The effect of temperature and transmembrane potentials on the rates of electron transfer between membrane-bound biological redox components

We have investigated rate data for the temperature and free energy dependence of the primary electron-transfer processes in bacterial photosynthesis. Rather than representing the whole electronic-nuclear coupling by a frequently applied discrete single-mode model, we have incorporated a continuum of modes characterized by a certain distribution function. In this way, we can illuminate the role of both a broad distribution of low-frequency modes representing the medium and a narrow distribution representing local nuclear modes. Furthermore, it emerges from the calculations that both sets are important in the overall scheme of primary photosynthetic electron-transfer processes. By means of this model and quantum-mechanical rate theory, we can reproduce a number of important features of the primary photosynthetic processes concerning in particular the temperature (tunnelling or thermally activated nuclear motion) and free energy dependence (‘normal’, ‘activationless’, or ‘inverted’ regions) of the rate constants and estimate such parameters as nuclear-reorganization energy, electron-exchange integrals and electron-transfer distances. We have finally considered some of the important factors which determine the potential drop across the membrane and estimated the extent to which variations in the potential drop affect the rate constants of the electron-transfer processes.     

Subpicosecond and picosecond studies of electron transfer intermediates in Rhodopseudomonas sphaeroides reaction centers

The primary electron transfer processes in isolated reaction centers of Rhodopseudomonas sphaeroides have been investigated with subpicosecond and picosecond spectroscopic techniques. Spectra and kinetics of the absorbance changes following excitation with 0.7-ps 610-nm pulses, absorbed predominantly by bacteriochlorophyll (BChl), indicate that the radical pair state P⁺BPh^?, in which an electron has been transferred from the BChl dimer (P) to a bacteriopheophytin (BPh), is formed with a time constant no greater than 4 ps. The initial absorbance changes also reveal an earlier state, which could be an excited singlet state, or a P⁺BChl^? radical pair.The bleaching at 870 nm produced by 7 ps excitation pulses at 530 nm (absorbed by BPh) or at 600 nm (absorbed predominantly by BChl) shows no resolvable delay with respect to standard compounds in solution, suggesting that the time for energy transfer from BPh to P is less than 7 ps. However, the bleaching in the BPh band at 545 nm following 7-ps 600-nm excitation, exhibits an 8- to 10-ps lag with respect to standard compounds. This finding is qualitatively similar to the 35-ps delay previously observed at 760 nm by Shuvalov at al. (Shuvalov, V.A., Klevanik, A.V., Sharkov, A.V., Matveetz, Y.A. and Kryukov, P.G. (1978) FEBS Lett. 91, 135–139) when 25-ps 880-nm excitation flashes were used. A delay in the bleaching approximately equal to the width of the excitation flash can be explained in terms of the opposing effects of bleaching due to the reduction of BPh, and absorbance increases due to short-lived excited states (probably of BChl) that turn over rapidly during the flash.The decay of the initial bleaching at 800 nm produced by 7-ps 530- or 600-nm excitation flashes shows a fast component with a 30-ps time constant, in addition to a slower component having the 200-ps kinetics expected for the decay of P⁺BPh^?. The dependence on excitation intensity of the absorbance changes due to the 30-ps component indicate that the quantum yield of the state responsible for this step is lower than that observed for the primary electron transfer reactions. This suggests that at least part of the transient bleaching at 800 nm is due to a secondary process, possibly caused by excitation with an excessive number of photons. If the 800-nm absorbing BChl (B) acts as an intermediate electron carrier in the primary photochemical reaction, electron transfer between B and the BPh must have a time constant no greater than 4 ps.     

