Attenuated total reflection Fourier transform infrared spectroscopy of redox transitions in photosynthetic reaction centers: comparison of perfusion- and light-induced difference spectra

Chemically induced Fourier transform infrared difference spectra associated with redox transitions of several primary electron donors and acceptors in photosynthetic reaction centers (RCs) have been compared with the light-induced FTIR difference spectra involving the same cofactors. The RCs are deposited on an attenuated total reflection (ATR) prism and form a film that is enclosed in a flow cell. Redox transitions in the film of RCs can be repetitively induced either by perfusion of buffers poised at different redox potentials or by illumination. The perfusion-induced ATR-FTIR difference spectra for the oxidation of the primary electron donor P in the RCs of the purple bacteria Rb. sphaeroides and Rp. viridis and P700 in the photosystem 1 of Synechocystis 6803, as well as the Q(A)/Q(A) transition of the quinone acceptor (Q(A)) in Rb. sphaeroides RCs are reported for the first time. They are compared with the light-induced ATR-FTIR difference spectra P+Q(A)/PQ(A) for the RCs of Rb. sphaeroides and P700+/P700 for photosystem 1. It is shown that the perfusion-induced and light-induced ATR-FTIR difference spectra recorded on the same RC film display identical signal to noise ratios when they are measured under comparable conditions. The ATR-FTIR difference spectra are very similar to the equivalent FTIR difference spectra previously recorded upon photochemical or electrochemical excitation of these RCs in the more conventional transmission mode. The ATR-FTIR technique requires a smaller amount of sample compared with transmission FTIR and allows precise control of the aqueous environment of the RC films.     

Electron transfer kinetics in photosynthetic reaction centers embedded in polyvinyl alcohol films

The coupling between electron transfer and protein dynamics has been studied at room temperature in isolated reaction centers (RCs) from the photosynthetic bacterium Rhodobacter sphaeroides by incorporating the protein in polyvinyl alcohol (PVA) films of different water/RC ratios. The kinetic analysis of charge recombination shows that dehydration of RC-containing PVA films causes reversible, inhomogeneous inhibition of electron transfer from the reduced primary quinone acceptor (Q(A)(-)) to the secondary quinone Q(B). A more extensive dehydration of solid PVA matrices accelerates electron transfer from Q(A)(-) to the primary photooxidized electron donor P(+). These effects indicate that incorporation of RCs into dehydrated PVA films hinders the conformational dynamics gating Q(A)(-) to Q(B) electron transfer at room temperature and slows down protein relaxation which stabilizes the primary charge-separated state P(+)Q(A)(-). A comparison with analogous effects observed in trehalose-coated RCs suggests that protein motions are less severely reduced in PVA films than in trehalose matrices at comparable water/RC ratios.     

Inter- and intraspecific variation in excited-state triplet energy transfer rates in reaction centers of photosynthetic bacteria

In protein-cofactor reaction center (RC) complexes of purple photosynthetic bacteria, the major role of the bound carotenoid (C) is to quench the triplet state formed on the primary electron donor (P) before its sensitization of the excited singlet state of molecular oxygen from its ground triplet state. This triplet energy is transferred from P to C via the bacteriochlorophyll monomer B(B). Using time-resolved electron paramagnetic resonance (TREPR), we have examined the temperature dependence of the rates of this triplet energy transfer reaction in the RC of three wild-type species of purple nonsulfur bacteria. Species-specific differences in the rate of transfer were observed. Wild-type Rhodobacter capsulatus RCs were less efficient at the triplet transfer reaction than Rhodobacter sphaeroides RCs, but were more efficient than Rhodospirillum rubrum RCs. In addition, RCs from three mutant strains of R. capsulatus carrying substitutions of amino acids near P and B(B) were examined. Two of the mutant RCs showed decreased triplet transfer rates compared with wild-type RCs, whereas one of the mutant RCs demonstrated a slight increase in triplet transfer rate at low temperatures. The results show that site-specific changes within the RC of R. capsulatus can mimic interspecies differences in the rates of triplet energy transfer. This application of TREPR was instrumental in defining critical energetic and coupling factors that dictate the efficiency of this photoprotective process.     

