Sulfhydryl modifying reagents inhibit Q A − oxidation in reaction centers from Rhodobacter sphaeroides and Capsulatus,but not Rhodopseudomonas viridis

The effects of various sulfhydryl-modifying reagents on reaction centers (RCs) from purple photosynthetic bacteria have been examined, with particular emphasis on the activity of the acceptor quinones, Q_A and Q_B, comprising the two electron gate. Mercurial reagents, especially p-chloromercuribenzenesulfonate (pCMBS), were effective in inhibiting Q_B function in RCs from Rhodobacter sphaeroides and Rb. capsulatus, but not in Rhodopseudomonas viridis. The inhibition was fully reversible by dialysis against dithiothreitol (DTT). The effect on Q_B function was not an apparent one mediated by an alteration in the redox potential of Q_A. N-ethylmaleimide (NEM) had no effect on any of the quinone functions, even at very high concentrations. Comparison of the X-ray structures of the RCs from Rb. sphaeroides and Rp. viridis and the known amino acid sequences for all three bacterial RCs suggest that a cysteine residue at position 108 in the L subunit of the Rhodobacter species is the most likely candidate for the site of action of the mercurial reagents. This was strongly supported by the absence of any effect of pCMBS on a site specific mutation of Rb. sphaeroides (L108CS) with residue L108 changed from cysteine to serine. These results imply a long distance (>20 Å) effect on the functioning of Q_B, perhaps involving a relatively gross structural alteration.     

Formation and decay of radical-pair state P+I− in Chloroflexus aurantiacus reaction centers

We have examined the room temperature kinetics of the absorption changes associated with the formation of state P⁺I⁻ (P⁺BPh⁻) and its subsequent decay to state P⁺Q⁻_A in reaction centers from Chloroflexus aurantiacus. Our data, acquired using 30-ps excitation flashes, strongly suggest that formation of P⁺I⁻ (P⁺BPh⁻) takes longer in Chloroflexus than in reaction centers from Rhodopseudomonas sphaeroides. The reduction of the photoactive bacteriopheophytin (BPh) could take as long as 13 ps. Absorption changes different from those due to P⁺I⁻ are observed early in the excitation flash, but the detailed identity of the transient remains unclear. We also find that the kinetics observed subsequent to P⁺I⁻ formation differ with detection wavelength. The time constant measured in the anion band (I⁻) at 655 nm is 324 ± 20 ps and probably reflects the rate of electron transfer from I⁻ (BPh⁻) to Q_A. However, the kinetics measured in the BPh ground-state absorption bands are slightly longer: 365 ± 19 and 367 ± 21 ps at 538 and 760 nm, respectively. At 810 nm, a wavelength normally associated with the monomeric bacteriochlorophyll (BChl) in the Chloroflexus reaction center, a slightly faster (281 ± 19 ps) time constant is observed. This detection-wavelength dependence of the kinetics is similar to that observed recently in Rps. sphaeroides reaction centers. Comparison of these results suggests that the kinetics observed in the ground-state absorption bands of the BPhs and BChls in Chloroflexus may contain contributions from readjustments of the pigments and/or protein in response to the charge separation process.     

Effects of zwitterionic detergents on the electronic structure of the primary donor and the charge recombination kinetics of P+Q A − in native and mutant reaction centers from Rhodobacter sphaeroides

The effects of detergents on the electronic structure of the oxidized primary donor P⁺ and the time constant _AP of the P⁺Q _A ^– charge recombination at ambient temperatures have been investigated in native and mutant reaction centers (RCs) from Rhodobacter sphaeroides. It is shown that N-lauryl-N,N-dimethyl-3-ammonio-1-propane sulfonate (SB12) induces a transition to a second distinct conformation of the RC. In the case of the wild type and the mutant FY(M197), in which a hydrogen bond is introduced to the 2-acetyl group of the dimer half of P that is associated with the M-subunit of the RC, the conformational change causes a more asymmetric spin density distribution between the two bacteriochlorophyll moieties of P⁺ in favor of the L-half. For both types of RCs the time constant _AP depends on the SB12/RC ratio as does the position of the long-wavelength band of P, _max. The increase of _AP by 30 ms and the shift of _max from 866 nm to 851 nm are indicative for the conformational change. In addition, a smaller linear increase of _AP with increasing SB12/RC ratio is superimposed on the variation of _AP due to the conformational change. Similar effects of SB12 on the optical spectra as well as on _AP are also observed for the two heterodimer mutants HL(L173) and HL(M202), in which one of the bacteriochlorophylls of P is replaced by a bacteriopheophytin. There are no clear indications for a correlation of _AP with the localization of the positive charge in P⁺. Furthermore, it is concluded from the dependence of _AP on the SB12/RC ratio that the single-site mutations do not affect the standard free energy difference of the two conformations to a measurable extent.     