A possible new mechanism of temperature dependence of electron transfer in photosynthetic systems

A new theory for the electron transfer by the non-adiabatic process is formulated taking into account the origin shift and the frequency change of the vibration. The resultant formulas are quite similar to those of Jortner (Jortner, J. (1976) J. Chem. Phys. 64, 4860–4867) except that the free energy gap ΔG is used instead of the energy gap ΔE. By applying this theory to the photosynthetic electron transfer, the role of the remarkable temperature dependence of the electron transfer from cytochrome to P⁺ in Chromatium vinosum and the experimental data were reproduced very well using a small value of the coupling strength in contrast with the previous theory. This implies that proteins play a role to exclude many of the solvent molecules from the region of the electron transfer reaction between the donor and acceptor molecules. The negative activation process in the back electron transfer from Q^?_A to P⁺, the very slow back electron transfer from I^? to P⁺ and the solvent isotope effect on the cytochrome oxidation are also successfully explained by this new theory. It is shown that even a qualitative conclusion as to the molecular parameters obtained from the temperature dependence of the electron transfer is different between the present theory and that of Jortner.     

Nanosecond fluorescence from isolated photosynthetic reaction centers of Rhodopseudomonas sphaeroides

The time-course of fluorescence from reaction centers isolated from Rhodopseudomonas sphaeroides was measured using single-photon counting techniques. When electron transfer is blocked by the reduction of the electron-accepting quinones, reaction centers exhibit a relatively long-lived (delayed) fluorescence due to back reactions that regenerate the excited state (P*) from the transient radical-pair state, P^F. The delayed fluorescence can be resolved into three components, with lifetimes of 0.7, 3.2 and 11 ns at 295 K. The slowest component decays with the same time-constant as the absorbance changes due to P^F, and it depends on both temperature and magnetic fields in the same way that the absorbance changes do. The time-constants for the two faster components of delayed fluorescence are essentially independent of temperature and magnetic fields. The fluorescence also includes a very fast (prompt) component that is similar in amplitude to that obtained from unreduced reaction centers. The prompt fluorescence presumably is emitted mainly during the period before the initial charge-transfer reaction creates P^F from P*. From the amplitudes of the prompt and delayed fluorescence, we calculate an initial standard free-energy difference between P* and P^F of about 0.16 eV at 295 K, and 0.05 eV at 80 K, depending somewhat on the properties of the solvent. The multiphasic decay of the delayed fluorescence is interpreted in terms of relaxations in the free energy of P^F with time, totalling about 0.05 eV at 295 K, possibly resulting from nuclear movements in the electron-carriers or the protein.     

Effect of inhibitors,redox state and isoprenoid chain length on the affinity of ubiquinone for the secondary acceptor binding site in the reaction centers of photosynthetic bacteria

Quinone and inhibitor binding to Rhodopseudomonas sphaeroides (R-26 and GA) reaction centers were studied using spectroscopic methods and by direct adsorption of reaction centers onto anion exchange filters in the presence of ¹⁴C-labelled quinone or inhibitor. These measurements show that as secondary acceptor, Q_B, ubiquinone (UQ) is tightly bound in the semiquinone form and loosely bound in the quinone and quinol forms. The quinol is probably more loosely bound than the quinone. o-Phenanthroline and terbutryn, a triazine inhibitor, compete with UQ and with each other for binding to the reaction center. Inhibition by o-phenanthroline of electron transfer from the primary to the secondary quinone acceptor (Q_A to Q_B) occurs via displacement of UQ from the Q_B binding site. Displacement of UQ by terbutryn is apparently accessory to the inhibition of electron transfer. Terbutryn binding is lowered by reduction of Q_B to Q^?_B but is practically unaffected by reduction of Q_A to Q^?_A in the absence of Q_B. UQ-9 and UQ-10 have a 5- to 6-fold higher binding affinity to the Q_B site than does UQ-1, indicating that the long isoprenoid chain facilitates the binding to the Q_B site.     

