Mutant reaction centers of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> I(L177)H with strongly bound bacteriochlorophyll <Emphasis Type="Italic">a</Emphasis>: Structural properties and pigment-protein interactions

Methods of photoinduced Fourier transform infrared (FTIR) difference spectroscopy and circular dichroism were employed for studying features of pigment-protein interactions caused by replacement of isoleucine L177 by histidine in the reaction center (RC) of the site-directed mutant I(L177)H of Rhodobacter sphaeroides. A functional state of pigments in the photochemically active cofactor branch was evaluated with the method of photo-accumulation of reduced bacteriopheophytin H _A ^? . The results are compared with those obtained for wild-type RCs. It was shown that the dimeric nature of the radical cation of the primary electron donor P was preserved in the mutant RCs, with an asymmetric charge distribution between the bacteriochlorophylls P_A and P_B in the P⁺ state. However, the dimers P in the wild-type and mutant RCs are not structurally identical due probably to molecular rearrangements of the P_A and P_B macrocycles and/or alterations in their nearest amino acid environment induced by the mutation. Analysis of the electronic absorption and FTIR difference P⁺Q^?/PQ spectra suggests the 17³-ester group of the bacteriochlorophyll P_A to be involved in covalent interaction with the I(L177)H RC protein. Incorporation of histidine into the L177 position does not modify the interaction between the primary electron acceptor bacteriochlorophyll B_A and the bacteriopheophytin H_A. Structural changes are observed in the monomer bacteriochlorophyll B_B binding site in the inactive chromophore branch of the mutant RCs.     

FTIR spectroscopy of the reaction center of Chloroflexus aurantiacus: Photooxidation of the primary electron donor

Photochemical oxidation of the primary electron donor P in reaction centers (RCs) of the filamentous anoxygenic phototrophic bacterium Chloroflexus (C.) aurantiacus was examined by light-induced Fourier transform infrared (FTIR) difference spectroscopy at 95 K in the spectral range of 4000–1200 cm⁻¹. The light-induced P⁺Q_A⁻/PQ_A IR spectrum of C. aurantiacus RCs is compared to the well-characterized FTIR difference spectrum of P photooxidation in the purple bacterium Rhodobacter (R.) sphaeroides R-26 RCs. The presence in the P⁺Q_A⁻/PQ_A FTIR spectrum of C. aurantiacus RCs of specific low-energy electronic transitions at ∼2650 and ∼2200 cm⁻¹, as well as of associated vibrational (phase-phonon) bands at 1567, 1481, and 1294–1285 cm⁻¹, indicates that the radical cation P⁺ in these RCs has dimeric structure, with the positive charge distributed between the two coupled bacteriochlorophyll a molecules. The intensity of the P⁺ absorbance band at ∼1250 nm (upon chemical oxidation of P at room temperature) in C. aurantiacus RCs is approximately 1.5 times lower than that in R. sphaeroides R-26 RCs. This fact, together with the decreased intensity of the absorbance band at ∼2650 cm⁻¹, is interpreted in terms of the weaker coupling of bacteriochlorophylls in the P⁺ dimer in C. aurantiacus compared to R. sphaeroides R-26. In accordance with the previous (pre)resonance Raman data, FTIR measurements in the carbonyl stretching region show that in C. aurantiacus RCs (i) the 13¹-keto C=O groups of P_A and P_B molecules constituting the P dimer are not involved in hydrogen bonding in either neutral or photooxidized state of P and (ii) the 3¹-acetyl C=O group of P_B forms a hydrogen bond (probably with tyrosine M187) absorbing at 1635 cm⁻¹. Differential signals at 1757(+)/1749(−) and 1741(+)/1733(−) cm⁻¹ in the FTIR spectrum of C. aurantiacus RCs are attributed to the 13³-ester C=O groups of P in different environments.     

