Spectroscopic characterization of a semi-stable, charge-separated state in Cu(2+)-substituted reaction centers from Rhodobacter sphaeroides

In reaction centers from Rhodobacter sphaeroides exposed to continuous illumination in the presence of an inhibitor of the Q(A)(-) to Q(B) electron transfer, a semi-stable, charge-separated state was formed with halftimes of formation and decay of several minutes. When the non-heme iron was replaced by Cu(2+), the decay of the semi-stable, charge-separated state became much slower than in centers with bound Fe(2+) with about the same rate constant for formation. In Cu(2+)-substituted reaction centers, the semi-stable state was associated with an EPR signal, significantly different from that observed after chemical reduction of the acceptor-side quinone or after illumination at low temperature, but similar to that of an isolated Cu(2+) in the absence of magnetic interaction. The EPR results, obtained with Cu(2+)-substituted reaction centers, suggest that the slow kinetics of formation and decay of the charge-separated, semi-stable state is associated with a structural rearrangement of the acceptor side and the immediate environment of the metal-binding site.     

Characterization of a semi-stable,charge-separated state in reaction centers from Rhodobacter sphaeroides

In reaction centers from Rhodobacter sphaeroides, subjected to continuous illumination in the presence of an inhibitor of the Q_A to Q_B electron transfer, the oxidation of P870 consisted of several kinetic phases with a fast initial reaction followed by very slow accumulation of P870⁺ with a halftime of several minutes. When the light was turned off, a phase of fast charge recombination was followed by an equally slow reduction of P870⁺. In reaction centers depleted of Q_B, where forward electron transfer from Q_A is also prevented, the slow reactions were also observed but with different kinetic properties. The kinetic traces of accumulation and decay of P870⁺ could be fitted to a simple three-state model where the initial, fast charge separation is followed by a slow reversible conversion to a long-lived, charge-stabilized state. Spectroscopic examination of the charge-separated, semi-stable state, using optical absorbance and EPR spectroscopy, suggests that the unpaired electron on the acceptor side is located in an environment significantly different from normal. The activation parameters and enthalpy and entropy changes, determined from the temperature dependence of the slow conversion reaction, suggest that this might be coupled to changes in the protein structure of the reaction centers, supporting the spectroscopic results. One model that is consistent with the present observations is that reaction centers, after the primary charge separation, undergo a slow, light-induced change in conformation affecting the acceptor side. This revised version was published online in June 2006 with corrections to the Cover Date.     

Localization of light-induced structural changes in bacterial photosynthetic reaction centers

Light-induced structural changes in photosynthetic reaction centers from Rhodobacter sphaeroides were investigated using two approaches. Cu²⁺ was used as a paramagnetic structural probe. The EPR spectrum of Cu²⁺ incorporated into the metal-depleted reaction centers was affected by 1,10-phenanthroline, an electron transfer inhibitor substituting Q_B, which suggests a localization of Cu²⁺ in a vicinity of the Q _B ^– site. However, the spectrum was not influenced by low temperature (77 K) illumination of the sample which suggests that the copper ion position is not exactly the same as that of the iron ion. Freezing the reaction centers under illumination in the presence of potassium ferricyanide and 1,10-phenanthroline caused a change in the shape of the Cu²⁺ EPR spectrum in comparison to that of a sample frozen in darkness. These data indicate a change of the Cu²⁺ ligand symmetry owing to light-induced structural changes which are probably located near the acceptor side of the reaction center. Partial trypsinolysis of reaction centers was also used to locate the structural changes. Trypsin treatment in the dark and under illumination resulted in different peptide patterns as detected by gel electrophoresis and reverse-phase high-performance liquid chromatography. Partial amino-acid sequence analysis of a number of peptides, characteristic of either light- or dark-treated reaction centers, showed that they originated from the acceptor sides of the H and M subunits. The occurrence of light-induced structural differences in the H-subunit is consistent with the suggestion that it may be involved in regulating electron transfer in this part of the reaction center.     

