Relationship between the oxidation potential of the bacteriochlorophyll dimer and electron transfer in photosynthetic reaction centers

The primary electron donor in the photosynthetic reaction center from purple bacteria is a bacteriochlorophyll dimer containing four conjugated carbonyl groups that may form hydrogen bonds with amino acid residues. Spectroscopic analyses of a set of mutant reaction centers confirm that hydrogen bonds can be formed between each of these carbonyl groups and histidine residues in the reaction center subunits. The addition of each hydrogen bond is correlated with an increase in the oxidation potential of the dimer, resulting in a 355-mV range in the midpoint potential. The resulting changes in the free-energy differences for several reactions involving the dimer are related to the electron transfer rates using the Marcus theory. These reactions include electron transfer from cytochrome c₂ to the oxidized dimer, charge recombination from the primary electron acceptor quinone, and the initial forward electron transfer.     

Primary photochemistry of reaction centers from the photosynthetic purple bacteria

Photosynthetic organisms transform the energy of sunlight into chemical potential in a specialized membrane-bound pigment-protein complex called the reaction center. Following light activation, the reaction center produces a charge-separated state consisting of an oxidized electron donor molecule and a reduced electron acceptor molecule. This primary photochemical process, which occurs via a series of rapid electron transfer steps, is complete within a nanosecond of photon absorption. Recent structural data on reaction centers of photosynthetic bacteria, combined with results from a large variety of photochemical measurements have expanded our understanding of how efficient charge separation occurs in the reaction center, and have changed many of the outstanding questions.Abbreviations BChl bacteriochlorophyll - P a dimer of BChl molecules - BPh bacteriopheophytin - Q_A and Q_B quinone molecules - L, M and H light, medium and heavy polypeptides of the reaction center     

Secondary electron transfer in reaction centers of Rhodopseudomonas sphaeroides. Out-of-phase periodicity of two for the formation of ubisemiquinone and fully reduced ubiquinone.

Electron transfer between purified reaction centers from Rhodopseudomonas sphaeroides and exogenous ubiquinone has been studied in the presence of electron donors by measurements of light-induced absorbance changes following a sequence of short actinic light flashes. Each odd flash promotes the formation of a molecule of ubisemiquinone; after each even flash the semiquinone disappears and a molecule of the fully reduced quinone appears. We interpret these results by means of a model where a specialized molecule of ubiquinone is reduced by the primary electron acceptor in a one-electron transfer reaction after each flash, and is reoxidized by a molecule of the ubiquinone pool in a two-electron transfer reaction every two flashes.     

Photoelectric currents across planar bilayer membranes containing bacterial reaction centers: the response under conditions of multiple reaction-center turnovers

The characteristics of the photocurrent response activated by continuous illumination of planar bilayer membranes containing bacterial reaction centers have been resolved by voltage clamp methods. The photocurrent response to a long light pulse consists of an initial spike arising from the fast, quasi-synchronous electron transfer from the reaction center bacteriochlorophyll dimer, BChl2, to the primary quinone QA. This is followed by a slow relaxation of the current to that promoted by secondary, asynchronous multiple electron transfers from the reduced cytochrome c through the reaction centers to the ubiquinone-10 pool. Currents derived from cytochrome c oxidation that occurs when cytochrome c is associated with the reaction center or when limited by diffusional interaction from solution are recognized. Changes of the ionic strength and pH in the aqueous phase, and the clamped membrane potential (+/- 150 mV), affect the electron-transfer rate between cytochrome c and BChl2. In contrast, the primary light-induced charge separation between BChl2 and QA, or electron transfer between QA on the ubiquinone pool are unaffected. During illumination of reaction center membranes supplemented with cytochrome c and a ubiquinone pool, there is a small but significant steady-state current which is considered to be caused by the re-oxidation of photoreduced quinone by molecular oxygen. In the dark, after illumination of reaction centers supplemented with cytochrome c and a ubiquinone pool, there is a small amount of reverse current resulting from the movement of charges back across the membrane. This reverse current is observed maximally after 400 ms illumination while prolonged illumination diminishes the effect. The source of this current is uncertain, but it is considered to be due to the flux of anionic semiquinone within the membrane profile; this may also be the species that interacts with oxygen giving rise to the steady-state current. It is postulated that when the reaction centers are contained in an alkane-containing phospholipid membrane, in contrast to the in vivo situation, the semiquinone anion formed in the QB site is not tightly bound to the site and can, by exchange-diffusion with the membrane-quinone pool, move away from the site and accumulate in the membrane. However, in the absence, more quantitative work superoxide anion, resulting from O2 interaction with semiquinone of QA, QB or pool cannot be excluded.     