B-branch electron transfer in reaction centers of Rhodobacter sphaeroides assessed with site-directed mutagenesis

Mutants of Rhodobacter (Rba.) sphaeroides are described which were designed to study electron transfer along the so-called B-branch of reaction center (RC) cofactors. Combining the mutation L(M214)H, which results in the incorporation of a bacteriochlorophyll, β, for H_A [Kirmaier et al. (1991) Science 251: 922–927] with two mutations, G(M203)D and Y(M210)W, near B_A, we have created a double and a triple mutant with long lifetimes of the excited state P^* of the primary donor P, viz. 80 and 160 ps at room temperature, respectively. The yield of P⁺Q_A ⁻ formation in these mutants is reduced to 50 and 30%, respectively, of that in wildtype RCs. For both mutants, the quantum yield of P⁺H_B ⁻ formation was less than 10%, in contrast to the 15% B-branch electron transfer demonstrated in RCs of a similar mutant of Rba. capsulatus with a P^* lifetime of 15 ps [Heller et al. (1995) Science 269: 940–945]. We conclude that the lifetime of P^* is not a governing factor in switching to B-branch electron transfer. The direct photoreduction of the secondary quinone, Q_B, was studied with a triple mutant combining the G(M203)D, L(M214)H and A(M260)W mutations. In this triple mutant Q_A does not bind to the reaction center [Ridge et al. (1999) Photosynth Res 59: 9–26]. It is shown that B-branch electron transfer leading to P⁺Q_B ⁻ formation occurs to a minor extent at both room temperature and at cryogenic temperatures (about 3% following a saturating laser flash at 20 K). In contrast, in wildtype RCs P⁺Q_B ⁻ formation involves the A-branch and does not occur at all at cryogenic temperatures. Attempts to accumulate the P⁺Q_B ⁻ state under continuous illumination were not successful. Charge recombination of P⁺Q_B ⁻ formed by B-branch electron transfer in the new mutant is much faster (seconds) than has been previously reported for charge recombination of P⁺Q_B ⁻ trapped in wildtype RCs (10⁵ s) [Kleinfeld et al. (1984b) Biochemistry 23: 5780–5786]. This difference is discussed in light of the different binding sites for Q_B and Q_B ⁻ that recently have been found by X-ray crystallography at cryogenic temperatures [Stowell et al. (1997) Science 276: 812–816]. We present the first low-temperature absorption difference spectrum due to P⁺Q_B ⁻. This revised version was published online in June 2006 with corrections to the Cover Date.     

Femtosecond primary charge separation in Synechocystis sp. PCC 6803 photosystem I

The ultrafast (< 100 fs) conversion of delocalized exciton into charge-separated state between the primary donor P700 (bleaching at 705 nm) and the primary acceptor A₀ (bleaching at 690 nm) in photosystem I (PS I) complexes from Synechocystis sp. PCC 6803 was observed. The data were obtained by application of pump-probe technique with 20-fs low-energy pump pulses centered at 720 nm. The earliest absorbance changes (close to zero delay) with a bleaching at 690 nm are similar to the product of the absorption spectrum of PS I complex and the laser pulse spectrum, which represents the efficiency spectrum of the light absorption by PS I upon femtosecond excitation centered at 720 nm. During the first ∼ 60 fs the energy transfer from the chlorophyll (Chl) species bleaching at 690 nm to the Chl bleaching at 705 nm occurs, resulting in almost equal bleaching of the two forms with the formation of delocalized exciton between 690-nm and 705-nm Chls. Within the next ∼ 40 fs the formation of a new broad band centered at ∼ 660 nm (attributed to the appearance of Chl anion radical) is observed. This band decays with time constant simultaneously with an electron transfer to A₁ (phylloquinone). The subtraction of kinetic difference absorption spectra of the closed (state P700⁺A₀A₁) PS I reaction center (RC) from that of the open (state P700A₀A₁) RC reveals the pure spectrum of the P700⁺A₀⁻ ion-radical pair. The experimental data were analyzed using a simple kinetic scheme: An* [(PA₀)*A₁ P⁺A₀⁻A₁] P⁺A₀A₁⁻, and a global fitting procedure based on the singular value decomposition analysis. The calculated kinetics of transitions between intermediate states and their spectra were similar to the kinetics recorded at 694 and 705 nm and the experimental spectra obtained by subtraction of the spectra of closed RCs from the spectra of open RCs. As a result, we found that the main events in RCs of PS I under our experimental conditions include very fast (< 100 fs) charge separation with the formation of the P700⁺A₀⁻A₁ state in approximately one half of the RCs, the ∼ 5-ps energy transfer from antenna Chl* to P700A₀A₁ in the remaining RCs, and ∼ 25-ps formation of the secondary radical pair P700⁺A₀A₁⁻.     