Pressure effects on the photochemistry of bacterial photosynthetic reaction centers from Rhodobacter sphaeroides R-26 and Rhodopseudomonas viridis

Electron-transfer reactions and triplet decay rates have been studied at pressures up to 300 MPa. In reaction centers from Rhodobacter sphaeroides R-26, high pressure hastened the electron transfers from both the primary and secondary quinones (Q_A and Q_B) to the primary electron donor bacteriochlorophyll, P. Motion of Q_A between two sites, one nearer to P and the other nearer to Q_B, could account for these pressure effects. In reaction centers from Rhodopseudomonas viridis, charge recombination was slowed by high pressure. Decay rates were also studied for the triplet state, P^R. In Rb. sphaeroides R-26 with Q_A reduced with Na₂S₂O₄, the decay was hastened by pressure. This could be explained if P^R decays through a charge-transfer triplet state, or if the decay kinetics of P^R are sensitive to the distance between P and Q_A⁻. In Rps. viridis reaction centers, and in Rb. sphaeroides reaction centers that were depleted of Q_A, the lifetime of P^R was not altered by pressure.     

A mechanism stabilizing a long-lived charge-separated state of photosynthetic reaction centers frozen under intense illumination

It is experimentally shown that slow chilling of photosynthetic reaction centers from purple bacteria Rhodobacter sphaeroides to <230 K under intense illumination leads to appearance of long-lived chargeseparated states (P⁺Q _A ^? ). This longevity implies that the recombination is blocked or the charge-separated state is stabilized. The longevity effect is caused by structural relaxation of reaction centers to a new equilibrium state that diminishes the free energy difference of recombination. The possible mechanism of such relaxation involves orientation of the polar water molecules in the semiquinone local electrostatic field. Detailed analysis of the longevity effect has been carried out, and its outcome supports the idea that many electron transfer reactions in biological systems are non-equilibrium.     

The S1 split signal of photosystem II; a tyrosine-manganese coupled interaction

Detailed optical and EPR analyses of states induced in dark-adapted PS II membranes by cryogenic illumination permit characterization and quantification of all pigment derived donors and acceptors, as well as optically silent (in the visible, near infrared) species which are EPR active. Near complete turnover formation of Q_A⁻ is seen in all centers, but with variable efficiency, depending on the donor species. In minimally detergent-exposed PS II membranes, negligible (< 5%) oxidation of chlorophyll or carotenoid centers occurs for illumination temperatures 5-20 K. An optically silent electron donor to P680⁺ is observed with the same decay kinetics as the S₁ split signal. Cryogenic donors to P680⁺ seen are: (i) transient (t_1/2 ∼ 150 s) tyrosine related species, including ‘split signals’ (∼ 15% total centers), (ii) reduced cytochrome b₅₅₉ (∼ 30-50% centers), and (iii) an organic donor, possibly an amino acid side chain, (∼ 30% centers).     

Mechanism of photoinduced electron transfer in photosystem II reaction centers

The shape of the EPR spectrum of the triplet state of photosystem II reaction centers with a singly reduced primary acceptor complex Q_AFe²⁺ was studied. It was shown that the spectroscopic properties do not significantly change when the relaxation of the primary acceptor is accelerated and when the magnetic interaction between the reduced quinone molecule Q_A and the nonheme iron ion Fe²⁺ is disrupted. This observation confirmed the earlier conclusion that the anisotropy of the quantum yield of the triplet state is the main cause of the anomalous shape of the EPR spectrum. A scheme of primary processes in photosystem II that is consistent with the observed properties of the EPR spectrum of the triplet state is discussed.     

Influence of magnetic fields on the P-870 triplet state in Rps. sphaeroides reaction centers

Magnetic fields influence two properties of the P-870 triplet state observed in Rps. sphaeroides reaction centers: the yield of formation and the kinetics of decay. These effects have been studied in reaction centers which were prepared in three different states: state Q_A ^–, state Q_A ^2– and state (– Q_A) (Q_A depleted). The triplet yields decrease with increasing magnetic fields, with B1/2's of about 140, 41 and 57 Gauss, respectively. The half-time of ³P-870 decay is not influenced by the field in state Q_A ^–; it increases at increasing fields, in state Q_A ^2– and state (– Q_A), with the same B1/2 as the triplet yield. These results are discussed in the framework of current theories of the radical-pair dynamics and of the mechanism of triplet decay.Abbreviations I primary electron acceptor - LDAO lauryldimethylamine oxide - P-870 primary electron donor - Q_A first quinone acceptor - SDS sodium dodecylsulfate - YAG Yttrium Aluminum Garnet     