Light-induced charge separation across the photosynthetic membrane: a proposed structure for the photosystem I reaction center

The photosystem I reaction center is reviewed from the standpoint of electron acceptors, polypeptide composition, and structural organization. Experimental difficulties in the current model are highlighted, and current results on the polypeptide location of each acceptor are reviewed. The amount of iron and labile sulfide, the types of iron-sulfur clusters, and the number of ∼ 64-kDa polypeptides are considered in light of a model for the reaction center. In particular, the presence of [2Fe-2S] clusters in photosystem I has significance in the structure and organization of F_x on the reaction center. Since four cysteinyl ligands are assumed to hold an iron-sulfur cluster, a photosystem I subunit may consist of two ∼ 64-kDa polypeptides bridged by a [2Fe-2S] cluster. The reaction center would consist of a symmetrical pair of these subunits positioned so that two [2Fe-2S] clusters are in magnetic interaction, thereby constituting F_x. The reaction center core would therefore incorporate four ∼ 64-kDa polypeptides and have a molecular weight in excess of 250 kDa     

Electrochemical approach to the development of a photoelectrode on the basis of photosynthetic reaction centers

Experimental approaches to the coupling of photoinduced charge separation in reaction centers (RCs) of the photosynthetic bacterium Rhodobacter sphaeroides R-26 with electron transfer to an electrode are discussed. Exogenous quinones are used as an electron transfer mediator. With the use of photopolarography it is shown that water-soluble ubiquinone can serve as a diffusionally mobile mediator of electron transfer. Some methods of quinone immobilization at an electrode have been developed to obtain a non-diffusional mediator of electron transfer. Quasi-reversible electrochemical kinetics were observed for aminonaphthoquinone immobilized as a monolayer at a Pt electrode. The ubiquinone-depleted RCs were subjected to affinity immobilization at these chemically modified electrodes probably due to the insertion of the immobilized quinone into the primary quinone Q_A binding site. The quantum efficiency of photocurrent formation was ca. 5% for the photoelectrode obtained. The electrochemical process of the immobilized quinone is shown to be the stage that limits electron transfer from RCs to the electrode.     

Chemical activity of iron in [2Fe-2S]-protein centers and FeS2(100) surfaces

Iron atoms bonded to sulfur play an important role in proteins, heterogeneous catalysts, and gas sensors. First-principles density functional calculations were used to investigate the structure and chemical activity of a unique [2Fe-2S] center in the split-Soret cytochrome c (Ssc) from Desulfovibrio desulfuricans. In agreement with a previously proposed structural model [Abreu et al., J. Biol. Inorg. Chem. 2003, 8, 360], it is found that the [2Fe-2S] cluster is located in a surface pocket of the Ssc and bonded to only three cysteines. The [2Fe-2S] center in the Ssc is nonplanar and somewhat distorted with respect to canonical [2Fe-2S] centers seen in proteins where the iron-sulfur unit is bonded to four cysteines. In the Ssc, the lack of one Fe-cysteine bond is partially compensated by the separation between the cysteines that minimizes electrostatic repulsion among these ligands. The unique structure of the [2Fe-2S] center in the Ssc makes the center more chemically active than canonical [2Fe-2S] centers in proteins, (RS)(4)[2Fe-2S] inorganic complexes, and an FeS2(100) surface. A [2Fe-2S] center in the Ssc interacts efficiently with electron acceptors (O2, NO, CO) and poorly with a Lewis base such as H2O. The interaction with molecular oxygen is so strong that eventually oxidatively destroys the [2Fe-2S] unit. The bonding energy of the ligands to the [2Fe-2S] centers and FeS2(100) surface increases following the sequence: H2O < CO < NO < O2. The higher the electron affinity of the ligand, the larger its bonding energy. A relatively large positive charge on the Fe cations in FeS2(100) makes this sulfide surface less reactive toward O2, CO, and NO than the [2Fe-2S] centers in proteins and inorganic complexes.     

