Functional Reaction Centers Research Articles

Biomimetic modeling of photosynthetic reaction center function: long-lived, spin-polarized radical ion pair formation in chlorophyll-porphyrin-quinone triads

Biomimetic modeling of photosynthetic reaction center function: long-lived, spin-polarized radical ion pair formation in chlorophyll-porphyrin-quinone triads

Visualization of coherent nuclear motion in a membrane protein by femtosecond spectroscopy

Multicolour near-infrared femtosecond experiments of a genetically modified bacterial reaction centre show that the phase of two excited-state vibrational modes is conserved for a period of picoseconds over a large range of temperatures. The direct visualization of low-frequency nuclear vibrations in this protein, which is embedded in its natural membrane, implicates coherent nuclear motion in the primary electron transfer reaction in functional reaction centres.

History of concepts of the comparative biochemistry of oxygenic and anoxygenic photosyntheses

Experiments of Hans Molisch in 1907 demonstrated that purple bacteria do not evolve molecular oxygen during photosynthetic metabolism, and can use organic compounds as sources of cell carbon for anaerobic 'photoheterotrophic' growth. Molisch's conclusion that he discovered a new photosynthetic growth mode was not accepted for some 30 years because of the prevailing definition of photosynthesis as light-dependent conversion of carbon dioxide and inorganic reductants to cell materials. Meanwhile, during the decade of the 1930s, Cornelis van Niel formulated the 'comparative biochemical watercleavage hypothesis' of photosynthesis, which enjoyed great popularity for about 20 years. According to this concept, photolysis of water yielded 'H' and 'OH', the former acting as the hydrogen donor for CO2 reduction in all modes of photosynthesis. Oxygenic organisms were presumed to contain a unique biochemical system capable of converting 'OH' to water and O2. To explain the absence of O2 formation by purple and green photosynthetic bacteria, it was supposed that such organisms lacked the oxygen-forming system and, instead, 'OH' was disposed of by reduction with an inorganic H(e) donor (other than water) according to the general equation:[Formula: see text] where H2A is H2 or an inorganic sulfur compound.Critical tests of van Niel's hypothesis could not be devised, and his proposal was abandoned soon after the discovery of in vitro photophosphorylation by green plant chloroplasts and membranes of purple bacteria in 1954. Photophosphorylation was then viewed as one key common denominator of oxygenic and anoxygenic photosyntheses. From later research it became clear that light-dependent phosphorylation of adenosine diphosphate was a consequence of photochemical charge separation and electron flow in reaction centers embedded in membranes of all photosynthetic organisms. The similarities, as well as the differences, in fine structure and function of reaction centers in anoxygenic and oxygenic organisms are now believed to reflect the course of evolution of oxygenic organisms from anoxygenic photosynthetic precursors. Thus, with the acquisition of new knowledge, concepts of the comparative biochemistry of photosynthetic processes have been radically altered during the past several decades. This paper describes highpoints of the history of these changes.

Biochemical characterization and electron-transfer reactions of sym1, a Rhodobacter capsulatus reaction center symmetry mutant which affects the initial electron donor

A 51 bp section of the Rhodobacter capsulatus photosynthetic reaction center M subunit gene (nucleotides M562-M612 of the pufM structural sequence) encoding amino acids M187-M203 was replaced by the homologous region of the L subunit gene. This resulted in the symmetrization of much of the amino acid environment of the reaction center initial electron donor, P. This is the first in a series of large-scale symmetry mutations and is referred to as sym1. The sym1 mutant was able to grow photosynthetically, indicating that reaction center function was largely intact. Isolated reaction centers showed an approximately 10-nm blue shift in the QY band of P. The standard free energy change between P* and P+BphA- determined from analysis of the long-lived fluorescence from quinone-reduced reaction centers decreased from about -120 meV in the wild-type to about -75 meV in the sym1 mutant. A 65-70% quantum yield of electron transfer from P* to P+QA- was observed, most of the yield loss occurring between P* and P+BphA-. The decay of the stimulated emission from P* was about 3-fold slower in this mutant than in the wild-type. Time-resolved spectral analysis of the charge-separated intermediates formed in sym1 reaction centers indicated that the major product was P+BphA-. A model-dependent analysis of the observed rates and electron-transfer yields gave the following microscopic rate constants for sym1 reaction centers (wild-type values under the same conditions are given in parentheses): [formula: see text] Analysis of the sym1 mutant, mutants near P made by other groups, and interspecies variation of amino acids in the vicinity of P suggests that the protein asymmetry in the environment of the initial electron donor is important for optimizing the rate and yield of electron transfer, but is not strictly required for overall reaction center function.

