Sequential assembly of photosynthetic units in Rhodobacter sphaeroides as revealed by fast repetition rate analysis of variable bacteriochlorophyll a fluorescence

The development of functional photosynthetic units in Rhodobacter sphaeroides was followed by near infra-red fast repetition rate (IRFRR) fluorescence measurements that were correlated to absorption spectroscopy, electron microscopy and pigment analyses. To induce the formation of intracytoplasmic membranes (ICM) (greening), cells grown aerobically both in batch culture and in a carbon-limited chemostat were transferred to semiaerobic conditions. In both aerobic cultures, a low level of photosynthetic complexes was observed, which were composed of the reaction center and the LH1 core antenna. Interestingly, in the batch cultures the reaction centers were essentially inactive in forward electron transfer and exhibited low photochemical yields F_V/F_M, whereas the chemostat culture displayed functional reaction centers with a rather rapid (1-2 ms) electron transfer turnover, as well as a high F_V/F_M of ∼0.8. In both cases, the transfer to semiaerobiosis resulted in rapid induction of bacteriochlorophyll a synthesis that was reflected by both an increase in the number of LH1-reaction center and peripheral LH2 antenna complexes. These studies establish that photosynthetic units are assembled in a sequential manner, where the appearance of the LH1-reaction center cores is followed by the activation of functional electron transfer, and finally by the accumulation of the LH2 complexes.     

Free-energy change accompanying the reduction of the reaction center secondary quinone in Rhodopseudomonas sphaeroides chromatophores

The standard free-energy change accompanying the electron transfer from Q_A to Q_B was estimated from the intensity of the delayed fluorescence in chromatophores of Rhodopseudomonas sphaeroides. The value of 120 meV (at pH 8) suggests that Q_B⁻ is more stable in the chromatophore membrane than in the isolated reaction center.     

Singlet and triplet absorption spectra of carotenoids bound in the reaction centers of Rhodopseudomonas sphaeroides R26

The singlet and triplet state absorption spectra are reported for two carotenoids, methoxyneurosporene and spheroidene, incorporated into the reaction center protein of the photosynthetic bacterial carotenoidless mutant Rhodopseudomonas sphaeroides R26. The spectra for the two different carotenoid molecules are identical suggesting a strong interaction between the protein and the different chromophores. Combined effects of electrochromic band shifts and carotenoid structural changes are invoked to account for the spectral observations.     

Triplet state of the primary donor in reaction centers of the phototrophic bacterium <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> R26 with active photoinduced electron transfer

EPR characteristics of transient paramagnetic states photoinduced in isolated reaction centers of Rhodobacter sphaeroides R26 with intact electron transfer have been studied. It was demonstrated that the detected weak triplet state EPR signal belongs to the primary donor molecule and is populated via the conventional mechanism of radical pair S-T₀ mixing. The distortion of the spectral shape of this signal is explained by the triplet quantum yield anisotropy brought about by the short lifetime of precursor radical pairs. The angular dependence of the anisotropy was evaluated. It was shown that the spectral shape of the triplet state of photosystem II reaction center observed in the case of singly-reduced primary quinone acceptor can also be described by the anisotropic quantum yield of the triplet, with practically the same angular dependence. These properties confirm the conclusions on the mechanism of photoinduced electron transfer in photosystem II, made in previous publications. The peculiarities in the functioning of photosystem II reaction centers are probably determined by strict limitations on the triplet state generation.     

High-resolution optical absorption-difference spectra of the triplet state of the primary donor in isolated reaction centers of the photosynthetic bacteria Rhodopseudomonas sphaeroides R-26 and Rhodopseudomonas viridis measured with optically detected magnetic resonance at 1.2 K

