Thermodynamic properties of the reaction center of Rhodopseudomonas viridis in vivo measurement of the reaction center bacteriochlorophyll-primary acceptor intermediary electron carrier

The thermodynamic properties of redox components associated with the reaction center of Rhodopseudomonas viridis have been characterized with respect to their midpoint potentials and relationship with protons. In particular a midpoint potential for the intermediary electron carrier acting between the reaction center bacteriochlorophyll and the primary acceptor has been determined. The rationale for this measurement was that the light-induced triplet/biradical EPR signal would not be observed if this intermediate was chemically reduced before activation. The midpoint potential of the intermediary at pH 10.8 is about −400 mV (n = 1).     

Structural and functional characterization of the unusual triheme cytochrome bound to the reaction center of Rhodovulum sulfidophilum

The cytochrome bound to the photosynthetic reaction center of Rhodovulum sulfidophilum presents two unusual characteristics with respect to the well characterized tetraheme cytochromes. This cytochrome contains only three hemes because it lacks the peptide motif CXXCH, which binds the most distal fourth heme. In addition, we show that the sixth axial ligand of the third heme is a cysteine (Cys-148) instead of the usual methionine ligand. This ligand exchange results in a very low midpoint potential (-160 +/- 10 mV). The influence of the unusual cysteine ligand on the midpoint potential of this distal heme was further investigated by site-directed mutagenesis. The midpoint potential of this heme is upshifted to +310 mV when cysteine 148 is replaced by methionine, in agreement with the typical redox properties of a His/Met coordinated heme. Because of the large increase in the midpoint potential of the distal heme in the mutant, both the native and modified high potential hemes are photooxidized at a redox poise where only the former is photooxidizable in the wild type. The relative orientation of the three hemes, determined by EPR measurements, is shown different from tetraheme cytochromes. The evolutionary basis of the concomitant loss of the fourth heme and the down-conversion of the third heme is discussed in light of phylogenetic relationships of the Rhodovulum species triheme cytochromes to other reaction center-associated tetraheme cytochromes.     

P+HA- charge recombination reaction rate constant in Rhodobacter sphaeroides reaction centers is independent of the P/P+ midpoint potential.

The kinetics of the P+HA- (oxidized donor, reduced bacteriopheophytin acceptor) recombination reaction was measured in a series of reaction center mutants of Rhodobacter sphaeroides with altered P/P+ midpoint potentials between 410 and 765 mV. The time constant for P+HA- recombination was found to range between 14 and 26 ns and was essentially independent of P/P+ midpoint potential. Previous work has shown that the time constant for initial electron transfer in these mutants at room temperature is also only weakly dependent on the P/P+ midpoint potential, ranging from about 2.5 ps to about 50 ps. These results, taken together, imply that heterogeneity in the P/P+ midpoint potential within the reaction center population is not likely the dominant cause of the substantial kinetic complexity observed in the decay of the excited singlet state of P on the picosecond to nanosecond time scale. In addition, the pathway of P+HA- decay appears to be direct or via P+BA- rather than proceeding back through P, even in the highest-potential mutant, as is evident from the fact that the rate of P+HA- recombination is unaltered by pushing P+HA- much closer to P in energy. Finally, the midpoint potential independence of the P+HA- recombination rate constant suggests that the slow rate of P+HA- recombination arises from an inherent limitation in the maximum rate of this process rather than because it occurs in the inverted region of a classical Marcus rate vs free energy curve.     

EPR properties of the reaction center of Rhodopseudomas gelatinosa in situ and in a detergent-solubilized form.