Drastic changes in the ligand structure of the oxygen-evolving Mn cluster upon Ca depletion as revealed by FTIR difference spectroscopy

A Fourier transform infrared (FTIR) difference spectrum of the oxygen-evolving Mn cluster upon the S₁-to-S₂ transition was obtained with Ca²⁺-depleted photosystem II (PSII) membranes to investigate the structural relevance of Ca²⁺ to the Mn cluster. Previously, Noguchi et al. [Biochim. Biophys. Acta 1228 (1995) 189] observed drastic changes in the carboxylate stretching region of the S₂/S₁ FTIR spectrum upon Ca²⁺ depletion, whereas Kimura and co-workers [Biochemistry 40 (2001) 14061; ibid. 41 (2002) 5844] later claimed that these changes were not ascribed to Ca²⁺ depletion itself but caused by the interaction of EDTA to the Mn cluster and/or binding of K⁺ at the Ca²⁺ site. In the present study, the preparation of the Ca²⁺-depleted PSII sample and its FTIR measurement were performed in the absence of EDTA and K⁺. The obtained S₂/S₁ spectrum exhibited the loss of carboxylate bands at 1587/1562 and 1364/1403 cm^− 1 and diminished amide I intensities, which were identical to the previous observations in the presence of EDTA and K⁺. This result indicates that the drastic FTIR changes are a pure effect of Ca²⁺ depletion, and provides solid evidence for the general view that Ca²⁺ is strongly coupled with the Mn cluster.     

Comparison of the electron spin polarized spectrum found in plant photosystem I and in iron-depleted bacterial reaction centers with time-resolved K-band EPR; evidence that the photosystem I acceptor A1 is a quinone

The suggestion that the electron acceptor A₁ in plant photosystem I (PSI) is a quinone molecule is tested by comparisons with the bacterial photosystem. The electron spin polarized (ESP) EPR signal due to the oxidized donor and reduced quinone acceptor (P ₈₇₀ ⁺ Q^-) in iron-depleted bacterial reaction centers has similar spectral characteristics as the ESP EPR signal in PSI which is believed to be due to P ₇₀₀ ⁺ A ₁ ^- , the oxidized PSI donor and reduced A₁. This is also true for better resolved spectra obtained at K-band (24 GHz). These same spectral characteristics can be simulated using a powder spectrum based on the known g-anisotropy of reduced quinones and with the same parameter set for Q^- and A₁ ^-. The best resolution of the ESP EPR signal has been obtained for deuterated PSI particles at K-band. Simulation of the A₁ ^- contribution based on g-anisotropy yields the same parameters as for bacterial Q^- (except for an overall shift in the anisotropic g-factors, which have previously been determined for Q^-). These results provide evidence that A₁ is a quinone molecule. The electron spin polarized signal of P₇₀₀ ⁺ is part of the better resolved spectrum from the deuterated PSI particles. The nature of the P₇₀₀ ⁺ ESP is not clear; however, it appears that it does not exhibit the polarization pattern required by mechanisms which have been used so far to explain the ESP in PSI.Abbreviations hf hyperfine - A₀ A₀ acceptor of photosystem I - A₁ A₁ acceptor of photosystem I - Brij-58 polyoxyethylene 20 cetyl ether - CP1 photosystem I particles which lack ferridoxin acceptors - ESP electron spin polarized - EPR electron paramagnetic resonance - I intermediary electron acceptor, bacteriopheophytin - LDAO lauryldimethylamine - N-oxide, P₇₀₀ primary electron donor of photosystem I - PSI photosystem I - P₇₀₀ ^T triplet state of primary donor of photosystem I - P₈₇₀ primary donor in R. sphaeroides reaction center - Q quinore-acceptor in photosynthetic bacteria - RC reaction center     