We have recorded triplet optical absorption-difference spectra of the reaction center triplet state of isolated reaction centers from Rhodopseudomonas sphaeroides R-26 and Rps. viridis with optical absorption-detected electron spin resonance in zero magnetic field (ADMR) at 1.2 K. This technique is one to two orders of magnitude more sensitive than conventional flash absorption spectroscopy, and consequently allows a much higher spectral resolution. Besides the relatively broad bleachings and appearances found previously (see, e.g., Shuvalov V.A. and Parson W.W. (1981) Biochim. Biophys. Acta 638, 50–59) we have found strong, sharp oscillations in the wavelength regions 790–830 nm (Rps. sphaeroides) and 810–890 nm (Rps. viridis). For Rps. viridis these features are resolved into two band shifts (a blue shift at about 830 nm and a red shift at about 855 nm) and a strong, narrow absorption band at 838 nm. For Rps. sphaeroides R-26 the features are resolved into a red shift at about 810 nm and a strong absorption band at 807 nm. We conclude that the appearance of the absorption bands at 807 and 838 nm, respectively, is due to monomeric bacteriochlorophyll. Apparently, the exciton interaction between the pigments constituting the primary donor is much weaker in the triplet state than in the singlet state, and at low temperature the triplet is localized on one of the bacteriochlorophylls on an optical time scale. The fact that for Rps. sphaeroides the strong band shift and the monomeric band found at 1.2 K are absent at 293 K and very weak at 77 K indicates that these features are strongly temperature dependent. It seems, therefore, premature to ascribe the temperature dependence between 293 and 77 K of the intensity of the triplet absorption-difference spectrum at 810 nm (solely) to a delocalization of the triplet state on one of the accessory bacteriochlorophyll pigments.     

Coupling of collective motions of the protein matrix to vibrations of the non-heme iron in bacterial photosynthetic reaction centers

Non-heme iron is a conservative component of type II photosynthetic reaction centers of unknown function. We found that in the reaction center from Rba. sphaeroides it exists in two forms, high and low spin ferrous states, whereas in Rsp. rubrum mostly in a low spin state, in line with our earlier finding of its low spin state in the algal photosystem II reaction center (Burda et al., 2003). The temperature dependence of the non-heme iron displacement studied by Mössbauer spectroscopy shows that the surrounding of the high spin iron is more flexible (Debye temperature ~ 165 K) than that of the low spin atom (~ 207 K). Nuclear inelastic scattering measurements of the collective motions in the Rba. sphaeroides reaction center show that the density of vibrational states, originating from non-heme iron, has well-separated modes between lower (4-17 meV) and higher (17-25 meV) energies while in the one from Rsp. rubrum its distribution is more uniform with only little contribution of low energy (~ 6 meV) vibrations. It is the first experimental evidence that the fluctuations of the protein matrix in type II reaction center are correlated to the spin state of non-heme iron. We propose a simple mechanism in which the spin state of non-heme iron directly determines the strength of coupling between the two quinone acceptors (Q_A and Q_B) and fast collective motions of protein matrix that play a crucial role in activation and regulation of the electron and proton transfer between these two quinones. We suggest that hydrogen bond network on the acceptor side of reaction center is responsible for stabilization of non-heme iron in different spin states.     

Early Bacteriopheophytin Reduction in Charge Separation in Reaction Centers of Rhodobacter sphaeroides

A question at the forefront of biophysical sciences is, to what extent do quantum effects and protein conformational changes play a role in processes such as biological sensing and energy conversion? At the heart of photosynthetic energy transduction lie processes involving ultrafast energy and electron transfers among a small number of tetrapyrrole pigments embedded in the interior of a protein. In the purple bacterial reaction center (RC), a highly efficient ultrafast charge separation takes place between a pair of bacteriochlorophylls: an accessory bacteriochlorophyll (B) and bacteriopheophytin (H). In this work, we applied ultrafast spectroscopy in the visible and near-infrared spectral region to Rhodobacter sphaeroides RCs to accurately track the timing of the electron on B_A and H_A via the appearance of the B_A and H_A anion bands. We observed an unexpectedly early rise of the H_A⁻ band that challenges the accepted simple picture of stepwise electron transfer with 3 ps and 1 ps time constants. The implications for the mechanism of initial charge separation in bacterial RCs are discussed in terms of a possible adiabatic electron transfer step between B_A and H_A, and the effect of protein conformation on the electron transfer rate.     