In bacterial reaction centers protons can diffuse to the secondary quinone by alternative pathways

The mechanisms of proton conduction to the reduced secondary quinone in bacterial reaction centers were studied in wild-type and genetically modified reaction centers from Rhodobaster capsulatus. In the L212–213AA double mutant (L212Glu → Ala, L213Asp → Ala), reaction center function is severely altered. However, a photocompetent revertant of this strain which carries a third ‘compensating’ mutation. M231Arg → Leu, at about 15Åfrom the secondary quinone, displays the normal proton binding function of the reaction center. Furthermore, the apparent p K values of group(s) involved in the stabilization of the semiquinone anion are restored by that mutation. We conclude that L212Glu and L213Asp are not obligatory residues for proton donation to Q B in Rb. capsulatus. We suggest that protons can be delivered to the Q B site from the cytoplasm via a network of proton channels activated by compensatory mutations, possibly involving water molecules bound in the interior of the reaction center.

Some speculations concerning the evolution of photosynthetic function

Following a brief review of the light-driven reactions in photosynthetic membranes, two questions are addressed. (1) Why is the first charge separation reaction in photosynthetic reaction centers so fast; and (2) given what we know about the contemporary structure and function of reaction centers, can we develop a simple model for a much more primitive reaction center? It is proposed that the primary charge separation step in reaction centers is optimized to be ultra-fast principally in order to compete with detrapping into the antenna complex, rather than to compete with radiative and non-radiative losses in the special pair. This leads to a notion of kinetic perfection analogous to that developed for enzymes which operate under diffusion-limited conditions, but elaborated to permit even more 'perfect' function. This hypothesis is testable by changing components in photosynthetic membranes and subjecting them to selective pressures. We speculate that the reaction center is far too complex to have served as an early functional unit, and consider possible roles for the iron-quinone part of the reaction center as a very primitive photosynthetic unit. It is suggested that this working end later became associated with primitive antenna complexes, which then evolved into the elaborate structure we find today.The role of photosynthesis in the origin of life has been a topic of speculation for many years. It is evident that photosynthetic function is ancient and central. As a person who does not work in the field of evolution. I am not very familiar with much of the speculation that precedes this paper. Proposals and speculation by others are likely based on much firmer ground, and therefore I apologize in advance if some of these ideas have already been suggested by others. In this chapter I take the liberty to speculate on how a structure as complex as the contemporary photosynthetic reaction center (RC) could have evolved from more primitive units, and why it retains some of its remarkable properties and seemingly unnecessary components. Both subjects lead to specific predictions and testable hypotheses.

Construction and characterization of a mutant of Rhodobacter sphaeroides with the reaction center as the sole pigment-protein complex.

A strain of Rhodobacter sphaeroides has been constructed in which the photosynthetic reaction center is the sole pigment-protein complex. The strain, named RCO1, is capable of photoheterotrophic growth and possesses assembled and functional reaction centers which can undergo photochemical charge separation and are reduced by electrons derived from the cytochrome b/c1 complex. The circular dichroism and linear dichroism of reaction centers in membranes from strain RCO1 are similar to those described previously for reaction centers isolated in detergent solution. A second strain, named RCLH11, which is devoid of the peripheral LH2 antenna complex has also been constructed. A description of the properties of these strains is presented.

Inhibition of Water Splitting Increases the Susceptibility of Photosystem II to Photoinhibition

Photosystem II (PSII)-enriched membrane particles were isolated from peas (Pisum sativum L.) and treated in several different ways to inhibit the water oxidation reactions, but not reaction center function itself, as judged by the light-induced rate of reduction of 2,6-dichlorophenol indophenol with and without the artificial electron donor, diphenyl carbazide. It was shown that such treatments increased the susceptibility of the PSII-enriched membranes to photoinhibition. This trend was further observed if 2,6-dichlorophenol indophenol was present during the illumination with photoinhibitory light. On the other hand, protection against the enhanced photoinhibition was found when the water-splitting activity was reconstituted or when the artificial electron donor diphenyl carbazide was present during the preillumination. The results indicate that irreversible photodamage occurred within the PSII reaction center as a consequence of illumination with strong light and that the rate of this damage was enhanced under conditions that are expected to give rise to a photoaccumulation of oxidizing species such as P680(+) on the donor side of PSII. This mechanism of photoinhibitory damage occurred under both aerobic and anaerobic conditions.