Vibrational mode frequency calculations of chlorophyll-<Emphasis Type="Italic">d</Emphasis> for assessing (P740<Superscript>+</Superscript>-P740) FTIR difference spectra obtained using photosystem I particles from <Emphasis Type="Italic">Acaryochloris marina</Emphasis>

Acaryochloris marina is an oxygen-evolving organism that utilizes chlorophyll-d for light induced photochemistry. In photosystem I particles from Acaryochloris marina, the primary electron donor is called P740, and it is thought that P740 consist of two chlorophyll-d molecules. (P740⁺-P740) FTIR difference spectra have been produced, and vibrational features that are specific to chlorophyll-d (and not chlorophyll-a) were observed, supporting the idea that P740 consists chlorophyll-d molecules. Although bands in the (P740⁺-P740) FTIR difference spectra were assigned specifically to chlorophyll-d, how these bands shifted, and how their intensities changed, upon cation formation was never considered. Without this information it is difficult to draw unambiguous conclusions from the FTIR difference spectra. To gain a more detailed understanding of cation induced shifting of bands associated with vibrational modes of P740 we have used density functional theory to calculate the vibrational properties of a chlorophyll-d model in the neutral, cation and anion states. These calculations are shown to be of considerable use in interpreting bands in (P740⁺-P740) FTIR difference spectra. Our calculations predict that the 3¹ formyl C–H mode of chlorophyll-d upshifts/downshifts upon cation/anion formation, respectively. The mode intensity also decreases/increases upon cation/anion formation, respectively. The cation induced bandshift of the 3¹ formyl C–H mode of chlorophyll-d is also strongly dependant on the dielectric environment of the chlorophyll-d molecules. With this new knowledge we reassess the interpretation of bands that were assigned to 3¹ formyl C–H modes of chlorophyll-d in (P740⁺-P740) FTIR difference spectra. Considering our calculations in combination with (P740⁺-P740) FTIR DS we find that the most likely conclusions are that P740 is a dimeric Chl-d species, in an environment of low effective dielectric constant (∼2–8). In the P740⁺ state, charge is asymmetrically distributed over the two Chl-d pigments in a roughly 60:40 ratio. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Structural and functional studies on the tetraheme cytochrome subunit and its electron donor proteins: the possible docking mechanisms during the electron transfer reaction

The photosynthetic reaction centers (RCs) classified as the group II possess a peripheral cytochrome (Cyt) subunit, which serves as the electron mediator to the special-pair. In the cycle of the photosynthetic electron transfer reactions, the Cyt subunit accepts electrons from soluble electron carrier proteins, and re-reduces the photo-oxidized special-pair of the bacteriochlorophyll. Physiologically, high-potential cytochromes such as the cytochrome c₂ and the high-potential iron–sulfur protein (HiPIP) function as the electron donors to the Cyt subunit. Most of the Cyt subunits possess four heme c groups, and it was unclear which heme group first accepts the electron from the electron donor. The most distal heme to the special-pair, the heme-1, has a lower redox potential than the electron donors, which makes it difficult to understand the electron transfer mechanism mediated by the Cyt subunit. Extensive mutagenesis combined with kinetic studies has made a great contribution to our understanding of the molecular interaction mechanisms, and has demonstrated the importance of the region close to the heme-1 in the electron transfer. Moreover, crystallographic studies have elucidated two high-resolution three-dimensional structures for the RCs containing the Cyt subunit, the Blastochloris viridis and Thermochromatium tepidum RCs, as well as the structures of their electron donors. An examination of the structural data also suggested that the binding sites for both the cytochrome c₂ and the HiPIP are located adjacent to the solvent-accessible edge of the heme-1. In addition, it is also indicated by the structural and biochemical data that the cytochrome c₂ and the HiPIP dock with the Cyt subunit by different mechanisms although the two electron donors utilize the same region for the interactions; cytochrome c₂ is recognized through electrostatic interactions while hydrophobic interactions are important in the HiPIP docking.     