Reaction center complex from an aerobic photosynthetic bacterium,Erythrobacter species OCh 114

A photosynthetic reaction center complex has been purified from an aerobic photosynthetic bacterium, Erythrobacter species OCh 114. The reaction center was solubilized with 0.45% lauryldimethylamine N-oxide and purified by DEAE-Sephacel column chromatography. Absorption spectra of both reduced and oxidized forms of the reaction center were very similar to those of the reaction center from Rhodopseudomonas sphaeroides R-26 except for the contributions due to cytochrome and carotenoid. 1 mol reaction center contained 4 mol bacteriochlorophyll a, 2 mol bacteriopheophytin a, 4 mol cytochrome c-554, 2 mol ubiquinone-10, and carotenoid. The reaction center consisted of four different polypeptides of 26, 30, 32 and 42 kDa. The last one retained heme c. Absorbance at 450 nm oscillated with the period of two on consecutive flashes. The light-minus-dark difference spectrum had two peaks at 450 nm and 420 nm, indicating that odd flashes generated a stable ubisemiquinone anion and even flashes generated quinol. o-Phenanthroline accelerated the re-reduction of flash-oxidized reaction centers, indicating that o-phenanthroline inhibited the electron transfer between Q_A and Q_B. The cytochrome (cytochrome c-554) in the reaction center was oxidized on flash activation. The midpoint potential of the primary electron acceptor (Q_A) was determined by measuring the extent of oxidation of cytochrome c-554 at various ambient potentials. The mid-point potential of Q_A was −44 mV, irrespective of pH between 5.5 and 5.9.     

Blocking the K-pathway still allows rapid one-electron reduction of the binuclear center during the anaerobic reduction of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides

The K-pathway is one of the two proton-input channels required for function of cytochrome c oxidase. In the Rhodobacter sphaeroides cytochrome c oxidase, the K-channel starts at Glu101 in subunit II, which is at the surface of the protein exposed to the cytoplasm, and runs to Tyr288 at the heme a₃/Cu_B active site. Mutations of conserved, polar residues within the K-channel block or inhibit steady state oxidase activity. A large body of research has demonstrated that the K-channel is required to fully reduce the heme/Cu binuclear center, prior to the reaction with O₂, presumably by providing protons to stabilize the reduced metals (ferrous heme a₃ and cuprous Cu_B). However, there are conflicting reports which raise questions about whether blocking the K-channel blocks both electrons or only one electron from reaching the heme/Cu center. In the current work, the rate and extent of the anaerobic reduction of the heme/Cu center were monitored by optical and EPR spectroscopies, comparing the wild type and mutants that block the K-channel. The new data show that when the K-channel is blocked, one electron will still readily enter the binuclear center. The one-electron reduction of the resting oxidized (“O”) heme/Cu center of the K362M mutant, results in a partially reduced binuclear center in which the electron is distributed about evenly between heme a₃ and Cu_B in the R. sphaeroides oxidase. Complete reduction of the heme/Cu center requires the uptake of two protons which must be delivered through the K-channel.     

Problems of the theory of electron transfer in biological systems

Based on comparative analysis, it is shown that the electron transfer theory traditionally used in biophysics is often unable to explain the electron transfer regularities observed in biological molecular systems. The data for seven electron transfer reactions (direct and reverse) that occur in bacterial photosynthetic reaction centers (mainly, purple bacteria Rhodobacter sphaeroides) have been analyzed. Conceivable reasons for the discrepancy between the theoretical and experimental data are discussed and some approaches to overcoming this contradiction are offered.     

Partial purification,subunit structure and thermal stability of the photochemical reaction center of the thermophilic green bacterium Chloroflexus aurantiacus

Spectrally pure reaction center preparations from Chloroflexus aurantiacus have been obtained in a stable form; however, the product contained several contaminating polypeptides. The reaction center pigment molecules (probably three bacteriochlorophyll a and three bacteriopheophytin a molecules) are associated with two polypeptides (M_r = 30000 and 28000) in a reaction center complex of M_r = 52000. No carotenoid is present in the complex. These data together with previous spectral data suggest that the Chloroflexus reaction center represents a more primitive evolutionary form of the purple bacterial reaction center, and that it has little if any relationship to the green bacterial component. A reaction center preparation from Rhodopseudomonas sphaeroides R26 was fully denatured at 50°C while the Chloroflexus reaction center required higher temperatures (70–75°C) for complete denaturation. Thus, an intrinsic membrane protein of a photosynthetic thermophile has been demonstrated to have greater thermal stability than the equivalent component of a mesophile.     