Electron transfer in reaction centers of Rhodopseudomonas sphaeroides. I. Determination of the charge recombination pathway of D+QAQ−B and free energy and kinetic relations between Q−AQB and QAQ−B

The electron-transfer reactions and thermodynamic equilibria involving the quinone acceptor complex in bacterial reaction centers from R. sphaeroides were investigated. The reactions are described by the scheme: We found that the charge recombination pathway of D⁺Q_AQ^?_B proceeds via the intermediate state D⁺Q^?_AQ_B, the direct pathway contributing less than approx. 5% to the observed recombination rate. The method used to obtain this result was based on a comparison of the kinetics predicted for the indirect pathway (given by the product k_AD-times the fraction of reaction centers in the Q^?_AQ_B state) with the observed recombination rate, k^obs_{D⁺ →D}. The kinetic measurements were used to obtain the pH dependence (6.1 ? pH ? 11.7) of the free energy difference between the states Q^?_AQ_B and Q_AQ^?_B. At low pH (less than 9) Q_AQ^?_B is stabilized relative to Q^?_AQ_B by 67 meV, whereas at high pH Q^?_AQ_B is energetically favored. Both Q^?_A and Q^?_B associate with a proton, with pK values of 9.8 and 11.3, respectively. The stronger interaction of the proton with Q^?_B provides the driving force for the forward electron transfer.     

The thermodynamic characteristic of four-heme cytochrome c in Rhodopseudomonas viridis reaction centers,as derived from a quantitative analysis of the differential absorption spectra in α-domain

A method of decomposing of the absorption spectrum of four-heme cytochrome of a Rhodopseudomonas viridis reaction center preparation into spectra of individual components was used to estimate the degree of the reduction of hemes as a function of redox potential in the medium. The method enables an evaluation of the shape of redox-titration curves of each heme. The redox-titration curves derived by this approach are approximated well by a Nernst equation with n = 1 and E_m-values of 360 mV, 312 mV, 20 mV and less than −50 mV. For all of the redox species the values of midpoint potential estimates by the above method are in good agreement with those determined earlier using another procedure [Dracheva et al. (1988) Eur. J. Biochem. 171, 253-264]. The accuracy of deconvolution of data is within the experimental errors of the redox potential measurement.     

Delayed fluorescence study on P*QA→ P+QA − charge separation energetics linked to protons and salt in reaction centers from Rhodobacter sphaeroides

The energetics of the first stable charge separated state, P⁺Q_A– relative to that of P–Q_A was examined in isolated RC from Rhodobacter sphaeroides by delayed fluorescence. The temperature dependence of the delayed fluorescence indicates that the charge separation is a highly enthalpy-driven process (H = – 818 ± 20 meV at pH 8) and the free energy gap between P–Q_A and P⁺Q_A– drops with increasing pH (40 ± 4 meV between pH 6 and 10). The pH-dependence of the free energy change of the P⁺Q_A– state runs parallel to the (integrated) net proton uptake due to the PQ_A/P⁺Q_A– redox change in a wide pH range and under different ionic conditions. Elevation of the ionic strength increases the delayed fluorescence intensity and decreases the (dark and light) pK_a values as well as the light-induced pK_a changes of the protonatable groups of the protein. The observed dependence of the energetics of P⁺Q_A– on the concentration and composition of mobile ions is discussed in terms of binding and screening of protonatable groups and surface charges as dominant modes of electrostatic interaction between RC and salt.     

Comparative ENDOR study at 34 GHz of the triplet state of the primary donor in bacterial reaction centers of Rb. sphaeroides and Bl. viridis