The identity of a new copper(II) electron paramagnetic resonance signal in cytochrome c oxidase

In reoxidation experiments with cytochrome c oxidase (EC 1.9.3.1) in the presence of both reducing substrate and molecular oxygen, a new EPR signal from Cu²⁺ has been observed. The new signal corresponds to 0.45 Cu per functional unit. It is concluded that the new EPR signal originates from Cu²⁺_B, the copper which is EPR-nondetectable in the resting enzyme.Optical absorption changes in the 500–700 nm region accompanies the decay of the new Cu²⁺ EPR signal.Based on the results in this investigation a catalytic cycle for cytochrome oxidase is proposed.     

Oxidation-reduction properties of the electron acceptors of Photosystem II. II. Redox titration at various pH values of the flash-induced formation of C550 in Chlamydomonas Photosystem II particles lacking the secondary quinone electron acceptor

Redox titrations of the flash-induced formation of C550 (a linear indicator of Q^?) were performed between pH 5.9 and 8.3 in Chlamydomonas Photosystem II particles lacking the secondary electron acceptor, B. One-third of the reaction centers show a pH-dependent midpoint potential (E_m,7.5) = ? 30 mV) for redox couple $\text{Q}\text{Q}{}^{\mathrm{?}}$, which varies by ?60 mV/pH unit. Two-thirds of the centers show a pH-independent midpoint potential (E_mm = + 10 mV) for this couple. The elevated pH-independent E_m suggests that in the latter centers the environment of Q has been modified such as to stabilize the semiquinone anion, Q^?. The midpoint potentials of the centers having a pH-dependent E_m are within 20 mV of those observed in chloroplasts having a secondary electron acceptor. It appears therefore that the secondary electron acceptor exerts little influence on the E_m of $\text{Q}\text{Q}{}^{\mathrm{?}}$. An EPR signal at g 1.82 has recently been attributed to a semiquinone-iron complex which comprises Q^?. The similar redox behavior reported here for C550 and reported by others (Evans, M.C.W., Nugent, J.H.A., Tilling, L.A. and Atkinson, Y.E. (1982) FEBS Lett. 145, 176–178) for the g 1.82 signal in similar Photosystem II particles confirm the assignment of this EPR signal to Q^?. At below ?200 mV, illumination of the Photosystem II particles produces an accumulation of reduced pheophytin (Ph^?). At ?420 mV Ph^? appears with a quantum yield of 0.006–0.01 which in this material implies a lifetime of 30–100 ns for the radical pair P-680⁺Ph^?.     

The iron-quinone acceptor complex in Rhodospirillum rubrum chromatophores studied by EPR

A new EPR signal is reported in Rhodospirillum rubrum chromatophores. The signal is attributed to Q⁻_BFe²⁺, the semiquinone-iron complex of the secondary quinone electron acceptor, on the basis of the following observations. (1) It is induced by a single laser flash given a room temperature and is stable. (2) It is present after odd-numbered flashes and absent after even-numbered flashes when a series of flashes is given. (3) When it is already present, low-temperature illumination results in the disappearance of the signal due to formation of the Q⁻_AFe²⁺Q⁻_B state. (4) Its formation is inhibited by the presence of orthophenanthroline at normal values of pH. The Q⁻_BFe²⁺ signal has two main features, one at g = 1.93 and the other at g = 1.82. The two features have different microwave power and temperature dependences, with the g = 1.82 signal being more difficult to saturate and requiring lower temperatures to be observable. Raising the pH leads to an increase in the g = 1.82 feature, while the g = 1.93 signal decreases in amplitude. It is suggested that the two parts of the signal may represent two EPR forms due to structural heterogeneity. The low-field feature of the Q⁻_BFe²⁺ signal shifts to lower field as the pH is raised and a pK for this change seems to occur at pH 9.4. The Q⁻_AFe²⁺ signal at g = 1.88 also shifts as the pH is increased; however, the shift is less marked than that seen for Q⁻_BFe²⁺, the shift is to higher field and the range over which it occurs is wider and depends upon the temperature of Q⁻_AFe²⁺ formation. This effect may be due to a pK on a protein group being shifted to higher pH by the presence of Q⁻_A. ortho-Phenanthroline broadens and shifts the Q⁻_AFe²⁺ signal. The inhibition of electron transfer between Q⁻_A and Q_B by ortho-phenanthroline becomes less effective at high pH. The new Q⁻_BFe²⁺ signal is unlike other semiquinone-iron signals reported in the literature in bacteria; however, it is remarkably similar to the Q⁻_BFe²⁺ signal reported in Photosystem II.     