Photochemistry and electron transfer in borohydride-treated photosynthetic reaction centers

The photochemistry and electron-transfer activities of sodium-borohydride-treated reaction centers from the purple photosynthetic bacterium Rhodopseudomonas sphaeroides R26 have been investigated by both milliand picosecond absorption techniques. Separation from the treated reaction center of the reduction product, apparently a reduced form of one of the two molecules of bacteriochlorophyll contributing to the 800 nm ground-state absorption band, is also reported. In the near-infrared region, differences between treated and untreated reaction centers are observed in both milli- and picosecond light-induced difference spectra. However, borohydride-treated reaction centers exhibit photochemistry and electron transfer which are indistinguishable from those observed in untreated reaction centers. These results indicate that normal activity occurs in reaction centers that contain both molecules of bacteriopheophytin, but only three of the usual four molecules of bacteriochlorophyll.     

The structure and function of the cytochrome <Emphasis Type="Italic">c</Emphasis><Subscript>2</Subscript>: reaction center electron transfer complex from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

In the photosynthetic bacterium, Rhodobacter sphaeroides, the mobile electron carrier, cytochrome c₂ (cyt c₂) transfers an electron from reduced heme to the photooxidized bacteriochlorophyll dimer in the membrane bound reaction center (RC) as part of the light induced cyclic electron transfer chain. A complex between these two proteins that is active in electron transfer has been crystallized and its structure determined by X-ray diffraction. The structure of the cyt:RC complex shows the cyt c₂ (cyt c₂) positioned at the center of the periplasmic surface of the RC. The exposed heme edge from cyt c₂ is in close tunneling contact with the electron acceptor through an intervening bridging residue, Tyr L162 located on the RC surface directly above the bacteriochlorophyll dimer. The binding interface between the two proteins can be divided into two regions: a short-range interaction domain and a long-range interaction domain. The short-range domain includes residues immediately surrounding the tunneling contact region around the heme and Tyr L162 that display close intermolecular contacts optimized for electron transfer. These include a small number of hydrophobic interactions, hydrogen bonds and a pi-cation interaction. The long-range interaction domain consists of solvated complementary charged residues; positively charged residues from the cyt and negatively charged residues from the RC that provide long range electrostatic interactions that can steer the two proteins into position for rapid association.     

Spectrophotometric and voltage clamp characterization of monolayers of bacterial photosynthetic reaction centers

Bacterial photosynthetic reaction centers from Rhodopseudomonas sphaeroides have been spread on an air/aqueous interface, compressed, and transferred quantitatively to either glass or transparent, tin oxide-coated slides. These assemblies permit the concomitant measurement of both optical and electrical activities to be made on protein films under voltage-clamp conditions. Optical spectra of the monolayer-coated slides reveal characteristic reaction center absorptions. Linear dichroism spectra of the monolayers indicate that the reaction center is aligned on the air/aqueous interface with an angle of inclination which is essentially the same as it is with respect to the membrane plane in vivo. The kinetics of the light-induced absorbance changes of the reaction center in the deposited films are essentially unaltered from those in solution; however, there is some loss in the extent of photochemical activity. Measurement of the light-induced electrical transients shows capacitative charging and discharging currents, which can be readily associated with the reaction center bacteriochlorophyll dimer to ubiquinone electron transfer. The extent of the photochemical activity detected by the voltage-clamp is at best only 10–12% of that measured by optical assay. This suggests that on the air/aqueous interface, the reaction centers must be predominately oriented with opposing directions of electron transfer, having only a slight, variable tendency to align with the ubiquinone directed toward the aqueous phase. In spite of the present shortcomings, these assemblies appear to be uniquely useful to study the effect of clamped potentials on the kinetics and mechanisms of electron transfer.     