Secondary electron transfer in reaction centers of Rhodopseudomonas sphaeroides. Out-of-phase periodicity of two for the formation of ubisemiquinone and fully reduced ubiquinone

Electron transfer between purified reaction centers from Rhodopseudomonas sphaeroides and exogenous ubiquinone has been studied in the presence of electron donors by measurements of light-induced absorbance changes following a sequence of short actinic light flashes. Each odd flash promotes the formation of a molecule of ubisemiquinone; after each even flash the semiquinone disappears and a molecule of the fully reduced quinone appears.We interpret these results by means of a model where a specialized molecule of ubiquinone is reduced by the primary electron acceptor in a one-electron transfer reaction after each flash, and is reoxidized by a molecule of the ubiquinone pool in a two-electron transfer reaction every two flashes.     

Photochemical Electron Transport in Photosynthetic Reaction Centers from Rhodopseudomonas spheroides: III. Effects of Orthophenanthroline and Other Chemicals

Reaction centers from Rhodopseudomonas spheroides mediate the photochemical oxidation of cytochrome c (cyt c), and show a time-varying fluorescence of P870. Analyses of these effects indicate that the reaction centers contain a primary photochemical electron acceptor capable of holding one electron. Native or added ubiquinone (UQ) can act as a secondary electron acceptor. Orthophenanthroline (o-phen) blocks electron transfer from primary to secondary acceptors, and allows the primary acceptor to be exhibited in the foregoing experiments. Other chelators (with the possible exception of 8-hydroxyquinoline) and dichlorophenyldimethylurea (DCMU) are without apparent effect on reaction centers. o-Phen also inhibits the primary photochemical act in reaction centers; this effect is prevented by the presence of UQ. 2-n-Nonyl-4-hydroxyquinoline-N-oxide (NQNO) inhibits the primary photochemistry in reaction centers but does not affect secondary electron transfer.     

Design of a redox-linked active metal site: manganese bound to bacterial reaction centers at a site resembling that of photosystem II

Metals bound to proteins perform a number of crucial biological reactions, including the oxidation of water by a manganese cluster in photosystem II. Although evolutionarily related to photosystem II, bacterial reaction centers lack both a strong oxidant and a manganese cluster for mediating the multielectron and proton transfer needed for water oxidation. In this study, carboxylate residues were introduced by mutagenesis into highly oxidizing reaction centers at a site homologous to the manganese-binding site of photosystem II. In the presence of manganese, light-minus-dark difference optical spectra of reaction centers from the mutants showed a lack of the oxidized bacteriochlorophyll dimer, while the reduced primary quinone was still present, demonstrating that manganese was serving as a secondary electron donor. On the basis of these steady-state optical measurements, the mutant with the highest-affinity site had a dissociation constant of approximately 1 microM. For the highest-affinity mutant, a first-order rate with a lifetime of 12 ms was observed for the reduction of the oxidized bacteriochlorophyll dimer by the bound manganese upon exposure to light. The dependence of the amplitude of this component on manganese concentration yielded a dissociation constant of approximately 1 muM, similar to that observed in the steady-state measurements. The three-dimensional structure determined by X-ray diffraction of the mutant with the high-affinity site showed that the binding site contains a single bound manganese ion, three carboxylate groups (including two groups introduced by mutagenesis), a histidine residue, and a bound water molecule. These reaction centers illustrate the successful design of a redox active metal center in a protein complex.     

Anti-cooperativity in the two electron oxidation of the S118C disulfide dimer of azurin

We have constructed a disulfide dimer of S118C azurin, in which two copper centers are coupled through a relatively short covalent pathway, and studied its electron transfer properties. The dimer exhibits intriguing mechanistic properties. Due to the strain in the molecule, caused by the limited accessibility of Cys118, anti-cooperativity occurs in the two step oxidation of the dimer with a difference in redox potential between the two half reactions of 33 mV. Upon oxidation, the dimer favours the semi-reduced over the fully oxidized state, as the Cu(I) site in the semi-reduced dimer is able to stabilize the strained dimer complex. The internal electron transfer is surprisingly slow, which could be partially due to an increase in reorganization energy.     