Probing the intrinsic electronic structure of the cubane [4Fe-4S] cluster: nature's favorite cluster for electron transfer and storage

The cubane [4Fe-4S] is the most common multinuclear metal center in nature for electron transfer and storage. Using electrospray, we produced a series of gaseous doubly charged cubane-type complexes, [Fe4S4L4]2- (L = -SC2H5, -SH, -Cl, -Br, -I) and the Se-analogues [Fe4Se4L4]2- (L = -SC2H5, -Cl), and probed their electronic structures with photoelectron spectroscopy and density functional calculations. The photoelectron spectral features are similar among all the seven species investigated, revealing a weak threshold feature due to the minority spins on the Fe centers and confirming the low-spin two-layer model for the [4Fe-4S](2+) core and its "inverted level scheme". The measured adiabatic detachment energies, which are sensitive to the terminal ligand substitution, provide the intrinsic oxidation potentials of the [Fe4S4L4]2- complexes. The calculations revealed a simple correlation between the electron donor property of the terminal thiolate as well as the bridging sulfide with the variation of the intrinsic redox potentials. Our data provide intrinsic electronic structure information of the [4Fe-4S] cluster and the molecular basis for understanding the protein and solvent effects on the redox properties of the [4Fe-4S] active sites.     

Dipyridamole and its derivatives modify the kinetics of the electron transport in reaction centers from Rhodobacter sphaeroides

A well known vasodilator dipyridamole (DIP), 2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido[5,4-d]pyrim idine, and its derivatives have recently been shown as potential co-activators (modulators) in the phenomenon of multidrug resistance (MDR) in cancer therapy. They inhibit the specific function of a transmembrane P-glycoprotein responsible for the ex-flux of anti-cancer drugs from tumor cells. To clarify molecular mechanisms of the anti-MDR activity of DIP and its two derivatives, RA25 and RA47, we have studied their effects on electron transport in reaction centers (RC) from purple photosynthetic bacteria Rb. sphaeroides, using RC as a model system. Increasing concentrations of DIP and RA47 progressively accelerate the back electron transfer from the primary quinone acceptor QA to the bacteriochlorophyll dimer Bchl2 (Bchl2+ -QA- recombination). In the absence of o-phenantroline, when both quinone acceptors QA and QB are involved in the electron transport, RA47 is more effective than DIP. DIP stabilizes the electron on the secondary quinone acceptor QB, the effect manifested as the retardation of Bchl2+ -QB- recombination. Effects of RA25 are negligible in all cases. The drugs are proposed to change the electron transport affecting the RC structural dynamics and the stabilization of the electron on quinone acceptors through modification of H-bonds in the system.     

Transmembrane charge transfer in photosynthetic reaction centers: some similarities and distinctions

This mini review presents a general comparison of structural and functional peculiarities of three types of photosynthetic reaction centers (RCs)--photosystem (PS) II, RC from purple bacteria (bRC) and PS I. The nature and mechanisms of the primary electron transfer reactions, as well as specific features of the charge transfer reactions at the donor and acceptor sides of RCs are considered. Comparison of photosynthetic RCs shows general similarity between the core central parts of all three types, between the acceptor sides of bRC and PS II, and between the donor sides of bRC and PS I. In the latter case, the similarity covers thermodynamic, kinetic and dielectric properties, which determine the resemblance of mechanisms of electrogenic reduction of the photooxidized primary donors. Significant distinctions between the donor and acceptor sides of PS I and PS II are also discussed. The results recently obtained in our laboratory indicate in favor of the following sequence of the primary and secondary electron transfer reactions: in PS II (bRC): Р(680)(Р(870)) → Chl(D1)(В(А)) → Phe(bPhe) → Q(A); and in PS I: Р(700) → А(0А)/A(0B) → Q(A)/Q(B).     