The carotenoid band shift in reaction centers from Rhodopseudomonas sphaeroides

A specific carotenoid associated with reaction centers purified from Rhodopseudomonas sphaeroides shows an optical absorbance change in response to photochemical activity, at temperatures down to 35 K. The change corresponds to a bathochromic shift of 1 nm of each absorption band. The same change is induced by either chemical oxidation or photo-oxidation of reaction center bacteriochlorophyll (P-870). Reduction of the electron acceptor of the reaction center, either chemically or photochemically, does not cause a carotenoid absorbance change or modify a change already induced by oxidation of P-870. The change of the carotenoid spectrum can therefore be correlated with the appearance of positive charge in the reaction center. In these studies we observed that at 35 K the absorption band of reaction center bacteriochlorophyll near 600 nm exhibits a shoulder at 605 nm. The resolution into two components is more pronounced in the light-dark difference spectrum. This observation is consistent with our earlier finding, that the “special pair” of bacteriochlorophyll molecules that acts as photochemical electron donor has a dimer-like absorption spectrum in the near infrared.     

Some experiments on the primary electron acceptor in reaction centres from Rhodopseudomonas sphaeroides

The bacterial reaction center absorbance change at 450 nm (A-450) assigned to an anionic semiquinone, has been suggested as a candidate for the reduced form of the primary electron acceptor in bacterial photosynthesis. In reaction centers of Rhodopseudomonas sphaeroides we have found kinetic discrepancies between the decay of A-450 and the recovery of photochemical competence. In addition, no proton uptake is measurable on the first turnover, although subsequent ones elicit one proton bound per electron. These results are taken to indicate that the acceptor reaction after a long dark period may be different for the first turnover than for subsequent ones. It is suggested that A-450 is still a likely candidate for the acceptor function but that in reaction centers, additional quinone may act as an adventitious primary acceptor when the “true” primary acceptor is reduced. Alternatively, the primary acceptor may act in a “ping-pong” fashion with respect to subsequent photoelectrons.     

Kinetics of electron transfer between the primary and the secondary electron acceptor in reaction centers from Rhodopseudomonas sphaeroides

Photoreduction of the two ubiquinone molecules, UQ₁ and UQ₂, bound to purified reaction center from Rhodopseudomonas sphaeroides induces different absorption band shifts of bacteriochlorophyll and bacteriopheophytin molecules depending on which ubiquinone is photoreduced. This allows us to study electron transfer between UQ₁ and UQ₂ directly by absorption spectrometry. The results support a model in which electrons are transferred one by one from UQ₁ to UQ₂ with a half-time of 200 μs, and two by two from fully reduced UQ₂ to the secondary acceptor pool.     

Reaction centers from triazine-resistant strains of Rhodopseudomonas sphaeroides: Localization of the mutation site by protein hybridization experiments

A procedure for dissociation and reconstitution of reaction centers has been used to hybridize reaction centers from three different herbicide-resistant mutant strains of Rhodopseudomonas sphaeroides with LM or H subunits derived from the native (susceptible) strains. All three mutant strains exhibited low rates of electron transfer. Hybridization of mutant reaction centers with native LM restored the high rates of electron transfer. Hybridization with native H did not. This procedure shows that the site of mutations in these mutant strains are on the LM unit.     

Secondary electron transfer in reaction centers of Rhodopseudomonas sphaeroides. Out-of-phase periodicity of two for the formation of ubisemiquinone and fully reduced ubiquinone

Electron transfer between purified reaction centers from Rhodopseudomonas sphaeroides and exogenous ubiquinone has been studied in the presence of electron donors by measurements of light-induced absorbance changes following a sequence of short actinic light flashes. Each odd flash promotes the formation of a molecule of ubisemiquinone; after each even flash the semiquinone disappears and a molecule of the fully reduced quinone appears.We interpret these results by means of a model where a specialized molecule of ubiquinone is reduced by the primary electron acceptor in a one-electron transfer reaction after each flash, and is reoxidized by a molecule of the ubiquinone pool in a two-electron transfer reaction every two flashes.