The photochemical reaction centers from a variety of purple photosynthetic bacteria are composed of a trimer of protein subunits. However, the recently isolated reaction center from Rhodopseudomonas gelatinosa appears to have only two subunits. In this paper we examine the EPR characteristics of the primary photochemical reactants in this species, and compare them with those of other species. Despite of the differences in protein composition, no dramatic differences in EPR properties are seen in vivo, although some interesting effects are seen upon solubilization of the reaction center, which may be related to the unusual lability of the isolated preparation. Perhaps the most noteworthy phenomenon seen in Rps. gelatinosa is the apparent ability of electrons on the reduced intermediary electron carrier to tunnel at low temperatures to the oxidized c-type cytochrome, which has not been seen in other species studied to date.     

Effect of reduction of the reaction center intermediate upon the picosecond oxidation reaction of the bacteriochlorophyll dimer in Chromatium vinosum and Rhodopseudomonas viridis

The photo-oxidation of the reaction center bacteriochlorophyll dimer or special pair was monitored at 1235 nm in Chromatium vinosum and at 1301 nm in Rhodopseudomonas viridis. In both species, the photo-oxidation was apparently complete within 10 ps after light excitation and proceeded unimpeded at low temperatures regardless of the prior state of reduction of the traditional primary electron acceptor, a quinone-iron complex. Thus the requirement for an intermediary electron carrier (I), previously established by picosecond measurements in Rps. sphaeroides (see ref. 4), is clearly a more general phenomenon.

The intermediary carrier, which involves bacteriopheophytin, was examined from the standpoint of its role as the direct electron acceptor from the photo-excited reaction center bacteriochlorophyll dimer. To accomplish this, the extent of light induced bacteriochlorophyll dimer oxidation was measured directly by the picosecond response of the infrared bands and indirectly by EPR assay of the triplet/biradical, as a function of the state of reduction of the I/I couple (measured by EPR) prior to activation. Two independent methods of obtaining I in a stably reduced form were used: chemical equilibrium reduction, and photochemical reduction. In both cases, the results demonstrated that the intermediary carrier, which we designate I, alone governs the capability for reaction center bacteriochlorophyll photooxidation, and as such I appears to be the immediate and sole electron acceptor from the light excited reaction center bacteriochlorophyll dimer.     

Pumping capacity of bacterial reaction centers and backpressure regulation of energy transduction.

Transduction of free-energy by Rhodobacter sphaeroides reaction-center-light-harvesting-complex-1 (RCLH1) was quantified. RCLH1 complexes were reconstituted into liposomal membranes. The capacity of the RCLH1 complex to build up a proton motive force was examined at a range of incident light intensities, and induced proton permeabilities, in the presence of artificial electron donors and acceptors. Experiments were also performed with RCLH1 complexes in which the midpoint potential of the reaction center primary donor was modified over an 85-mV range by replacement of the tyrosine residue at the M210 position of the reaction center protein by histidine, phenylalanine, leucine or tryptophan. The intrinsic driving force with which the reaction center pumped protons tended to decrease as the midpoint potential of the primary donor was increased. This observation is discussed in terms of the control of the energetics of the first steps in light-driven electron transfer on the thermodynamic efficiency of the bacterial photosynthetic process. The light intensity at which half of the maximal proton motive force was generated, increased with increasing proton permeability of the membrane. This presents the first direct evidence for so-called backpressure control exerted by the proton motive force on steady-state cyclic electron transfer through and coupled proton pumping by the bacterial reaction center.     

Energy trapping and detrapping in reaction center mutants from Rhodobacter sphaeroides

Time-resolved fluorescence of chromatophores isolated from strains of Rhodobacter sphaeroides containing light harvesting complex I (LHI) and reaction center (RC) (no light harvesting complex II) was measured at several temperatures between 295 K and 10 K. Measurements were performed to investigate energy trapping from LHI to the RC in RC mutants that have a P/P(+) midpoint potential either above or below wild-type (WT). Six different strains were investigated: WT + LHI, four mutants with altered RC P/P(+) midpoint potentials, and an LHI-only strain. In the mutants with the highest P/P(+) midpoint potentials, the electron transfer rate decreases significantly, and at low temperatures it is possible to directly observe energy transfer from LHI to the RC by detecting the fluorescence kinetics from both complexes. In all mutants, fluorescence kinetics are multiexponential. To explain this, RC + LHI fluorescence kinetics were analyzed using target analysis in which specific kinetic models were compared. The kinetics at all temperatures can be well described with a model which accounts for the energy transfer between LHI and the RC and also includes the relaxation of the charge separated state P(+)H(A)(-), created in the RC as a result of the primary charge separation.     