Light-induced fluorescence quenching and loss of photochemistry in chromatophores of photosynthetic purple bacteria

Exposure of chromatophores of Rhodobacter capsulatus to a high light intensity (10 000 μE m −2 s −1) in the presence of oxygen results in non-photochemical quenching of bacteriochlorophyll fluorescence and a decrease in the quantum yield of charge separation at the reaction centre. Several observations support the hypothesis that this is due to the formation of a quencher in the light-harvesting antennae. The relationship between quenching of the maximum fluorescence yield F m and the dark-level fluorescence yield F o fits theoretical predictions for antenna quenching, while the decrease in photochemical quantum yield is proportional to the decrease in F v/ F m (where F v= F m— F o). The number of functional reaction centres appears to be unchanged. No change in redox dependence of charge separation can be detected, suggesting that no damage of the reaction centre occurs. It is suggested that the quencher is formed by the oxidation of some species. Consistent with this, quenching is inhibited by the removal of oxygen. Aerobic quenching can be observed in mutants lacking either the outer light-harvesting complex LHII or both the reaction centre and the inner light-harvesting complex LHI indicative of quenching throughout the light-harvesting apparatus. Under anaerobic conditions there is still some light-dependent inhibition of quantum yield of charge separation by a distinct process that does not effect F v/ F m. The mechanism of this process is unknown

Light-regulated expression of the psbD gene family in Synechococcus sp. strain PCC 7942: evidence for the role of duplicated psbD genes in cyanobacteria.

The genome of the cyanobacterium Synechococcus sp. strain PCC 7942 contains two psbD genes encoding the D2 protein of the photosystem II reaction center: psbDI, which is cotranscribed as a discistronic message with psbC (the gene encoding CP43, a chlorophyll-a binding protein), and psbDII, which is monocistronic. Northern blot analysis of psbD transcripts showed that the two genes responded differently when wild-type cells were shifted from moderate to high light intensity. Whereas psbDII transcripts increased 500% relative to unshifted control cells, psbDI-psbC transcripts remained unchanged. The beta-galactosidase activities expressed from translational fusions between the psbD genes and the Escherichia coli lacZ reporter gene displayed responses similar to those seen in the RNA. D2 protein levels in thylakoid membranes from wild-type cells increased to 250% of those of the unshifted control cells 12 h after a shift to high light intensities. In contrast, in a mutant strain (AMC016) that carries an inactive psbDII gene, D2 levels decreased by 50% under identical conditions. These results suggested that induction of psbDII gene expression by light can serve as a supplementary system for maintaining a functional photosystem II reaction center at high light intensity. This hypothesis was corroborated by mixed-culture experiments, in which AMC016 cells competed poorly with wild-type cells at high light intensity. These data suggest for the first time that differential expression of members of a cyanobacterial gene family serves to maintain a functional PSII reaction center under diverse environmental conditions.

Photoinhibition of photosynthesis represents a mechanism for the long-term regulation of photosystem II

The obligate shade plant, Tradescantia albiflora Kunth grown at 50 μmol photons · m(-2) s(-1) and Pisum sativum L. acclimated to two photon fluence rates, 50 and 300 μmol · m(-2) · s(-1), were exposed to photoinhibitory light conditions of 1700 μmol · m(-2) · s(-1) for 4 h at 22° C. Photosynthesis was assayed by measurement of CO2-saturated O2 evolution, and photosystem II (PSII) was assayed using modulated chlorophyll fluorescence and flash-yield determinations of functional reaction centres. Tradescantia was most sensitive to photoinhibition, while pea grown at 300 μmol · m(-2) · s(-1) was most resistant, with pea grown at 50 μmol · m(-2) · s(-1) showing an intermediate sensitivity. A very good correlation was found between the decrease of functional PSII reaction centres and both the inhibition of photosynthesis and PSII photochemistry. Photoinhibition caused a decline in the maximum quantum yield for PSII electron transport as determined by the product of photochemical quenching (qp) and the yield of open PSII reaction centres as given by the steady-state fluorescence ratio, F'vF'm, according to Genty et al. (1989, Biochim. Biophys. Acta 990, 81-92). The decrease in the quantum yield for PSII electron transport was fully accounted for by a decrease in F'vF'm, since qp at a given photon fluence rate was similar for photoinhibited and noninhibited plants. Under lightsaturating conditions, the quantum yield of PSII electron transport was similar in photoinhibited and noninhibited plants. The data give support for the view that photoinhibition of the reaction centres of PSII represents a stable, long-term, down-regulation of photochemistry, which occurs in plants under sustained high-light conditions, and replaces part of the regulation usually exerted by the transthylakoid ΔpH gradient. Furthermore, by investigating the susceptibility of differently lightacclimated sun and shade species to photoinhibition in relation to qp, i.e. the fraction of open-to-closed PSII reaction centres, we also show that irrespective of light acclimation, plants become susceptible to photoinhibition when the majority of their PSII reaction centres are still open (i.e. primary quinone acceptor oxidized). Photoinhibition appears to be an unavoidable consequence of PSII function when light causes sustained closure of more than 40% of PSII reaction centres.