The primary electron donor (P) in the photosynthetic bacterial reaction center of Rhodobacter sphaeroides and Blastochloris viridis consists of a dimer of bacteriochlorophyll a and b cofactors, respectively. Its photoexcited triplet state in frozen solution has been investigated by time resolved ENDOR spectroscopy at 34 GHz. The observed ENDOR spectra for ³P₈₆₅ and ³P₉₆₀ are essentially the same, indicating very similar spin density distributions. Exceptions are the ethylidene groups unique to the bacteriochlorophyll b dimer in ³P₉₆₀. Strikingly, the observed hyperfine coupling constants of the ethylidene groups are larger than in the monomer, which speaks for an asymmetrically delocalized wave function over both monomer halves in the dimer. The latter observation corroborates previous findings of the spin density in the radical cation states P ₈₆₅ ^?+ (Lendzian et al. in Biochim Biophys Acta 1183:139–160, 1993) and P ₉₆₀ ^?+ (Lendzian et al. in Chem Phys Lett 148:377–385, 1988). As compared to the bacteriochlorophyll monomer, the hyperfine coupling constants of the methyl groups 2¹ and 12¹ are reduced by at least a factor of two, and quantitative analysis of these couplings gives rise to a ratio of approximately 3:1 for the spin density on the halves P_L:P_M. Our findings are discussed in light of the large difference in photosynthetic activity of the two branches of cofactors present in the bacterial reaction center proteins.     

The influence of electrostatic interactions and intramolecular dynamics on electron transfer from the cytochrome subunit to the cation —radical of the bacteriochlorophyll dimer in reaction centers from Rps. viridis

Interheme electrostatic interaction can explain the acceleration of the electron transfer (ET) rate from the highest potential heme (C38o) to the photooxidized bacteriochlorophyll dimer (P⁺) which takes place after the reduction of neighbouring heme(s) of the cytochrome subunit in the reaction center of Rps. viridis. The electrostatic interaction energies calculated for neighbouring hemes, 7.0 Å apart (edge-to-edge), and for two high potential hemes, 21.5 Å apart are found to be 0.110 eV and 0.040 eV respectively. The reorganisation energy of the C₃₈₀-P⁺ transition of about 0.290±0.030 eV is calculated using the Marcus theory of electron tunneling. An empirical relation for the rate of ET is given. The low temperature restriction of the C₃₈₀-⁺ transition is caused by an energetic inhibition which originates from an opposite shifting of the energy levels of C₃₈₀ and P⁺ due to the freezing of protein dynamics and protein-bound water mobility. The freezing of the protein dynamics is revealed by the Mössbauer effect and correlates with the efficiency of the ET.Abbreviations RC reaction center - P⁺ cation-radical of bacteriochlorophyll dimer - C₃₈₀, C20, C310, C_–60, hemes indexed by the values of their individual redox potentials (in mV) - ET electron transfer     

Protein recognition in ferredoxin–P450 electron transfer in the class I CYP199A2 system from Rhodopseudomonas palustris

CYP199A2 from Rhodopseudomonas palustris CGA009 is a heme monooxygenase that catalyzes the oxidation of para-substituted benzoic acids. CYP199A2 activity is reconstituted by a class I electron transfer chain consisting of the associated [2Fe–2S] ferredoxin palustrisredoxin (Pux) and a flavoprotein palustrisredoxin reductase (PuR). Another [2Fe–2S] ferredoxin, palustrisredoxin B (PuxB; RPA3956) has been identified in the genome. PuxB shares sequence identity and motifs with vertebrate-type ferredoxins involved in Fe–S cluster assembly but also 50% identity with Pux and it mediates electron transfer from PuR to CYP199A2, albeit with lower steady-state turnover activity: 99 nmol (nmol P450)^?1min^?1 for 4-methoxybenzoic acid oxidation compared with 1,438 nmol (nmol P450)^?1 min^?1 for Pux. This difference mainly arises from weak CYP199A2–PuxB binding (K _m 34.3 vs. 0.45 μM for Pux) rather than slow electron transfer (k _cat 19.1 vs. 37.9 s^?1 for Pux). Comparison of the 2.0-Å-resolution crystal structure of the PuxB A105R mutant with other vertebrate-type, P450-associated ferredoxins revealed similar protein folds but also significant differences in some loop regions. Therefore, PuxB offers a platform for studying ferredoxin–P450 recognition in class I P450 systems. Substitution of PuxB residues at key locations with those in Pux shows that Ala42, Cys43, and Ala44 in the [2Fe–2S] cluster binding loop and Met66 are important in electron transfer from PuxB to CYP199A2, whereas Phe73 and the C-terminal Ala105 were involved in both protein binding and electron transfer.     