Preferential binding of equine ferricytochrome c to the bacterial photosynthetic reaction center from Rhodobacter sphaeroides

Redox titration of horse heart cytochrome c (cyt c), in the presence of varying concentrations of detergent-solubilized photosynthetic reaction center (RC) from Rhodobacter sphaeroides, revealed an RC concentration-dependent decrease in the measured cyt c midpoint potential that is indicative of a 3.6 +/- 0.2-fold stronger binding affinity of oxidized cytochrome to a single binding site. This effect was correlated with preferential binding in the functional complex by redox titration of the fraction of RCs exhibiting microsecond, first-order, special pair reduction by cytochrome. A binding affinity ratio of 3.1 +/- 0.4 was determined by this second technique, confirming the result. Redox titration of flash-induced intracomplex electron transfer also showed the association in the electron transfer-active complex to be strong, with a dissociation constant of 0.17 +/- 0.03 microM. The tight binding is associated with a slow off-rate which, in the case of the oxidized form, can influence the kinetics of P(+) reduction. The pitfalls of the common use of xenon flashlamps to photoexcite fast electron-transfer reactions are discussed with relation to the first electron transfer from primary to secondary RC quinone acceptors. The results shed some light on the diversity of kinetic behavior reported for the cytochrome to RC electron-transfer reaction.     

EPR properties of the reaction center of Rhodopseudomas gelatinosa in situ and in a detergent-solubilized form

The photochemical reaction centers from a variety of purple photosynthetic bacteria are composed of a trimer of protein subunits. However, the recently isolated reaction center from Rhodopseudomonas gelatinosa appears to have only two subunits. In this paper we examine the EPR characteristics of the primary photochemical reactants in this species, and compare them with those of other species. Despite of the differences in protein composition, no dramatic differences in EPR properties are seen in vivo, although some interesting effects are seen upon solubilization of the reaction center, which may be related to the unusual lability of the isolated preparation. Perhaps the most noteworthy phenomenon seen in Rps. gelatinosa is the apparent ability of electrons on the reduced intermediary electron carrier to tunnel at low temperatures to the oxidized c-type cytochrome, which has not been seen in other species studied to date.     

Introduction of a [4Fe-4S (S-cys)4]+1,+2 iron-sulfur center into a four-alpha helix protein using design parameters from the domain of the Fx cluster in the Photosystem I reaction center.

We describe the insertion of an iron-sulfur center into a designed four alpha-helix model protein. The model protein was re-engineered by introducing four cysteine ligands required for the coordination of the mulinucleate cluster into positions in the main-chain directly analogous to the domain predicted to ligand the interpeptide [4Fe-4S (S-cys)4] cluster, Fx, from PsaA and PsaB of the Photosystem I reaction center. This was achieved by inserting the sequence, CDGPGRGGTC, which is conserved in PsaA and PsaB, into interhelical loops 1 and 3 of the four alpha-helix model. The holoprotein was characterized spectroscopically after insertion of the iron-sulfur center in vitro. EPR spectra confirmed the cluster is a [4Fe-4S] type, indicating that the cysteine thiolate ligands were positioned as designed. The midpoint potential of the iron-sulfur center in the model holoprotein was determined via redox titration and shown to be -422 mV (pH 8.3, n = 1). The results support proposals advanced for the structure of the domain of the [4Fe-4S] Fx cluster in Photosystem I based upon sequence predictions and molecular modeling. We suggest that the lower potential of the Fx cluster is most likely due to factors in the protein environment of Fx rather than the identity of the residues proximal to the coordinating ligands.     