Primary processes in plant photosynthesis: photosystem I reaction center

The photosystem I (PSI) pigment-protein complex of plants converts light energy into a transmembrane charge separation, which ultimately leads to the reduction of carbon dioxide. Recent studies on the dynamics of primary energy transfer, charge separation, and following electron transfer of the reaction center (RC) of the PSI prepared from spinach are reviewed. The main results of femtosecond transient absorption and fluorescence spectroscopies as applied to the P700-enchied PSI RC are summarized. This specially prepared material contains only 12–14 chlorophylls per P700, which is a special pair of chlorophyll a and has a significant role in primary charge separation. The P700-enriched particles are useful to study dynamics of cofactors, since about 100 light-harvesting chlorophylls are associated with wild PSI RC and prevent one from observing the elementary steps of the charge separation. In PSI RC energy and electron transfer were found to be strongly coupled and an ultrafast up-hill energy equilibration and charge separation were observed upon preferential excitation of P700. The secondary electron-transfer dynamics from the reduced primary electron acceptor chlorophyll a to quinone are described. With creating free energy differences (ΔG₀) for the reaction by reconstituting various artificial quinones and quinoids, the rate of electron transfer was measured. Analysis of rates versus ΔG₀ according to the quantum theory of electron transfer gave the reorganization energy, electronic coupling energy and other factors. It was shown that the natural quinones are optimized in the photosynthetic protein complexes. The above results were compared with those of photosynthetic purple bacteria, of which the structure and functions have been studied most.     

Pulsed electron paramagnetic resonance experiments identify the paramagnetic intermediates in the pyruvate ferredoxin oxidoreductase catalytic cycle

Pyruvate ferredoxin oxidoreductase (PFOR) is central to the anaerobic metabolism of many bacteria and amitochondriate eukaryotes. PFOR contains thiamine pyrophosphate (TPP) and three [4Fe-4S] clusters, which link pyruvate oxidation to reduction of ferredoxin. In the PFOR reaction, TPP reacts with pyruvate to form lactyl-TPP, which undergoes decarboxylation to form a hydroxyethyl-TPP (HE-TPP) intermediate. One electron is then transferred from HE-TPP to one of the three [4Fe-4S] clusters to form an HE-TPP radical and a [4Fe-4S]1+ intermediate. Pulsed EPR methods have been used to measure the distance between the HE-TPP radical and the [4Fe-4S]1+ cluster to which it is coupled. Computational analysis including the PFOR crystal structure and the spin distribution in the HE-TPP radical and in the reduced [4Fe-4S] cluster demonstrates that the distance between the HE-TPP radical and the medial cluster B matches the experimentally determined dipolar interaction, while one of the other two clusters is too close and the other is too far away. These results clearly demonstrate that it is the medial cluster (cluster B) that is reduced. Thus, rapid electron transfer occurs through the electron-transfer chain, which leaves an oxidized proximal cluster poised to accept an electron from the HE-TPP radical in the subsequent reaction step.     

Femtosecond spectroscopy of the primary charge separation in reaction centers of Chloroflexus aurantiacus with selective excitation in the QY and Soret bands