Acceptor and donor-side interactions of phenolic inhibitors in Photosystem II

Certain phenolic compounds represent a distinct class of Photosystem (PS) II Q_B site inhibitors. In this paper, we report a detailed study of the effects of 2,4,6-trinitrophenol (TNP) and other phenolic inhibitors, bromoxynil and dinoseb, on PS II energetics. In intact PS II, phenolic inhibitors bound to only 90-95% of Q_B sites even at saturating concentrations. The remaining PS II reaction centers (5-10%) showed modified Q_A to Q_B electron transfer but were sensitive to urea/triazine inhibitors. The binding of phenolic inhibitors was 30- to 300-fold slower than the urea/triazine class of Q_B site inhibitors, DCMU and atrazine. In the sensitive centers, the S₂Q_A⁻ state was 10-fold less stable in the presence of phenolic inhibitors than the urea/triazine herbicides. In addition, the binding affinity of phenolic herbicides was decreased 10-fold in the S₂Q_A⁻ state than the S₁Q_A state. However, removal of the oxygen-evolving complex (OEC) and associated extrinsic polypeptides by hydroxylamine (HA) washing abolished the slow binding kinetics as well as the destabilizing effects on the charge-separated state. The S₂-multiline electron paramagnetic resonance (EPR) signal and the ‘split’ EPR signal, originating from the S₂Y_Z state showed no significant changes upon binding of phenolic inhibitors at the Q_B site. We thus propose a working model where Q_A redox potential is lowered by short-range conformational changes induced by phenolic inhibitor binding at the Q_B niche. Long-range effects of HA-washing eliminate this interaction, possibly by allowing more flexibility in the Q_B site.     

Building reactive copper centers in human carbonic anhydrase II

Reengineering metalloproteins to generate new biologically relevant metal centers is an effective a way to test our understanding of the structural and mechanistic features that steer chemical transformations in biological systems. Here, we report thermodynamic data characterizing the formation of two type-2 copper sites in carbonic anhydrase and experimental evidence showing one of these new, copper centers has characteristics similar to a variety of well-characterized copper centers in synthetic models and enzymatic systems. Human carbonic anhydrase II is known to bind two Cu²⁺ ions; these binding events were explored using modern isothermal titration calorimetry techniques that have become a proven method to accurately measure metal-binding thermodynamic parameters. The two Cu²⁺-binding events have different affinities (K _a approximately 5 × 10¹² and 1 × 10¹⁰), and both are enthalpically driven processes. Reconstituting these Cu²⁺ sites under a range of conditions has allowed us to assign the Cu²⁺-binding event to the three-histidine, native, metal-binding site. Our initial efforts to characterize these Cu²⁺ sites have yielded data that show distinctive (and noncoupled) EPR signals associated with each copper-binding site and that this reconstituted enzyme can activate hydrogen peroxide to catalyze the oxidation of 2-aminophenol.     

Peroxynitrite inhibits electron transport on the acceptor side of higher plant photosystem II

Peroxynitrite is a strong oxidant that has been proposed to form in chloroplasts. The interaction between peroxynitrite and photosystem II (PSII) has been investigated to determine whether this oxidant could be a hazard for PSII. Peroxynitrite is shown to inhibit oxygen evolution in PSII membranes in a dose-dependent manner. Analyses by PAM fluorimetry and EPR spectroscopy have demonstrated that the inhibition target of peroxynitrite is on the PSII acceptor side. In the presence of the herbicide DCMU, the chlorophyll (Chl) a fluorescence induction curve is inhibited by peroxynitrite, but the slow phase of the Chl a fluorescence decay does not change. EPR studies demonstrate that the Signal II_slow and Signal II_fast of peroxynitrite-treated Tris-washed PSII membranes are induced at room temperature, implying that the redox active tyrosines Y_Z and Y_D of PSII are not significantly nitrated. A featureless EPR signal with a g value of approximately 2.0043 ± 0.0003 and a line width of 10 ± 1 G is induced under continuous illumination in the presence of peroxynitrite. This new EPR signal corresponds with the semireduced plastoquinone Q_A in the absence of magnetic interaction with the non-heme Fe²⁺. We conclude that peroxynitrite impairs PSII electron transport in the Q_AFe²⁺ niche.     