Characterization of a Rhodobacter capsulatus reaction center mutant that enhances the distinction between spectral forms of the initial electron donor

A large scale mutation of the Rhodobacter capsulatus reaction center M-subunit gene, sym2-1, has been constructed in which amino acid residues M205-M210 have been changed to the corresponding L subunit amino acids. Two interconvertable spectral forms of the initial electron donor are observed in isolated reaction centers from this mutant. Which conformation dominates depends on ionic strength, the nature of the detergent used, and the temperature. Reaction centers from this mutant have a ground-state absorbance spectrum that is very similar to wild-type when measured immediately after purification in the presence of high salt. However, upon subsequent dialysis against a low ionic strength buffer or the addition of positively charged detergents, the near-infrared spectral band of P (the initial electron donor) in sym2-1 reaction centers is shifted by over 30 nm to the blue, from 852 to 820 nm. Systematically varying either the ionic strength or the amount of charged detergent reveals an isobestic point in the absorbance spectrum at 845 nm. The wild-type spectrum also shifts with ionic strength or detergent with an isobestic point at 860 nm. The large spectral separation between the two dominant conformational forms of the sym2-1 reaction center makes detailed measurements of each state possible. Both of the spectral forms of P bleach in the presence of light. Electrochemical measurements of the P/P+ midpoint potential of sym2-1 reaction centers show an increase of about 30 mV upon conversion from the long-wavelength form to the short-wavelength form of the mutant. The rate constant of initial electron transfer in both forms of the mutant reaction centers is essentially the same, suggesting that the spectral characteristics of P are not critical for charge separation. The short-wavelength form of P in this mutant also converts to the long-wavelength form as a function of temperature between room temperature and 130 K, again giving rise to an isobestic point, in this case at 838 nm for the mutant. A similar, though considerably less pronounced spectral change with temperature occurs in wild-type reaction centers, with an isobestic point at about 855 nm, close to that found by titrating with ionic strength or detergent. Fitting the temperature dependence of the sym2-1 reaction center spectrum to a thermodynamic model resulted in a value for the enthalpy of the conformational interconversion between the short- and long-wavelength forms of about -6 kJ/mol and an entropy of interconversion of about -35 J/(K mol). Similar values of enthapy and entropy changes can be used to model the temperature dependence in wild-type. Thus, much of the temperature dependence of the reaction center special pair near-infrared absorbance band can be described as an equilibrium shift between two spectrally distinct conformations of the reaction center.     

Structural organization of the Chromatium vinosum reaction center associated c-cytochromes.

Magnetic interactions operating between the Chromatium vinosum reaction center associated c-cytochromes and the electron carriers of the reaction center have been assayed by comparing the magnetic properties of these components alone, and in various combinations with paramagnetic forms of the reaction center electron carriers. These studies have yielded the following results. 1. The oxidized paramagnetic forms of the high potential cytochromes c-555 produce no discernable alteration of the light-induced (BChl)2.+signal. 2. Similarly, analysis of the lineshape of the light-induced (BChl)2.+signal shows that a magnetic interaction with the oxidized low potential cytochromes c-553 is likely to produce less than a 1 gauss splitting of the (BChl)2.+signal, which corresponds to a minimum separation of 25 +/- 3 A between the unpaired spins if the heme and (BChl)2 are orientated in a coplanar arrangement, suggesting a minimum separation of 15+/- 3A between the heme edge and the (BChl)2 edge. 3. a prominent magnetic interaction is observed to operate between the cytochrome c-553 and c-555, which results in a 30-35 gauss splitting of these spectra, and suggests an iron to iron separation of about 8 A.4. Magnetic interactions are not observed between the c-cytochromes and the reaction center "primary acceptor" (the iron . quinone complex) nor with the reaction center intermediate electron carrier (which involves bacteriopheophytin) suggesting separations greater than 10 A. 5. Magnetic interactions are not discerned between the two cytochrome c-553 hemes, nor between the two cytochrome c-555 hemes, implying that the distance between the cytochromes of the same pair is greater than 10 A. 6. EPR studies of oriented chromatophores have demonstrated that the cytochrome c-553 and c-555 hemes are perpendicular to each other, and suggest that the cytochrome c-553 heme plane lies parallel to the plane of the membrane, while the cytochrome c-555 heme plane lies perpendicular to the plane of the membrane surface.     