Incorporation of light-harvesting complex I alpha and beta polypeptides into the intracytoplasmic membrane of Rhodobacter capsulatus

The light-harvesting complex I (LHI) of Rhodobacter capsulatus is an oligomer of basic subunits each consisting of the two different pigment-binding polypeptides LHI alpha and LHI beta, encoded by the pufA (LHI alpha) and pufB (LHI beta) genes. Pulse-labeling experiments showed that in the presence of the LHI alpha polypeptide, the LHI beta polypeptide was inserted earlier into the intracytoplasmic membrane than was the LHI alpha polypeptide. Each of the pufA and pufB genes was deleted to test whether the LHI alpha and beta polypeptides, respectively, are inserted into the intracytoplasmic membrane independently of the LHI partner polypeptide. Neither deletion mutant strain formed the LHI antenna, but a functional reaction center complex was present. Pulse-labeling experiments indicated that the LHI beta polypeptide was inserted into the intracytoplasmic membrane with the same kinetics and in the same amounts regardless of whether the LHI alpha polypeptide was present. However, the LHI beta polypeptide did not accumulate in the membrane in the absence of the LHI alpha protein but was degraded linearly within about 12 min. In contrast to the LHI beta protein, only trace amounts of the LHI alpha polypeptide were inserted into or attached to the membrane if the LHI beta polypeptide was not synthesized.

Identification of 8 1-hydroxychlorophyll a as a functional reaction center pigment in heliobacteria

Chromatographic analysis of membranes and antenna-reaction center complexes of Heliobacterium chlorum and membranes of Heliobacillus mobilis revealed the presence of a small amount of a polar pigment with an absorption spectrum which was essentially identical to that of chlrophyll (Chl) a. Its structure was elucidated by means of 1H-NMR Correlated Spectroscopy, 13C-NMR and 252Cf-Plasma Desorption Mass Spectrometry as 81-hydroxychlorophyll a (81-OH-Chl a), esterified with farnesol. It is concluded that 81-OH-Chl a is responsible for the band near 670 nm in the absorption spectrum of heliobacteria. The molar ratio bacteriochlorophyll (BChl) g: 81-OH-Chl a was approx. 17 for rapidly growing cells of H. chlorum and Hb. mobilis. On the assumption that the antenna consists of 35–40 BChls g, this means that two molecules of 81-OH-Chl a are present per reaction center. At least one of them functions as electron acceptor in the primary charge separation.

Genetic analysis of photosynthesis in Rhodospirillum centenum.

A genetic system has been developed for studying bacterial photosynthesis in the recently described nonsulfur purple photosynthetic bacterium Rhodospirillum centenum. Nonphotosynthetic mutants of R. centenum were obtained by enrichment for spontaneous mutations, by ethyl methanesulfonate mutagenesis coupled to penicillin selection on solid medium, and by Tn5 transposition mutagenesis with an IncP plasmid vector containing a temperature-sensitive origin of replication. In vivo and in vitro characterization of individual strains demonstrated that 38 strains contained mutations that blocked bacteriochlorophyll a biosynthesis at defined steps of the biosynthetic pathway. Collectively, these mutations were shown to block seven of eight steps of the pathway leading from protoporphyrin IX to bacteriochlorophyll a. Three mutants were isolated in which carotenoid biosynthesis was blocked early in the biosynthetic pathway; the mutants also exhibited pleiotropic effects on stability or assembly of the photosynthetic apparatus. Five mutants failed to assemble a functional reaction center complex, and seven mutants contained defects in electron transport as shown by an alteration in cytochromes. In addition, several regulatory mutants were isolated that acquired enhanced repression of bacteriochlorophyll in response to the presence of molecular oxygen. The phenotypes of these mutants are discussed in relation to those of similar mutants of Rhodobacter and other Rhodospirillum species of purple photosynthetic bacteria.