The primary charge separation and electron-transfer processes of photosynthesis occur in the reaction center (RC). Isolated RCs of the green filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus were studied at room temperature by using femtosecond transient absorption spectroscopy with selective excitation. Upon excitation in the Q(Y) absorbance band of the bacteriochlorophyll (BChl) dimer (P) at 865 nm, a 7.0 +/- 0.5 ps kinetic component was observed in the 538 nm region (Q(X) band of the bacteriopheophytin (BPheo)), 750 nm region (Q(Y) band of the BPheo), and 920 nm region (stimulated emission of the excited-state of P), indicating that this lifetime represents electron transfer from P to BPheo. The same time constant was also observed upon 740 nm or 800 nm excitation. A longer lifetime (300 +/- 30 ps), which was assigned to the time of reduction of the primary quinone, Q(A), was also observed. The transient absorption spectra and kinetics all indicate that only one electron-transfer branch is involved in primary charge separation under these excitation conditions. However, the transient absorption changes upon excitation in the Soret band at 390 nm reveal a more complex set of energy and electron-transfer processes. By comparison to studies on the RCs of the purple bacterium Rhodobacter sphaeroides, we discuss the possible mechanism of electron-transfer pathway dependence on excitation energy and propose a model of the Cf. aurantiacus RC that better explains the observed results.     

Function of the Reaction Center of Green Sulfur Bacteria

The reaction center (RC) of green sulfur bacteria belongs to the Fe-S type RC, as do the photosystem I of oxygenic photosynthetic organisms and the RC of heliobacteria. The core parts of the green sulfur bacterial and the heliobacterial RC are assumed to be homodimeric, in contrast to those of purple bacteria, photosystem I and photosystem II. This paper describes recent advances in the study of the function of the green sulfur bacterial RC.     

An approach based on quantum chemistry calculations and structural analysis of a [2Fe-2S] ferredoxin that reveal a redox-linked switch in the electron-transfer process to the Fd-NADP+ reductase

[2Fe-2S] ferredoxins act as electron carriers in photosynthesis by mediating the transfer of electrons from photosystem I to various enzymes such as ferredoxin:NADP(+):reductase (FNR). We have analyzed by density functional theory the possible variations of the electronic properties of the [2Fe-2S] ferredoxin, from the cyanobacterium Anabaena, depending on the redox-linked structural changes observed by X-ray diffraction at atomic resolution (Morales, R.; et al. Biochemistry 1999, 38, 15764-15773). The present results point out a specific and concerted role of Ser47, Phe65, and Glu94 located at the molecule surface, close to the iron-sulfur cluster. These residues were already known to be crucial for efficient electron transfer to FNR (e.g., Hurley, J. K.; et al. Biochemistry 1997, 36, 11100-11117). Our calculations suggest that the Glu94 carboxylate negative charge regulates the electron charge delocalization between the Ser47 CO group and the Phe65 aromatic ring, depending on the redox state. The Glu94 carboxylate is stabilized by a strong hydrogen bond implicating a hydroxyl-containing side chain (i.e., Ser or Thr) at location 47. We propose that the Phe65 ring acts as an intermediary carrier receiving the reducing electron prior to its transfer from the reduced Fd to FNR, in view of its central role in the Fd-FNR interaction.     

pH-sensitive fluorescent dye as probe for proton uptake in photosynthetic reaction centers

Isolated and purified reaction centers (RC) from Rhodobacter sphaeroides R-26.1 were solubilised in detergent with excess quinone and external electron donors and illuminated in the presence of pyranine. The pH change accompanying the reaction center photocycle was monitored by recording the variation of the pyranine fluorescence intensity. Using Q(B)-depleted reaction centers or blocking the photocycle with terbutryne strongly reduced the pH change. The usefulness and limits of this technique in monitoring the pH changes during the RC photocycle are also discussed.     

Degenerate electron exchange reaction of n-alkane radical cations in solution

The degenerate electron exchange (DEE) reaction involving radical cations (RCs) of n-nonane, n-dodecane, and n-hexadecane in n-hexane solution was studied over the temperature range 253-313 K using the method of time-resolved magnetic field effect in recombination fluorescence of spin-correlated radical ion pairs. In the dilute solutions the rate constant of DEE was found to be 200 times slower than the diffusion limit. Using n-nonane as an example, we showed that two reasons are responsible for the low value of the RC self-exchange rate: (1) conformational variability of molecules and RCs and (2) the activation barrier of DEE reaction. The calculations of the reaction enthalpy performed by the B3LYP/6-31G(d) method indicated that electron transfer can be effective only upon collision of RC with a neutral molecule either in the all-trans conformation or in the conformation differing from the latter by rotation of the end ethyl fragment. The activation barrier of the DEE reaction was estimated using the reorganization energy of the internal degrees of freedom calculated at the B3LYP level and was found to be about 6 kcal/mol. A possible influence of the interaction between RC and a neutral molecule in an encounter complex on DEE rate constant is also discussed.     

