllide a hydratase (BchF)-accumulates chlorophyllide a (Chlide a) and 3-vinyl bacteriochlorophyllide a (3V-Bchlide a). BF synthesizes 3-vinyl bacteriochlorophyll a (3V-Bchl a) through prenylation of 3V-Bchlide a and assembles a novel reaction center (V-RC) using 3V-Bchl a and Mg-free 3-vinyl bacteriopheophytin a (3V-Bpheo a) at a molar ratio of 2:1. We aimed to verify whether a bchF-deleted R. sphaeroides mutant produces a photochemically active RC that facilitates photoheterotrophic growth. The mutant grew photoheterotrophically-implying a functional V-RC-as confirmed by the emergence of growth-competent suppressors of bchC-deleted mutant (BC) under irradiation. Suppressor mutations in BC were localized to bchF, which diminished BchF activity and caused 3V-Bchlide a accumulation. bchF expression carrying the suppressor mutations in trans resulted in the coproduction of V-RC and wild-type RC (WT-RC) in BF. The V-RC had a time constant (τ) for electron transfer from the primary electron donor P (a dimer of 3V-Bchl a) to the A-side containing 3V-Bpheo a (HA) similar to that of the WT-RC and a 60% higher τ for electron transfer from HA to quinone A (QA). Thus, the electron transfer from HA to QA in the V-RC should be slower than that in the WT-RC. Furthermore, the midpoint redox potential of P/P+ of the V-RC was 33 mV more positive than that of the WT-RC. R. sphaeroides, thus, synthesizes the V-RC when 3V-Bchlide a accumulates. The V-RC can support photoheterotrophic growth; however, its photochemical activity is inferior to that of the WT-RC. IMPORTANCE 3V-Bchlide a is an intermediate in the bacteriochlorophyll a (Bchl a)-specific biosynthetic branch and prenylated by bacteriochlorophyll synthase. R. sphaeroides synthesizes V-RC that absorbs light at short wavelengths. The V-RC was not previously discovered because 3V-Bchlide a does not accumulate during the growth of WT cells synthesizing Bchl a. The levels of reactive oxygen species increased with the onset of photoheterotrophic growth in BF, resulting in a long lag period. Although the inhibitor of BchF is unknown, the V-RC may act as a substitute for the WT-RC when BchF is completely inhibited. Alternatively, it may act synergistically with WT-RC at low levels of BchF activity. The V-RC may broaden the absorption spectra of R. sphaeroides and supplement its photosynthetic ability at various wavelengths of visible light to a greater extent than that by the WT-RC alone.

The role of electron transfer in the enzymatic activity of a photoactivable adenylate cyclase.

OaPAC is a photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata and belongs to a class of photoactive flavoenzymes which are involved in the conversion of ATP (adenosine triphosphate) to the important second messenger cAMP (cyclic adenosine monophosphate). The photoactivation is controlled by a BLUF (Blue Light Sensing Using Flavin) domain; a well-known light-triggered switch present in many biological photoreceptors and able to control photochemical energy into a diverse set of cellular responses (e.g. phototaxis, photophobia, enzyme activity, gene expression). Blue-light excitation of the BLUF domain of OaPAC results in a proton-coupled electron transfer process where the primary electron and proton donor is a tyrosine residue in the vicinity of the flavin. The electron transfer to the flavin is accompanied by a reorganization of its hydrogen bond network, and the induced structural changes are believed to propagate towards the AC domain enabling the conversion of ATP to cAMP. In this work we studied the relation between the electron transfer and the activity of the enzyme using ultrafast and fluorescence spectroscopy as well as differential scanning calorimetry. A.L. acknowledges funding from the Hungarian National Research and Innovation Office (K-137557).