Physiological electron donors to the photochemical reaction center of Rhodobacter capsulatus

The nature and number of physiological electron donors to the photochemical reaction center of Rhodobacter capsulatus have been probed by deleting the genes for cytochromes c1 and b of the cytochrome bc1 complex, alone or in combination with deletion of the gene for cytochrome c2. Deletion of cytochrome c1 renders the organism incapable of photosynthetic growth, regardless of the presence or absence of cytochrome c2, because in the absence of the bc1 complex there is no cyclic electron transfer, nor any alternative source of electrons to rereduce the photochemically oxidized reaction center. While cytochrome c2 is capable of reducing the reaction center, there appears no alternative route for its rereduction other than the bc1 complex. The deletion of cytochromes c1 and c2 reveals previously unrecognized membrane-bound and soluble high potential c-type cytochromes, with Em7 = +312 mV and Em6.5 = +316 mV, respectively. These cytochromes do not donate electrons to the reaction center, and their roles are unknown.     

Extraction of oxidized bacteriochlorophyll from illuminated photosynthetic reaction center particles

Illuminated and dark-adapted reaction center particles from Rhodopseudomonas spheroides were extracted with methanol, and the spectra of the extracts were compared. Spectra of extracts of illuminated reaction center particles showed less absorption due to bacteriochlorophyll than did spectra of extracts of dark-adapted reaction center particles. The lost absorption in extracts of illuminated reaction center particles was restored by the addition of ascorbate. This showed that these extracts contained oxidized bacteriochlorophyll which could be rereduced in vitro. The spectrum of the restored absorption was different from that of the original bacteriochlorophyll, indicating that an alteration of the oxidized bacteriochlorophyll had taken place. This apparent alteration may have been due to the presence of the detergent lauryl-dimethylamine oxide in the extracts. However, equilibration of the protein with this detergent may have been a requirement for the extraction of photooxidized bacteriochlorophyll.     

Photo-induced cyclic electron transfer involving cytochrome bc1 complex and reaction center in the obligate aerobic phototroph Roseobacter denitrificans.

Flash-induced redox changes of b-type and c-type cytochromes have been studied in chromatophores from the aerobic photosynthetic bacterium Roseobacter denitrificans under redox-controlled conditions. The flash-oxidized primary donor P+ of the reaction center (RC) is rapidly re-reduced by heme H1 (Em,7 = 290 mV), heme H2 (Em,7 = 240 mV) or low-potential hemes L1/L2 (Em,7 = 90 mV) of the RC-bound tetraheme, depending on their redox state before photoexcitation. By titrating the extent of flash-induced low-potential heme oxidation, a midpoint potential equal to -50 mV has been determined for the primary quinone acceptor QA. Only the photo-oxidized heme H2 is re-reduced in tens of milliseconds, in a reaction sensitive to inhibitors of the bc1 complex, leading to the concomitant oxidation of a cytochrome c spectrally distinct from the RC-bound hemes. This reaction involves cytochrome c551 in a diffusional process. Participation of the bc1 complex in a cyclic electron transfer chain has been demonstrated by detection of flash-induced reduction of cytochrome b561, stimulated by antimycin and inhibited by myxothiazol. Cytochrome b561, reduced upon flash excitation, is re-oxidized slowly even in the absence of antimycin. The rate of reduction of cytochrome b561 in the presence of antimycin increases upon lowering the ambient redox potential, most likely reflecting the progressive prereduction of the ubiquinone pool. Chromatophores contain approximately 20 ubiquinone-10 molecules per RC. At the optimal redox poise, approximately 0.3 cytochrome b molecules per RC are reduced following flash excitation. Cytochrome b reduction titrates out at Eh < 100 mV, when low-potential heme(s) rapidly re-reduce P+ preventing cyclic electron transfer. Results can be rationalized in the framework of a Q-cycle-type model.     