Acceptor side effects on the electron transfer at cryogenic temperatures in intact photosystem II

In intact PSII, both the secondary electron donor (Tyr_Z) and side-path electron donors (Car/Chl_Z/Cyt_b559) can be oxidized by P₆₈₀⁺ at cryogenic temperatures. In this paper, the effects of acceptor side, especially the redox state of the non-heme iron, on the donor side electron transfer induced by visible light at cryogenic temperatures were studied by EPR spectroscopy. We found that the formation and decay of the S₁Tyr_Z EPR signal were independent of the treatment of K₃Fe(CN)₆, whereas formation and decay of the Car⁺/Chl_Z⁺ EPR signal correlated with the reduction and recovery of the Fe³⁺ EPR signal of the non-heme iron in K₃Fe(CN)₆ pre-treated PSII, respectively. Based on the observed correlation between Car/Chl_Z oxidation and Fe³⁺ reduction, the oxidation of non-heme iron by K₃Fe(CN)₆ at 0 °C was quantified, which showed that around 50-60% fractions of the reaction centers gave rise to the Fe³⁺ EPR signal. In addition, we found that the presence of phenyl-p-benzoquinone significantly enhanced the yield of Tyr_Z oxidation. These results indicate that the electron transfer at the donor side can be significantly modified by changes at the acceptor side, and indicate that two types of reaction centers are present in intact PSII, namely, one contains unoxidizable non-heme iron and another one contains oxidizable non-heme iron. Tyr_Z oxidation and side-path reaction occur separately in these two types of reaction centers, instead of competition with each other in the same reaction centers. In addition, our results show that the non-heme iron has different properties in active and inactive PSII. The oxidation of non-heme iron by K₃Fe(CN)₆ takes place only in inactive PSII, which implies that the Fe³⁺ state is probably not the intermediate species for the turnover of quinone reduction.     

Protein Dielectric Environment Modulates the Electron-Transfer Pathway in Photosynthetic Reaction Centers

The replacement of tyrosine by aspartic acid at position M210 in the photosynthetic reaction center of Rhodobacter sphaeroides results in the generation of a fast charge recombination pathway that is not observed in the wild-type. Apparently, the initially formed charge-separated state (cation of the special pair, P, and anion of the A-side bacteriopheophytin, H_A) can decay rapidly via recombination through the neighboring bacteriochlorophyll (B_A) soon after formation. The charge-separated state then relaxes over tens of picoseconds and recombination slows to the hundreds-of-picoseconds or nanosecond timescale. This dielectric relaxation results in a time-dependent blue shift of B_A^– absorption, which can be monitored using transient absorbance measurements. Protein dynamics also appear to modulate the electron transfer between H_A and the next electron carrier, Q_A (a ubiquinone). The kinetics of this reaction are complex in the mutant, requiring two kinetic terms, and the spectra associated with the two terms are distinct; a red shift of the H_A ground-state bleaching is observed between the shorter and longer H_A-to-Q_A electron-transfer phases. The kinetics appears to be pH-independent, suggesting a negligible contribution of static heterogeneity originating from protonation/deprotonation in the ground state. A dynamic model based on the energy levels of the two early charge-separated states, P⁺B_A^– and P⁺H_A^–, has been developed in which the energetics of these states is modulated by fast protein dielectric relaxations and this in turn alters both the kinetic complexity of the reaction and the reaction pathway.     