Manganese oxidation by modified reaction centers from Rhodobacter sphaeroides

The transfer of an electron from exogenous manganese (II) ions to the bacteriochlorophyll dimer, P, of bacterial reaction centers was characterized for a series of mutants that have P/P(+) midpoint potentials ranging from 585 to 765 mV compared to 505 mV for wild type. Light-induced changes in optical and EPR spectra of the mutants were measured to monitor the disappearance of the oxidized dimer upon electron donation by manganese in the presence of bicarbonate. The extent of electron transfer was strongly dependent upon the P/P(+) midpoint potential. The midpoint potential of the Mn(2+)/Mn(3+) couple was calculated to decrease linearly from 751 to 623 mV as the pH was raised from 8 to 10, indicating the involvement of a proton. The electron donation had a second order rate constant of approximately 9 x 10(4) M(-1) s(-1), determined from the linear increase in rate for Mn(2+) concentrations up to 200 microM. Weak dissociation constants of 100-200 microM were found. Quantitative EPR analysis of the six-line free Mn(2+) signal revealed that up to seven manganese ions were associated with the reaction centers at a 1 mM concentration of manganese. The association and the electron transfer between manganese and the reaction centers could be inhibited by Ca(2+) and Na(+) ions. The ability of reaction centers with high potentials to oxidize manganese suggests that manganese oxidation could have preceded water oxidation in the evolutionary development of photosystem II.     

The involvement of iron and ubiquinone in electron transfer reactions mediated by reaction centers from photosynthetic bacteria.

Reaction centers from Rhodopseudomonas sphaeroides strain R-26 were prepared with varying Fe and ubiquinone (Q) contents. The photooxidation of P-870 to P-870+ was found to occur with the same quantum yield in Fe-depleted reaction centers as in control samples. The kinetics of electron transfer from the initial electron acceptor (I) to Q also were unchanged upon Fe removal. We conclude that Fe has no measurable role in the primary photochemical reaction. The extent of secondary reaction from the first quinone acceptor (QA) to the second quinone acceptor (QB) was monitored by the decay kinetics of P-870+ after excitation of reaction centers with single flashes in the absence of electron donors, and by the amount of P-870 photooxidation that occurred on the second flash in the presence of electron donors. In reaction centers with nearly one iron and between 1 and 2 ubiquinones per reaction center, the amount of secondary electron transfer is proportional to the ubiquinone content above one per reaction center. In reaction centers treated with LiClO4 and o-phenanthroline to remove Fe, the amount of secondary reaction is decreased and is proportional to Fe content. Fe seems to be required for the secondary reaction. In reaction centers depleted of Fe by treatment with SDS and EDTA, the correlation between Fe content and secondary activity is not as good as that found using LiClO4. This is probably due in part to a loss of primary photochemical activity in samples treated with SDS; but the correlation is still not perfect after correction for this effect. The nature of the back reaction between P-870+ and Q-B was investigated using stopped flow techniques. Reaction centers in the P-870+ Q-B state decay with a 1-s half-time in both the presence and absence of o-phenanthroline, an inhibitor of electron transfer between Q-B and QB. This indicates that the back reaction between P-870+ and Q-A is direct, rather than proceeding via thermal repopulation of Q-A. The P-870+ Q-B state is calculated to lie at least 100 mV in free energy below the P-870+ Q-A state.     

Dynamics of electron transfer from high-potential cytochrome <Emphasis Type="Italic">c</Emphasis> to bacteriochlorophyll dimer in photosynthetic reaction centers as probed using laser-induced temperature jump

Laser-induced temperature jump experiments were used for testing the rates of thermoinduced conformational transitions of reaction center (RC) complexes in chromatophores of Chromatium minutissimum. The thermoinduced transition of the macromolecular RC complex to a state providing effective electron transport from the multiheme cytochrome c to the photoactive bacteriochlorophyll dimer within the temperature range 220–280 K accounts for tens of seconds with activation energy 0.166 eV/molecule. The rate of the thermoinduced transition in the cytochrome–RC complex was found to be three orders of magnitude slower than the rate of similar thermoinduced transition of the electron transfer reaction from the primary to secondary quinone acceptors studied in the preceding work (Chamorovsky et al. in Eur Biophys J 32:537–543, 2003). Parameters of thermoinduced activation of the electron transfer from the multiheme cytochrome c to the photoactive bacteriochlorophyll dimer are discussed in terms of cytochrome c docking onto the RC.     

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. The angular dependence of the anisotropy was evaluated. It was shown that the spectral shape of the triplet state of photosystem II reaction center observed in the case of singly-reduced primary quinone acceptor can also be described by the anisotropic quantum yield of the triplet, with practically the same angular dependence. These properties confirm the conclusions on the mechanism of photoinduced electron transfer in photosystem II, made in previous publications. The peculiarities in the functioning of photosystem II reaction centers are probably determined by strict limitations on the triplet state generation.     