Bidirectional electron transfer in photosystem I: direct evidence from high-frequency time-resolved EPR spectroscopy

Efficient charge separation occurring within membrane-bound reaction center proteins is the most important step of photosynthetic solar energy conversion. All reaction centers are classified into two types, I and II. X-ray crystal structures reveal that both types bind two symmetric membrane-spanning branches of potential electron-transfer cofactors. Determination of the functional roles of these pairs of branches is of fundamental importance. While it is established that in type II reaction centers only one branch functions in electron transfer, we present the first direct spectroscopic evidence that both cofactor branches are active in the type I reaction center, photosystem I.     

Thermal Effects and Structural Changes of Photosynthetic Reaction Centers Characterized by Wide Frequency Band Hydrophone: Effects of Carotenoids and Terbutryn

Photothermal characteristics and light‐induced structural (volume) changes of carotenoid‐containing and noncontaining photosynthetic reaction centers (RCs) were investigated by wide frequency band hydrophone. We found that the presence of carotenoid either does not play considerable role in the light‐induced conformational movements, or these rearrangements are too slow for inducing a photoacoustic (PA) signal. The kinetic component with a few tens of microseconds, exhibited by the carotenoid‐less RCs, appears to be similar to that of triplet state lifetimes, identified by other methods. The binding of terbutryn to the acceptor side is shown to affect the dynamics of the RC. Our results do not confirm large displacements or volume changes induced by the charge movements and by the charge relaxation processes in the RCs in few hundreds of microseconds time scale that accompanies the electron transfer between the primary and secondary electron acceptor quinones.     

Probing the contribution of electronic coupling to the directionality of electron transfer in photosynthetic reaction centers

Subpicosecond transient absorption studies are reported for a set of Rhodobacter (R.) capsulatus bacterial photosynthetic reaction centers (RCs) designed to probe the origins of the unidirectionality of charge separation via one of two electron transport chains in the native pigment-protein complex. All of the RCs have been engineered to contain a heterodimeric primary electron donor (D) consisting of a bacteriochlorophyll (BChl) and a bacteriopheophytin (BPh). The BPh component of the M heterodimer (Mhd) or L heterodimer (Lhd) is introduced by substituting a Leu for His M200 or His L173, respectively. Previous work on primary charge separation in heterodimer mutants has not included the Lhd RC from R. capsulatus, which we report for the first time. The Lhd and Mhd RCs are used as controls against which we assess RCs that combine the heterodimer mutations with a second mutation (His substituted for Leu at M212) that results in replacement of the native L-side BPh acceptor with a BChl (beta). The transient absorption spectra reveal clear evidence for charge separation to the normally inactive M-side BPh acceptor (H(M)) in Lhd-beta RCs to form D+H(M)- with a yield of approximately 6%. This state also forms in Mhd-beta RCs but with about one-quarter the yield. In both RCs, deactivation to the ground state is the predominant pathway of D decay, as it is in the Mhd and Lhd single mutants. Analysis of the results indicates an upper limit ofV2L/V2m < or = 4 for the contribution of the electronic coupling elements to the relative rates of electron transfer to the L versus M sides of the wild-type RC. In comparison to the L/M rate ratio (kL/kM) approximately 30 for wild-type RCs, our findings indicate that electronic factors contribute approximately 35% at most to directionality with the other 65% deriving from energetic considerations, which includes differences in free energies, reorganization energies, and contributions of one- and two-step mechanisms on the two sides of the RC.