Damping of oscillations in the semiquinone absorption in reaction centers after successive flashes. Determination of the equilibrium between QA−QB and QAQB−

A quantitative model for the damping of oscillations of the semiquinone absorption after successive light flashes is presented. It is based on the equilibrium between the states Q_A^?Q_B and Q_AQ_B^?. A fit of the model to the experimental results obtained for reaction centers from Rhodopseudomonas sphaeroides gave a value of $\text{α =}\text{[}\text{Q}{}_{\mathrm{<mtext>A</mtext>}}{}^{\mathrm{?}}\text{Q}{}_{\mathrm{<mtext>B</mtext>}}\text{]}\text{([}\text{Q}{}_{\mathrm{<mtext>A</mtext>}}{}^{\mathrm{?}}\text{Q}{}_{\mathrm{<mtext>B</mtext>}}\text{] + [}\text{Q}{}_{\mathrm{<mtext>A</mtext>}}\text{Q}{}_{\mathrm{<mtext>B</mtext>}}{}^{\mathrm{?}}\text{])}\text{= 0.065 ± 0.005}$ (T = 21°C, pH 8).     

The absence of a spectroscopically resolved intermediate state P+B− in bacterial photosynthesis

Reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides have been excited either in the bacteriopheophytin band at 760 nm or in the accessory bacteriochlorophyll (B) band around 800 nm with laser pulses of 150 fs duration. Upon monitoring in the absorption band of the primary donor (P) at 860 nm, ultrafast energy transfer is observed which leads to the excited state of P in less than 100 fs. A transient bleaching recovering in 400 ± 100 fs is specifically detected upon excitation and observation in the 800 nm absorption band of B. However, upon direct excitation of P in the near infrared and using either normal or borohydride-treated reaction centers, we have found no spectral or kinetic evidence indicating the presence of a transient intermediate state such as P⁺B⁻.     

Quest for minor but key chlorophyll molecules in photosynthetic reaction centers – unusual pigment composition in the reaction centers of the chlorophyll d-dominated cyanobacterium Acaryochloris marina

A short overview, based on our own findings, is given of the minor pigments that function as key components in photosynthesis. Recently, we found the presence of chlorophyll a, chlorophyll d′ and pheophytin a as minor pigments in the chlorophyll d-dominated cyanobacterium Acaryochloris marina. This revised version was published online in June 2006 with corrections to the Cover Date.     

Electron-paramagnetic-resonance measurements of the electron-transfer components of the reaction centre of Rhodopseudomonas viridis. Oxidation–reduction potentials and interactions of the electron acceptors

Oxidation-reduction potentiometry was carried out on Rhodopseudomonas viridis chromatophores. Measurements of e.p.r. signals of the semiquinone-iron type at g=1.82 have revealed a more complex situation than previously reported. The presence of three different components is indicated. The midpoint potential (E(m)) of the primary acceptor quinone/semiquinone couple was found to be approx. -165mV at pH10, with a pK being reached at around pH7.5. The primary acceptor also accepts a second electron with an E(m) of -525mV, but this redox transition exhibits a hysteresis effect. Interaction effects indicate the presence of another component with E(m) values at pH10 of approx. -165mV (pK reached at around pH7.5) for single reduction and -350mV (pK at pH10 or greater) for double reduction. It is suggested that this component is the secondary acceptor. Another semiquinone-iron-type component which gives a g=1.82 signal is also present. This component is distinguishable from the primary acceptor by its e.p.r. spectrum, which shows a double peak at g=1.82 and a g(x) line at g=1.76. This component has E(m) values at pH10 for single and double reduction of -15mV and approx. -150mV respectively. Both of these E(m) values are pH-dependent. The presence of an interaction between this component and the photoreduced primary acceptor indicates the close proximity of these components. However, the midpoint potential of this component indicates a function as a secondary electron-transport component rather than an electron acceptor in the reaction centre. The dependence of the bacteriopheophytin intermediate (I) doublet e.p.r. signal on the presence of the semiquinone-iron form of the primary acceptor is demonstrated. The midpoint potential of the I/I(-) couple is estimated to be lower than -600mV.     

The rate of Q(x)→Q(y) relaxation in bacteriochlorophylls of reaction centers from Rhodobacter sphaeroides determined by kinetics of the ultrafast carotenoid bandshift