Low-frequency resonance Raman studies of the H(M202)G cavity mutant of bacterial photosynthetic reaction centers

Low-frequency (90–435 cm⁻¹) NIR-excitation (875–900 nm) resonance Raman (RR) studies are reported for the H(M202)G cavity mutant of bacterial photosynthetic reaction centers (RCs) from Rb. sphaeroides that was first described by Goldsmith et al. [(1996) Biochemistry 35: 2421–2428]. In this mutant, the His residue that axially ligates the Mg ion of the M-side bacteriochlorophyll (BChl) of the special pair primary donor (P) is replaced by a non-ligating Gly residue. Regardless, the Mg ion of P_M in the H(M202)G RCs remains pentacoordinates and is presumably ligated by a water molecule, although this axial ligand has not been definitively identified. The low-frequency RR studies of the H(M202)G RCs are accompanied by studies of RCs exchanged with D₂O and incubated with imidazole (Im). The RR studies of the cavity mutant RCs reveal the following: (1) The structure of P_M in the H(M202)G RCs is different from that of the wild-type, consistent with an altered BChl core. (2) A water ligand for P_M in the H(M202)G RCs is generally consistent with the low-frequency RR spectra. The Mg-OH₂ stretching vibration is tentatively assigned to a band at 318 cm⁻¹, a frequency higher than that of the Mg-His stretch of the native pigment (∼ ∼235 cm⁻¹). (3) The BChl core structure of P_M in the cavity mutant is rendered similar (but not identical) to that of the wild-type when the adventitious water axial ligand is replaced by Im. (4) Exchange with D₂O results in more global structural changes, likely involving the protein, which in turn affect the structure of the BChls in P. (5) Assignment of the low-frequency vibrational spectrum of P is generally more complex than originally suggested.     

Direct evidence of structural changes in reaction centers of Rb. sphaeroides containing suppressor mutations for Asp L213 → Asn: A FTIR study of QB photoreduction

In bacterial reaction centers (RCs), changes of protonation state of carboxylic groups, of quinone-protein interactions as well as backbone rearrangements occuring upon Q_B photoreduction can be revealed by FTIR difference spectroscopy. The influence of compensatory mutations to the detrimental Asp L213 Asn replacement on Q_B ^–/Q_B FTIR spectra of Rb. sphaeroides RCs was studied in three double mutants carrying a Asn M44 Asp, Arg M233 Cys, or Arg H177 His suppressor mutation. The proton uptake by Glu L212 upon Q_B ^– formation, as reflected by the positive band at 1728 cm^–1, is increased in the Asn M44 Asp and Arg H177 His suppressor RCs with respect to native RCs, and remains comparable to that observed in Asp L213 Asn mutant RCs. Only the Arg M233 Cys suppressor mutation affected the 1728 cm^–1 band, reducing its amplitude to near native level. Thus, there is no clear correlation between the apparent extent of proton uptake by Glu L212 and the recovery of the proton transfer RC function. In all of the mutant spectra, several protein (amide I and amide II) and quinone anion (C...O/C...C) modes are perturbed compared to the spectrum of native RCs. These IR data show that all of the compensatory mutations alter the semiquinone-protein interactions and the backbone providing direct evidence of structural changes accompanying the restoration of efficient proton transfer in RCs containing the Asp L213 Asn lesion.     