Intramolecular dynamics and electron transfer in photosynthetic reaction centers. A study using luminescence

The temperature dependences of fluorescence and phosphorescence spectra maxima of chromophor labels--endogenic (tryptophan) and exogenic (eosinisothiocyanate)--were measured for the preparations of photosynthetic membranes and reaction centers from Rhodospirillum rubrum. It was found that the dipole mobility of protein-lipid matrix in the vicinity of the chromophores intensified markedly with a temperature rise from 150 to 300K resulting in the corresponding relaxation time tau r decrease from 10(0) to 10(-8) s. The efficiency of direct transfer of the photomobilized electron in the system of quinone acceptors (A1- leads to A2) of reaction centers (characteristic half-times of the process being 10(-3) divided by 10(-4) s) was shown also to increase sharply at temperatures higher than 200K parallel to the enhancement of molecular motions with tau r approximately 10(-8) s. Meanwhile, changes observed in the rate of recombination of primary photoproducts, i.e. an oxidized bacteriochlorophyll dimer, P+ and a reduced acceptor, A1- (characteristic half-time of 10(-1) divided by 10(-2) s) and the activization of low-frequency motions with tau r approximately 10(-3) s in the external layers and tau r less than 1 s in the internal parts of the reaction centers protein develop over the same range of low temperatures (150-220 K). The nature of interactions which determine the dependence of the photosynthetic electron transport on the molecular mobility of the membrane proteins is discussed.     

Triplet state of the primary donor in reaction centers of the phototrophic bacterium <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> 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. The angular dependence of the anisotropy was evaluated. It was shown that the spectral shape of the triplet state of photosystem II reaction center observed in the case of singly-reduced primary quinone acceptor can also be described by the anisotropic quantum yield of the triplet, with practically the same angular dependence. These properties confirm the conclusions on the mechanism of photoinduced electron transfer in photosystem II, made in previous publications. The peculiarities in the functioning of photosystem II reaction centers are probably determined by strict limitations on the triplet state generation.     

Spectral exhibition of electron-vibrational relaxation in P* state of Rhodobacter sphaeroides reaction centers

Photosynthesis Research - Electron-vibrational relaxation in the excited state of the primary electron donor, bacteriochlorophyll dimer P, in the reaction centers (RCs) of purple photosynthetic...     

Functional LH1 antenna complexes influence electron transfer in bacterial photosynthetic reaction centers

The effect of the light harvesting 1 (LH1) antenna complex on the driving force for light-driven electron transfer in the Rhodobacter sphaeroides reaction center has been examined. Equilibrium redox titrations show that the presence of the LH1 antenna complex influences the free energy change for the primary electron transfer reaction through an effect on the reduction potential of the primary donor. A lowering of the redox potential of the primary donor due to the presence of the core antenna is consistently observed in a series of reaction center mutants in which the reduction potential of the primary donor was varied over a 130 mV range. Estimates of the magnitude of the change in driving force for charge separation from time-resolved delayed fluorescence measurements in the mutant reaction centers suggest that the mutations exert their effect on the driving force largely through an influence on the redox properties of the primary donor. The results demonstrate that the energetics of light-driven electron transfer in reaction centers are sensitive to the environment of the complex, and provide indirect evidence that the kinetics of electron transfer are modulated by the presence of the LH1 antenna complexes that surround the reaction center in the natural membrane.     

Temperature and detection-wavelength dependence of the picosecond electron-transfer kinetics measured in Rhodopseudomonas sphaeroides reaction centers. Resolution of new spectral and kinetic components in the primary charge-separation process

We have examined the temperature dependence of the rate of electron transfer to ubiquinone from the bacteriopheophytin (BPh) that serves as an initial electron acceptor (I) in reaction centers of Rhodopseudomonas sphaeroides. The kinetics were measured from the decay of the 665-nm absorption band of the reduced BPh (BPh⁻ or I⁻) and from the recovery of the BPh band at 545 nm, following excitation of reaction centers in polyvinyl alcohol films with 30-ps flashes. The measured time constant decreases from 229 ± 25 ps at 295 K to 97 ± 8 ps near 100 K and then remains constant down to 5 K. The temperature dependence of the kinetics can be rationalized on the assumption that the reaction results in changes in the frequencies of numerous low-energy nuclear (vibrational) modes of the electron carriers and/or the protein. The kinetics measured in the absorption bands near 765 and 795 nm show essentially the same temperature dependence as those measured at 545 or 665 nm, but the time constants vary with detection wavelength. The time constant measured in the 795-nm region (70 ± 10 ps at 5 and 76 K) is shorter than that seen in the absorption bands of the BPh; the time constant measured at 758 nm is longer. Time constants measured with reaction centers in solution at 288 K also vary with the detection wavelength. These results can be explained on the assumption that the absorption changes measured at some wavelengths reflect nuclear relaxations rather than electron transfer. The absorption changes at 795 nm probably reflect a relaxation of the bacteriochlorophyll molecules that are near neighbors of the BPh and the primary electron donor (P). Those near 530 and 755 nm probably are due to the second BPh molecule, which does not appear to undergo oxidation or reduction.     