The Rieske FeS center from the gram-positive bacterium PS3 and its interaction with the menaquinone pool studied by EPR.

The Rieske 2Fe2S center from Bacillus PS3, a Gram-positive thermophilic eubacterium, has been studied by EPR spectroscopy. Its redox midpoint potential at pH 7.0 was determined to be +165 +/- 10 mV and was found to decrease with an apparent slope of -80 mV/pH unit above pH 7.9. The Qo-site inhibitor stigmatellin induced spectral changes analogous to those reported for Rieske centers from mitochondria and chloroplasts. The redox midpoint potential of the PS3 Rieske cluster was not affected by stigmatellin. The orientation of the g tensor was similar to other Rieske centers (gz and gy are oriented parallel, gx is oriented perpendicular to the membrane plane). The shape of the EPR spectrum of the Rieske cluster from PS3 changed as a function of the redox state of the menaquinone (MK) pool. This permitted the redox midpoint potential of the MK pool to be determined in the membrane. Values of -60 +/- 20 mV at pH 7.0 and of -130 +/- 20 mV at pH 8.0 were obtained. The results are compared with already published data from other Rieske centers. It is proposed that all Rieske centers that function in electron transport chains using MK as pool quinone show common features that distinguish them from Rieske centers operating in ubiquinone- or plastoquinone-based electron transfer chains.     

In vitro and in vivo electron transfer to the triheme cytochrome subunit bound to the photosynthetic reaction center complex in the purple bacterium Rhodovulum sulfidophilum.

The cytochrome subunit bound to the photosynthetic reaction center (RC) complex in Rhodovulum sulfidophilum lacks one heme-binding motif (CXXCH) out of four motifs found in other purple bacteria resulting in the absence of the most distal heme from the RC-core complex (S. Masuda et al., J. Biol. Chem. 274 (1999) 10795). Cytochrome c(2), which acts as the electron donor to the RC was purified, and its gene was cloned and sequenced. The redox midpoint potential of cytochrome c(2) was determined to be E(m)=357 mV. The photo-oxidation and re-reduction of purified cytochrome c(2) were observed in the presence of membrane preparations. Flash-induced photo-oxidation and re-reduction of the RC-bound cytochrome were also observed in intact cells. Despite the unusual nature of the RC-bound cytochrome subunit, the cyclic electron transfer system in Rdv. sulfidophilum was shown to be similar to those in other purple bacteria.     

Kinetics of electron transfer between soluble cytochrome c-554 and purified reaction center complex from the green sulfur bacterium Chlorobium tepidum

Soluble cytochrome c-554 (M _r 10 kDa) is purified from the green sulfur bacterium Chlorobium tepidum. Its midpoint redox potential is determined to be +148 mV from redox titration at pH 7.0. The kinetics of cytochrome c-554 oxidation by a purified reaction center complex from the same organism were studied by flash absorption spectroscopy at room temperature, and the results indicate that the reaction partner of cytochrome c-554 is cytochrome c-551 bound to the reaction center rather than the primary donor P840. The second-order rate constant for the electron donation from cytochrome c-554 to cytochrome c-551 was estimated to be 1.7×10⁷ M^–1 s^–1. The reaction rate was not significantly influenced by the ionic strength of the reaction medium.This revised version was published online in October 2005 with corrections to the Cover Date.     

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.