Equilibration kinetics in isolated and membrane-bound photosynthetic reaction centers upon illumination: a method to determine the photoexcitation rate

Kinetics of electron transfer, following variation of actinic light intensity, for photosynthetic reaction centers (RCs) of purple bacteria (isolated and membrane-bound) were analyzed by measuring absorbance changes in the primary photoelectron donor absorption band at 865 nm. The bleaching of the primary photoelectron donor absorption band in RCs, following a sudden increase of illumination from the dark to an actinic light intensity of I _exp, obeys a simple exponential law with the rate constant , in which α is a parameter relating the light intensity, measured in mW/cm², to a corresponding theoretical rate in units of reciprocal seconds, and k _rec is the effective rate constant of the charge recombination in the photosynthetic RCs. In this work, a method for determining the α parameter value is developed and experimentally verified for isolated and membrane-bound RCs, allowing for rigorous modeling of RC macromolecule dynamics under varied photoexcitation conditions. Such modeling is necessary for RCs due to alterations of the forward photoexcitation rates and relaxation rates caused by illumination history and intramolecular structural dynamics effects. It is demonstrated that the classical Bouguer–Lambert–Beer formalism can be applied for the samples with relatively low scattering, which is not necessarily the case with strongly scattering media or high light intensity excitation. An erratum to this article can be found at     

Primary electron transfer in reaction centers of YM210L and YM210L/HL168L mutants of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

The role of tyrosine M210 in charge separation and stabilization of separated charges was studied by analyzing of the femtosecond oscillations in the kinetics of decay of stimulated emission from P* and of a population of the primary charge separated state P⁺B_A⁻ in YM210L and YM210L/HL168L mutant reaction centers (RCs) of Rhodobacter sphaeroides in comparison with those in native Rba. sphaeroides RCs. In the mutant RCs, TyrM210 was replaced by Leu. The HL168L mutation placed the redox potential of the P⁺/P pair 123 mV below that of native RCs, thus creating a theoretical possibility of P⁺B_A⁻ stabilization. Kinetics of P* decay at 940 nm of both mutants show a significant slowing of the primary charge separation reaction in comparison with native RCs. Distinct damped oscillations in these kinetics with main frequency bands in the range of 90–150 cm⁻¹ reflect mostly nuclear motions inside the dimer P. Formation of a very small absorption band of B_A⁻ at 1020 nm is registered in RCs of both mutants. The formation of the B_A⁻ band is accompanied by damped oscillations with main frequencies from ∼10 to ∼150 cm⁻¹. Only a partial stabilization of the P⁺B_A⁻ state is seen in the YM210L/HL168L mutant in the form of a small non-oscillating background of the 1020-nm kinetics. A similar charge stabilization is absent in the YM210L mutant. A model of oscillatory reorientation of the OH-group of TyrM210 in the electric fields of P⁺ and B_A⁻ is proposed to explain rapid stabilization of the P⁺B_A⁻ state in native RCs. Small oscillatory components at ∼330–380 cm⁻¹ in the 1020-nm kinetics of native RCs are assumed to reflect this reorientation. We conclude that the absence of TyrM210 probably cannot be compensated by lowering of the P⁺B_A⁻ free energy that is expected for the double YM210L/HL168L mutant. An oscillatory motion of the HOH55 water molecule under the influence of P⁺ and B_A⁻ is assumed to be another potential contributor to the mechanism of P⁺B_A⁻ stabilization.     

Identification of the Intermediate Charge-Separated State PβL in a Leucine M214 to Histidine Mutant of the Rhodobacter sphaeroides Reaction Center Using Femtosecond Midinfrared Spectroscopy

Energy and electron transfer in a Leu M214 to His (LM214H) mutant of the Rhodobacter sphaeroides reaction center (RC) were investigated by applying time-resolved visible pump/midinfrared probe spectroscopy at room temperature. This mutant replacement of the Leu at position M214 resulted in the incorporation of a bacteriochlorophyll (BChl) in place of the native bacteriopheophytin in the L-branch of cofactors (denoted β_L). Purified LM214H RCs were excited at 600 nm (unselective excitation), at 800 nm (direct excitation of the monomeric BChl cofactors B_L and B_M), and at 860 nm (direct excitation of the primary donor (P) BChl pair (P_L/P_M)). Absorption changes associated with carbonyl (C=O) stretch vibrational modes (9-keto, 10a-ester, and 2a-acetyl) of the cofactors and of the protein were recorded in the region between 1600 cm⁻¹ and 1770 cm⁻¹, and the data were subjected to both a sequential analysis and a simultaneous target analysis. After photoexcitation of the LM214H RC, P^∗ decayed on a timescale of ∼6.3 ps to P⁺B_L⁻. The decay of P⁺B_L⁻ occurred with a lifetime of ∼2 ps, ∼3 times slower than that observed in wild-type and R-26 RCs (∼0.7 ps). Further electron transfer to the β_L BChl resulted in formation of the P⁺β_L⁻ state, and its infrared absorbance difference spectrum is reported for the first time, to our knowledge. The fs midinfrared spectra of P⁺B_L⁻ and P⁺β_L⁻ showed clear differences related to the different environments of the two BChls in the mutant RC.     