The rapid component of electron paramagnetic resonance signal II: A candidate for the physiological donor to Photosystem II in spinach chloroplasts

Rapid light-induced transients in EPR Signal IIf (F^?+) are observed in 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated, Tris-washed chloroplasts until the state F P680 Q^? is reached. In the absence of exogenous redox mediators several flashes are required to saturate this photoinactive state. However, the Signal IIf transient is observed on only the first flash following DCMU addition if an efficient donor to Signal IIf, phenylenediamine or hydroquinone, is present. Complementary polarographic measurements show that under these conditions oxidized phenylenediamine is produced only on the first flash of a series. The DCMU inhibition of Signal IIf can be completely relieved by oxidative titration of a one-electron reductant with E′_08.0 = +480 mV. At high reduction potentials the decay time of Signal IIf is constant at about 300 ms, whereas in the absence of DCMU the decay time is longer and increases with increasing reduction potential.A model is proposed in which Q^?, the reduced Photosystem II primary acceptor, and D, a one-electron 480 mV donor endogenous to the chloroplast suspension, compete in the reduction of Signal IIf (F^?+). At high potentials D is oxidized in the dark, and the (Q^? + F^?+) back reaction regenerates the photoactive F P680 Q state. The electrochemical and kinetic evidence is consistent with the hypothesis that the Signal IIf species, F, is identical with Z, the physiological donor to P680.     

Photoinhibition of carotenoidless reaction centers from Rhodobacter sphaeroides by visible light. Effects on protein structure and electron transport

Inhibition of electron transport and damage to the protein subunits by visible light has been studied in isolated reaction centers of the non-sulfur purple bacterium Rhodobacter sphaeroides. Illumination by 1100 μEm⁻² s⁻¹ light induced only a slight effect in wild type, carotenoid containing 2.4.1. reaction centers. In contrast, illumination of reaction centers isolated from the carotenoidless R26 strain resulted in the inhibition of charge separation as detected by the loss of the initial amplitude of absorbance change at 430 nm arising from the P⁺Q_B ⁻ → PQ_B recombination. In addition to this effect, the L, M and H protein subunits of the R26 reaction center were damaged as shown by their loss on Coomassie stained gels, which was however not accompanied by specific degradation products. Both the loss of photochemical activity and of protein subunits were suppressed in the absence of oxygen. By applying EPR spin trapping with 2,2,6,6-tetramethylpiperidine we could detect light-induced generation of singlet oxygen in the R26, but not in the 2.4.1. reaction centers. Moreover, artificial generation of singlet oxygen, also led to the loss of the L, M and H subunits. Our results provide evidence for the common hypothesis that strong illumination by visible light damages the carotenoidless reaction center via formation of singlet oxygen. This mechanism most likely proceeds through the interaction of the triplet state of reaction center chlorophyll with the ground state triplet oxygen in a similar way as occurs in Photosystem II. This revised version was published online in August 2006 with corrections to the Cover Date.     

Molecular interference of Cd with Photosystem II

Many heavy metals inhibit electron transfer reactions in Photosystem II (PSII). Cd²⁺ is known to exchange, with high affinity in a slow reaction, for the Ca²⁺ cofactor in the Ca/Mn cluster that constitutes the oxygen-evolving center. This results in inhibition of photosynthetic oxygen evolution. There are also indications that Cd²⁺ binds to other sites in PSII, potentially to proton channels in analogy to heavy metal binding in photosynthetic reaction centers from purple bacteria. In search for the effects of Cd²⁺-binding to those sites, we have studied how Cd²⁺ affects electron transfer reactions in PSII after short incubation times and in sites, which interact with Cd²⁺ with low affinity. Overall electron transfer and partial electron transfer were studied by a combination of EPR spectroscopy of individual redox components, flash-induced variable fluorescence and steady state oxygen evolution measurements. Several effects of Cd²⁺ were observed: (i) the amplitude of the flash-induced variable fluorescence was lost indicating that electron transfer from Y_Z to P₆₈₀⁺ was inhibited; (ii) Q_A⁻ to Q_B electron transfer was slowed down; (iii) the S₂ state multiline EPR signal was not observable; (iv) steady state oxygen evolution was inhibited in both a high-affinity and a low-affinity site; (v) the spectral shape of the EPR signal from Q_A⁻Fe²⁺ was modified but its amplitude was not sensitive to the presence of Cd²⁺. In addition, the presence of both Ca²⁺ and DCMU abolished Cd²⁺-induced effects partially and in different sites. The number of sites for Cd²⁺ binding and the possible nature of these sites are discussed.