Electron Transfer Dynamics in Rhodobacter sphaeroides Reaction Center Mutants with a Modified Ligand for the Monomer Bacteriochlorophyll on the Active Side

The histidine ligand of the monomer bacteriochlorophyll molecule on the active side (B_A) of the photosynthetic reaction center from Rhodobacter sphaeroides was mutated to a number of other amino acids. Histidine (H) at the position L153 was replaced with aspartic acid (D), glutamic acid (E), glutamine (Q), glycine (G), leucine (L), phenylalanine (F), serine (S), valine (V) and tyrosine (Y). These mutations were created to investigate how the alteration of the ligand residue affects the properties of the B_A molecule and changes the dynamics of the primary charge separation in reaction centers. In all of the mutants, the changes in the ligand result in a blue-shift of the B_A absorption spectrum, in both visible and near-infrared spectral regions, with the size of the shift varying between mutants. The primary electron transfer time constants in these mutant reaction centers range from 4.5 to 18 ps, being substantially slower than the wild-type value of 3 ps. The decrease in the electron transfer rate is interpreted as being due to an increase in the free energy of the initial charge-separated state P⁺B_A^–.This revised version was published online in October 2005 with corrections to the Cover Date.     

Dependence of tyrosine oxidation in highly oxidizing bacterial reaction centers on pH and free-energy difference

The pH and temperature dependences of tyrosine oxidation were measured in reaction centers from mutants of Rhodobacter sphaeroides containing a tyrosine residue near a highly oxidizing bacteriochlorophyll dimer. Under continuous illumination, a rapid increase in the absorption change at 420 nm was observed because of the formation of a charge-separated state involving the oxidized dimer and reduced primary quinone, followed by a slow absorption decrease attributed to tyrosine oxidation. Both the amplitude and rate of the slow absorption change showed a pH dependency, indicating that, at low pH, the rate of tyrosine oxidation is limited by the transfer of the phenolic proton to a nearby base. Below 17 degrees C, the rate of the slow absorption change had a strong exponential dependence on the temperature, indicating a high activation energy. At higher pH and temperature, the overall rate of tyrosyl formation appears to be limited by a proposed conformational change in the reaction center that is also observed in reaction centers that do not undergo tyrosine oxidation. The yield of tyrosyl formation measured using electron paramagnetic resonance spectroscopy decreased significantly at 4 degrees C compared to 20 degrees C and was lower at both temperatures in mutants expected to have a slightly smaller driving force for tyrosyl formation.     

Effects of mutations near the bacteriochlorophylls in reaction centers from Rhodobacter sphaeroides.

Mutations were made in four residues near the bacteriochlorophyll cofactors of the photosynthetic reaction center from Rhodobacter sphaeroides. These mutations, L131 Leu to His and M160 Leu to His, near the dimer bacteriochlorophylls, and M203 Gly to Asp and L177 Ile to Asp, near the monomer bacteriochlorophylls, were designed to result in the placement of a hydrogen bond donor group near the ring V keto carbonyl of each bacteriochlorophyll. Perturbations of the electronic structures of the bacteriochlorophylls in the mutants are indicated by additional resolved transitions in the bacteriochlorophyll absorption bands in steady-state low-temperature and time-resolved room temperature spectra in three of the resulting mutant reaction centers. The major effect of the two mutations near the dimer was an increase up to 80 mV in the donor oxidation-reduction midpoint potential. Correspondingly, the calculated free energy difference between the excited state of the primary donor and the initial charge separated state decreased by up to 55 mV, the initial forward electron-transfer rate was up to 4 times slower, and the rate of charge recombination between the primary quinone and the donor was approximately 30% faster in these two mutants compared to the wild type. The two mutations near the monomer bacteriochlorophylls had minor changes of 25 mV or less in the donor oxidation-reduction potential, but the mutation close to the monomer bacteriochlorophyll on the active branch resulted in a roughly 3-fold decrease in the rate of the initial electron transfer.