Time-resolved step-scan FTIR difference spectroscopy for the study of photosystem I with different benzoquinones incorporated into the A1 binding site

Time-resolved step-scan FTIR difference spectroscopy has been used to study photosystem I (PSI) with plastoquinone-9 (PQ) and two other benzoquinones (2,6-dimethyl-1,4-benzoquinone and 2,3,5,6-tetrachloro-1,4-benzoquinone) incorporated into the A₁ binding site. By subtracting a (P700⁺A₁^????P700A₁) FTIR difference spectrum for PSI with the native phylloquinone (PhQ) incorporated from corresponding spectra for PSI with different benzoquinones (BQs) incorporated, FTIR double difference spectra are produced, that display bands associated with vibrational modes of the quinones, without interference from features associated with protein vibrational modes.Molecular models for BQs involved in asymmetric hydrogen bonding were constructed and used in vibrational mode frequency calculations. The calculated data were used to aid in the interpretation and assignment of bands in the experimental spectra. We show that the calculations capture the general trends found in the experimental spectra.By comparing four different FTIR double difference spectra we are able to verify unambiguously bands associated with phyllosemiquinone in PSI at 1495 and 1415?cm^?1. We also resolve a previously unrecognized band of phyllosemiquinone at 1476?cm^?1 that calculations suggest is due in part to a C₄$\stackrel{?}{?}$O stretching mode.For PSI with PQ incorporated, calculations and experiment taken together indicate that the C₁$\stackrel{?}{?}$O and C₄$\stackrel{?}{?}$O vibrational modes of the semiquinone give rise to bands at 1487 and 1444?cm^?1, respectively. This is very distinct compared to PSI with PhQ incorporated.From the calculated and experimental spectra, we show that it is possible to distinguish between two possible orientations of PQ in the A₁ protein binding site.     

Charge separation in Rhodobacter sphaeroides mutant reaction centers with increased midpoint potential of the primary electron donor

Primary charge separation dynamics in four mutant reaction centers (RCs) of the purple bacterium Rhodobacter sphaeroides with increased midpoint potential of the primary electron donor P (M160LH, L131LH, M197FH, and M160LH + L131LH + M197FH) have been studied by femtosecond transient absorption spectroscopy at room temperature. The decay of the excited singlet state in the wild-type and mutant RCs is complex and has two main exponential components, which indicates heterogeneity of electron transfer rates or the presence of reverse electron transfer reactions. The radical anion band of monomeric bacteriochlorophyll B_A at 1020 nm was first observed in transient absorbance difference spectra of single mutants. This band remains visible, although with somewhat reduced amplitude, even at delays up to tens of picoseconds when stimulated emission is absent and the reaction centers are in the P⁺H _A ^? state. The presence of this band in this time period indicates the existence of thermodynamic equilibrium between the P⁺B _A ^? H_A and P⁺B_AH _A ^? states. The data give grounds for assuming that the value of the energy difference between the states P*, P⁺B _A ^? H_A, and P⁺B_AH _A ^? at early times is of the same order of magnitude as the energy kT at room temperature. Besides, monomeric bacteriochlorophyll B_A is found to be an immediate electron acceptor in the single mutant RCs, where electron transfer is hampered due to increased energy of the P⁺B _A ^? state with respect to P*.     