Photoreduction of menaquinone in the reaction center of the green photosynthetic bacterium Chloroflexus aurantiacus

Photochemically active reaction centers were isolated from the facultatively aerobic gliding green bacterium Chloroflexus aurantiacus. The absorption difference spectrum, obtained after a flash, reflected the oxidation of P-865, the primary donor, and agreed with that observed in a purified membrane preparation from the same organism (Bruce, B.D., Fuller, R.C. and Blankenship, R.E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6532–6536). By analysis of the kinetics in the presence of reduced N-methylphenazonium methosulfate to prevent accumulation of oxidized P-865, the absorption difference spectrum of an electron acceptor was obtained. The electron acceptor was identified as menaquinone (vitamin K-2), which is reduced to the semiquinone anion in a stoichiometry of approximately one molecule per reaction center. Reduction of menaquinone was accompanied by changes in pigment absorption in the infrared region. Our results indicate that the electron-acceptor chain of C. aurantiacus is very similar to that of purple bacteria.     

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.     

Bacteriopheophytin c in reaction center complexes of green photosynthetic bacteria

Two reaction center complexes prepared from cytoplasmic membranes of Chlorobium limicola f. thiosulfato-philum were compared by absorption and CD spectrophotometry. Bacteriopheophytin c (670 nm), which is optically active in one complex but not in the other, may serve as a secondary electron acceptor in the reaction center.     

Prompt and delayed fluorescence in pigment-protein complexes of a green photosynthetic bacterium

Excitation spectra were measured at 4 K of bacteriochlorophyll a fluorescence in reaction center containing pigment-protein complexes obtained from the green photosynthetic bacterium Prosthecochloris aestuarii. Excitation spectra for the longest-wave emission (838 nm) showed bands of bacteriochlorophyll a, carotenoid, and of a pigment with absorption bands at 670, 438 and possibly near 420 nm, which is probably identical to an unidentified porphyrin described in the preceding paper (Swarthoff, T., Kramer, H.J.M. and Amesz, J. (1982) Biochim. Biophys. Acta 681, 354–358). At room temperature the longest-wave emission is stimulated by a magnetic field, which indicates that at least part of the emission is delayed fluorescence brought about by a reversal of the primary charge separation. Below about 150 K no stimulation was observed. The excitation spectra for short-wave emission (828 nm) were very similar to the absorption spectrum of the isolated antenna bacteriochlorophyll a-protein complex, and showed bands of bacteriochlorophyll a only. This indicates that two forms of the antenna protein exist that are spectroscopically similar: a soluble form that is released by treatment with guanidine hydrochloride and a bound form that remains attached to the reaction center complex. The bands of the antenna complexes were weak in the excitation spectra of the 838 nm fluorescence, which indicates that the efficiency of energy transfer to the reaction center complex is low.     

Triplet states of bacteriochlorophyll and carotenoids in chromatophores of photosynthetic bacteria

Chromatophores from photosynthetic bacteria were excited with flashes lasting approx. 15 ns. Transient optical absorbance changes not associated with the photochemical electron-transfer reactions were interpreted as reflecting the conversion of bacteriochlorophyll or carotenoids into triplet states. Triplet states of various carotenoids were detected in five strains of bacteria; triplet states of bacteriochlorophyll, in two strains that lack carotenoids. Triplet states of antenna pigments could be distinguished from those of pigments specifically associated with the photochemical reaction centers. Antenna pigments were converted into their triplet states if the photochemical apparatus was oversaturated with light, if the primary photochemical reaction was blocked by prior chemical oxidation of P-870 or reduction of the primary electron acceptor, or if the bacteria were genetically devoid of reaction centers. Only the reduction of the electron acceptor appeared to lead to the formation of triplet states in the reaction centers.In the antenna bacteriochlorophyll, triplet states probably arise from excited singlet states by intersystem crossing. The antenna carotenoid triplets probably are formed by energy transfer from triplet antenna bacteriochlorophyll. The energy transfer process has a half time of approx. 20 ns, and is about 1 × 10³ times more rapid than the reaction of the bacteriochlorophyll triplet states with O₂. This is consistent with a role of carotenoids in preventing the formation of singlet O₂ in vivo. In the absence of carotenoids and O₂, the decay half times of the triplet states are 70 μs for the antenna bacteriochlorophyll and 6–10 μs for the reaction center bacteriochlorophyll. The carotenoid triplets decay with half times of 2–8 μs.With weak flashes, the quantum yields of the antenna triplet states are in the order of 0.02. The quantum yields decline severely after approximately one triplet state is formed per photosynthetic unit, so that even extremely strong flashes convert only a very small fraction of the antenna pigments into triplet states. The yield of fluorescence from the antenna bacteriochlorophyll declines similarly. These observations can be explained by the proposal that singlet-triplet fusion causes rapid quenching of excited singlet states in the antenna bacteriochlorophyll.     