Transient absorption changes induced by excitation of isolated reaction centers (RCs) from Rhodobacter sphaeroides with 600nm laser pulses of 20fs (full width at half maximum) were monitored in the wavelength region of 420-560nm. The spectral features of the spectrum obtained are characteristic for an electrochromic band shift of the single carotenoid (Car) molecule spheroidene, which is an integral constituent of these RCs. This effect is assigned to an electrochromic bandshift of Car due to the local electric field of the dipole moment formed by electronic excitation of bacteriochlorophyll (BChl) molecule(s) in the neighborhood of Car. Based on the known distances between the pigments, the monomeric BChl (B(B)) in the inactive B-branch is inferred to dominate this effect. The excitation of B(B) at 600nm leads to a transition into the S(2) state (Q(x) band), which is followed by rapid internal conversion to the S(1) state (Q(y) band), thus leading to a change of strength and orientation of the dipole moment, i.e., of the electric field acting on the Car molecule. Therefore, the time course of the electrochromic bandshift reflects the rate of the internal conversion from S(2) to S(1) of B(B). The evaluation of the kinetics leads to a value of 30fs for this relaxation process. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Triplet state EPR of reaction centers from the HisL173→LeuL173 mutant of Rhodobacter sphaeroides which contains a heterodimer primary donor

Electron paramagnetic resonance (EPR) spectroscopy has been used to examine the triplet states in reaction centers of Rhodobacter sphaeroides which have undergone a genetic modification affecting the primary donor. Reaction centers containing the His^L173Leu^L173 substitution in the amino acid sequence have a primary donor which consists of a BChl-BPh heterodimer. The triplets formed in this heterodimer reaction center were compared with those formed in the wild-type reaction center which contains the BChl-BChl homodimer. Both reaction centers transfer triplet energy to the carotenoid under illumination at liquid nitrogen temperatures (90 K). However, the intensity of the carotenoid triplet signal is significantly decreased in the Leu^L173 mutant compared with the wild-type reaction center. At 12 K, in wild-type reaction centers only the primary donor triplet is observed. The Leu^L173 mutant exhibits a signal similar to that observed by Bylina et al. (1990) in His^M200Leu^M200 mutant reaction centers from Rb. capsulatus. The values of the zero-field splitting parameters of this triplet are discussed within the context of various models for the primary donor triplet state. No alteration in the ability of the carotenoid to quench the primary donor triplet state results from mutations at these sites.Abbreviations BChl bacteriochlorophyll - BPh bacteriopheophytin - EPR electron paramagnetic resonance - LDAO lauryl-dimethylamine N-oxide     

Is bicarbonate in Photosystem II the equivalent of the glutamate ligand to the iron atom in bacterial reaction centers?

Photosystem II of oxygen-evolving organisms exhibits a bicarbonate-reversible formate effect on electron transfer between the primary and secondary acceptor quinones, QA and QB. This effect is absent in the otherwise similar electron acceptor complex of purple bacteria, e.g., Rhodobacter sphaeroides. This distinction has led to the suggestion that the iron atom of the acceptor quinone complex in PS II might lack the fifth and sixth ligands provided in the bacterial reaction center (RC) by a glutamate residue at position 234 of the M-subunit in Rb. sphaeroides RCs (M232 in Rps. viridis). By site-directed mutagenesis we have altered GluM234 in RCs from Rb. sphaeroides, replacing it with valine, glutamine and glycine to form mutants M234EV, M234EQ and M234EG, respectively. These mutants grew competently under phototrophic conditions and were tested for the formate-bicarbonate effect. In chromatophores there were no detectable differences between wild type (Wt) and mutant M234EV with respect to cytochrome b-561 reduction following a flash, and no effect of bicarbonate depletion (by incubation with formate). In isolated RCs, several electron transfer activities were essentially unchanged in Wt and M234EV, M234EQ and M234EG mutants, and no formate-bicarbonate effect was observed on: (a) the fast or slow phases of recovery of the oxidized primary donor (P+) in the absence of exogenous donor, i.e., the recombination of P+Q-A or P+Q-B, respectively; (b) the kinetics of electron transfer from Q-A to QB; or (c) the flash dependent oscillations of semiquinone formation in the presence of donor to P+ (QB turnover). The absence of a formate-bicarbonate effect in these mutants suggests that GluM234 is not responsible for the absence of the formate-bicarbonate effect in Wt bacterial RCs, or at least that other factors must be taken into account. The mutant RCs were also examined for the fast primary electron transfer along the active (A-)branch of the pigment chain, leading to reduction of QA. The kinetics were resolved to reveal the reduction of the monomer bacteriochlorophyll (tau = 3.5 ps), followed by reduction of the bacteriopheophytin (tau = 0.9 ps). Both steps were essentially unaltered from the wild type. However, the rate of reduction of QA was slowed by a factor of 2 (tau = 410 +/- 30 and 47 +/- 30 ps for M234EQ and M234EV, respectively, compared to 220 ps in the wild type).(ABSTRACT TRUNCATED AT 400 WORDS)