Electrogenicity at the donor/acceptor sides of cyanobacterial photosystem I

To study electrogenesis the photosystem I particles fromSynechococcus elongatus were incorporated into asolectin liposomes, and fast kinetics of laser flash-induced electric potential difference generation has been measured by a direct electrometric method in proteoliposomes adsorbed on a phospholipid-impregnated collodion film. The photoelectric response has been found to involve three electrogenic stages associated with (i) iron-sulfur center F_x reduction by the primary electron donor P700, (ii) electron transfer between iron-sulfur centers F_x and F_A/F_B, and (iii) reduction of photo-oxidized P700⁺ by reduced cytochromec ₅₅₃. The relative magnitudes of phases (ii) and (iii) comprised about 20% of phase (i).     

On the depletion and reconstitution of both QA and metal in reaction centers of the photosynthetic bacterium Rb. sphaeroides R-26

Four possible ways to prepare Q_A-depleted, Fe-depleted and Q_A-reconstituted RCs were studied: (1) first depleting the Fe, then depleting Q_A and finally reconstituting Q_A (D-Fe, D-Q, R-Q), (2) first depleting Q_A, then depleting the Fe and finally reconstituting Q_A (D-Q, D-Fe, R-Q), (3) first depleting Q_A, then reconstituting Q_A and finally depleting Fe (D-Q, R-Q, D-Fe), (4) first depleting Q_A, then depleting the Fe and reconstituting Q_A in the same step (D-Q, D-Fe-R-Q). Our results showed that: method (1) results in the irreversible loss of photochemical activity; method (2) and (3) result in low recovery of the photochemical activity and poor yield of Fe-depleted, Q_A-reconstituted RCs; method (4) gives surprisingly good results. This method allows for the first time to prepare the Q_A-depleted, Fe-depleted, Q_A-reconstituted RCs with high recovery of the photochemical activity and good yield. The sample has 98% of photochemical activity (yield of P⁺ Q_A ^-) compared with that of the native RCs and shows strong polarization of the EPR signal of Q_A ^- under continuous illumination at 5K. The decay halftime of I^- is slow (5 ns) compared with that of the native RCs, but it is the same as that measured for the RCs from which only iron is removed. These results indicate that the depletion of iron and the reconstitution of Q_A have been successful. Reconstitution of the Q_A-depleted, Fe-depleted and Q_A-reconstituted RCs with Zn²⁺ gives also the spin-polarized Q_A ^-, and yields the same decay of I^- (halftime 200 ps) as that of the native RCs.Abbreviations LDAO lauryldimethylamine N-oxide - EDTA ethylenediaminetetraacetic acid - BSA albumin bovine - TL buffer 10 mM Tris.HCl, 0.1% LDAO and 0.1 mM EDTA     

Light-induced Fourier transform infrared (FTIR) spectroscopic investigations of the primary donor oxidation in bacterial photosynthesis

Fourier transform infrared (FTIR) difference spectroscopy of the primary electron donor (P) photo-oxidation has been performed for reaction centers (RCs) and chromatophores of purple photosynthetic bacteria. In the 1800–650 cm⁻¹ spectral region highly reproducible absorbance changes were obtained that can be related to specific changes of individual bond absorption. Several bands in the difference spectra are tentatively assigned to changes of intensity and position of the keto and ester CO vibrations of the P bacteriochlorophylls, and a possible interpretation in terms of changes of their environment or type of bonding to the protein is given. Small difference bands in the amide I and II region allow only minor protein conformational changes.     

Period four oscillations in chlorophyll a fluorescence

The discovery of period four oscillations of the fluorescence yield under flashing light demonstrated that not only the redox state of the Photosystem II (PS II) electron acceptor Q_A, but also the oxygen evolving cycle (described by the S states) modulates the fluorescence yield of chlorophyll (Chl). The positive charges accumulated on the donor side of PS II act on the fluorescence yield (measured in the Q_A ⁻ state during a strong flash) through the concentration of the quencher P₆₈₀ ⁺, the oxidized form of PS II reaction center Chl a. However, the period four oscillations of the fluorescence yield detected 1 s after a strong flash (in the P₆₈₀Q_A state) have not yet been fully explained. This revised version was published online in June 2006 with corrections to the Cover Date.