Efficient photochemical activity and strong dichroism of single crystals of reaction centers from Rhodopseudomonas viridis

Crystallized reaction centers from Rhodopseudomonas viridis (i) are photochemically active with electron transfer from the special pair to the quinones, (ii) show dichroism giving valuable information on the orientation of the different chromophores and (iii) allow chemical treatment in the crystalline phase.     

Photoselection studies of the P-800 band in the photoreaction center of Rhodospirillum rubrum

Polarization measurements of light-induced absorption changes in photoreaction center prepared from Rhodospirillum rubrum indicate that the 870 nm band is most likely due to a single transition dipole. The 800 nm band appears to be formed by transition dipoles with at least three different orientations. In photoreaction center from strain G9, none of the transition dipoles of the 800 nm band appears to form an angle larger than 70° with the 870 nm transition dipole.     

Intermediate states between P* and Pf in bacterial reaction centers,as detected by the fluorescence kinetics

The fluorescence lifetimes of the reaction centers isolated from the wild-type Rhodopseudomonas sphaeroides purple bacterium and those from the R26 mutant strain, lacking the carotenoid, were measured at low redox potential. In addition to the prompt fluorescence occurring directly from P* and the long delayed emission related to the radical pair state Pf, two other components are present. We suggest that they may come from intermediate states between P* and Pf, or reflect the stabilization of Pf itself.     

Oligomers of bacteriochlorophyll and bacteriopheophytin with spectroscopic properties resembling those found in photosynthetic bacteria

Oligomers of bacteriopheophytin (BPh) and bacteriochlorophyll (BChl) were formed in mixed aqueous-organic solvent systems, and in aqueous micelles of the detergent lauryldimethylamine oxide (LDAO). Conditions were found that gave relatively homogeneous samples of oligomers and that allowed quantitative comparisons of the spectroscopic properties of the monomeric and oligomeric pigments. The formation of certain types of oligomers is accompanied by a large bathochromic shift of the long-wavelength (Q_y) absorption band of the BChl or BPh, and by a substantial increase in its dipole strength (hyperchromism). The hyperchromism of the Q_y band occurs at the expense of the Soret band, which loses dipole strength. The Q_x band shifts slightly to shorter wavelengths and also loses dipole strength. The CD spectrum in the near-infra-red (Q_y) region becomes markedly nonconservative. (The net rotational strength in the Q_y region is positive.) This also occurs at the expense of the bands at shorter wavelengths, which gain a net negative rotational strength. The spectroscopic properties of the oligomers resemble those of some of the BChl-protein complexes found in photosynthetic bacteria. The oligomerization of BPh in LDAO micelles is linked to the formation of large, cylindrical micelles that contain on the order of 10⁵ LDAO molecules. However, the spectral changes probably occur on the formation of small oligomers of BPh; they begin to be seen when the micelles contain about 10 molecules of BPh. The BPh oligomers formed in LDAO micelles fluoresce at 865 nm, but the fluorescence yield is decreased about 40-fold, relative to that of monomeric BPh. The fluorescence yield is insensitive to the BPh/LDAO molar ratio, suggesting that the oligomers formed under these conditions are predominantly dimers. When the oligomers are excited with a short flash of light, they are converted with a low quantum yield into a metastable form. This transformation probably involves alterations in the geometry of the oligomer, but not full dissociation.     

Picosecond photodichroism studies on reaction centers from the green photosynthetic bacterium Chloroflexus aurantiacus

Picosecond photodichroism (photoselection) measurements have been carried out on reaction centers from the facultative green photosynthetic bacterium Chloroflexus aurantiacus using weak 30 ps flashes in the long-wavelength band of the primary electron donor, P. Absorption changes due to the chemical and photochemical oxidation of P and the reduction of quinone also have been examined. Our results on Chloroflexus suggest that the Q_y transition-dipoles of the bacteriopheophytin molecules participating in, or affected by, the primary reactions are oriented essentially perpendicular to the 865 nm transition dipole of P. This is in agreement with previous work on reaction centers from purple bacteria, such as Rhodopseudomonas sphaeroides. The data also suggest that the 812 nm ground-state transition is oriented at an angle of 45–65° with respect to the 865 nm transition. The new band that appears near 800 nm upon oxidation of P is polarized mainly parallel to the 865 nm band. These relative polarizations of the absorption bands are in very good agreement with the results of recent linear dichroism studies (Vasmel, H., Meiburg, R.F., Kramer, H.J.M., De Vos, L.J. and Amesz, J. (1983) Biochim. Biophys. Acta 724, 333–339). Possible origins for the absorption changes and the photodichroism spectra are discussed. The data are consistent with either a monomeric or dimeric structure of P-865.     

Circular dichroism spectra of oriented photoreaction center from Rhodospirillum rubrum

The circular dichroism spectra of oriented and unoriented photoreaction centers of Rhodospirillum rubrum are compared. Orientation is achieved by pressing photoreaction center suspended in polyacrylamide gel. The biphasic bands at 870 and 810 nm and at 630 and 600 nm undergo a rotatory strength decrease when measured in the direction of the pressure, but not when measured in the direction normal to the pressure. Such a decrease in oriented photoreaction center is consistent with the model according to which these bands are dimer exciton bands of the special pair bacteriochlorophyll.     

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.     

Protein structure,electron transfer and evolution of prokaryotic photosynthetic reaction centers

Photosynthetic reaction centers from a variety of organisms have been isolated and characterized. The groups of prokaryotic photosynthetic organisms include the purple bacteria, the filamentous green bacteria, the green sulfur bacteria and the heliobacteria as anoxygenic representatives as well as the cyanobacteria and prochlorophytes as oxygenic representatives. This review focuses on structural and functional comparisons of the various groups of photosynthetic reaction centers and considers possible evolutionary scenarios to explain the diversity of existing photosynthetic organisms.Abbreviations BChl bacteriochlorophyll - Chl chlorophyll - Rb Rhodobacter - Rp Rhodopseudomonas     

High throughput engineering to revitalize a vestigial electron transfer pathway in bacterial photosynthetic reaction centers

Photosynthetic reaction centers convert light energy into chemical energy in a series of transmembrane electron transfer reactions, each with near 100% yield. The structures of reaction centers reveal two symmetry-related branches of cofactors (denoted A and B) that are functionally asymmetric; purple bacterial reaction centers use the A pathway exclusively. Previously, site-specific mutagenesis has yielded reaction centers capable of transmembrane charge separation solely via the B branch cofactors, but the best overall electron transfer yields are still low. In an attempt to better realize the architectural and energetic factors that underlie the directionality and yields of electron transfer, sites within the protein-cofactor complex were targeted in a directed molecular evolution strategy that implements streamlined mutagenesis and high throughput spectroscopic screening. The polycistronic approach enables efficient construction and expression of a large number of variants of a heteroligomeric complex that has two intimately regulated subunits with high sequence similarity, common features of many prokaryotic and eukaryotic transmembrane protein assemblies. The strategy has succeeded in the discovery of several mutant reaction centers with increased efficiency of the B pathway; they carry multiple substitutions that have not been explored or linked using traditional approaches. This work expands our understanding of the structure-function relationships that dictate the efficiency of biological energy-conversion reactions, concepts that will aid the design of bio-inspired assemblies capable of both efficient charge separation and charge stabilization.