Mimicking Photosynthetic Electron Transfer

AbstractThe photosynthetic reaction centers of plants and bacteria are photovoltaic devices on the molecular scale which convert light energy into chemical potential energy in the form of long-lived, energetic charge separated states. It is now possible to prepare synthetic multicomponent molecules which mimic important aspects of this process. For example, one of the keys to reaction center function is a multistep electron transfer strategy. In this paper, two general types of multistep electron transfer, sequential and parallel, are described and illustrated with several synthetic triad and pentad molecules.

Light and temperature dependent inhibition of photosynthesis in frost-hardened and un-hardened seedlings of pine

Needles of un-hardened and frost-hardended seedlings of Pinus sylvestris and Pinus contorta were exposed to photoinhibitory photon flux densities at temperatures between 0 and 35°C under laboratory conditions. Photoinhibition of photosynthesis was assayed by measuring oxygen evolution under saturating CO2 in a leaf disc oxygen electrode or by recording of photosystem II fluorescence induction kinetics at 77 K. It was demonstrated that frost hardening of pine did not affect the susceptibility of photosynthesis to short time (2 h) photoinhibition at 15°C. The two pine species irrespective of acclimative state were equally sensitive to photoinhibition as assayed by apparent photon yield analyses of photosynthetic oxygen evolution. Plots of the apparent photon yield of oxygen evolution vs. F v /F m revealed a non-linear relationship.In the temperature range of 15-20°C short term photoinhibition caused a loss of F v without effect on F 0 . However, photoinhibition at temperatures lower or higher caused F 0 to increase and decrease, respectively. In fact the decrease of F v v /F upon lowering the temperature was mainly caused by the temperature effect on F 0 . Besides photoinhibition causing the well established quenching of F v by increased radiationless decay somewhere in the reaction center-antenna complex, it is suggested that F 0 generally increases as a result of loss of functional reaction centers causing decreased trapping of excitation energy. However, the high temperature induced quenching of F 0 suggests that the quenching process (or processes) induced under photoinhibitory conditions is temperature dependent; i.e. it increases with the increase of temperature.In pine the photon yield of photosynthesis was much more sensitive to short term photoinhibition than was the rate of light saturated photosynthesis. This difference is explained by photosystem II and electron transport having surplus capacity relative to that of reductive carbon metabolism.

Control of photosynthetic membrane assembly in Rhodobacter sphaeroides mediated by puhA and flanking sequences.

A reaction center H- strain (RCH-) of Rhodobacter sphaeroides, PUHA1, was made by in vitro deletion of an XhoI restriction endonuclease fragment from the puhA gene coupled with insertion of a kanamycin resistance gene cartridge. The resulting construct was delivered to R. sphaeroides wild-type 2.4.1, with the defective puhA gene replacing the wild-type copy by recombination, followed by selection for kanamycin resistance. When grown under conditions known to induce intracytoplasmic membrane development, PUHA1 synthesized a pigmented intracytoplasmic membrane. Spectral analysis of this membrane showed that it was deficient in B875 spectral complexes as well as functional reaction centers and that the level of B800-850 spectral complexes was greater than in the wild type. The RCH- strain was photosythetically incompetent, but photosynthetic growth was restored by complementation with a 1.45-kilobase (kb) BamHI restriction endonuclease fragment containing the puhA gene carried in trans on plasmid pRK404. B875 spectral complexes were not restored by complementation with the 1.45-kb BamHI restriction endonuclease fragment containing the puhA gene but were restored along with photosynthetic competence by complementation with DNA from a cosmid carrying the puhA gene, as well as a flanking DNA sequence. Interestingly, B875 spectral complexes, but not photosynthetic competence, were restored to PUHA1 by introduction in trans of a 13-kb BamHI restriction endonuclease fragment carrying genes encoding the puf operon region of the DNA. The effect of the puhA deletion was further investigated by an examination of the levels of specific mRNA species derived from the puf and puc operons, as well as by determinations of the relative abundances of polypeptides associated with various spectral complexes by immunological methods. The roles of puhA and other genetic components in photosynthetic gene expression and membrane assembly are discussed.

Role of charge-transfer states in bacterial photosynthesis

Photon echo, photon-echo excitation, and "hole-burning" data recorded in the 800-990 nm region of Rhodobacter sphaeroides R26 and Rhodopseudomonas viridis reaction centers are reported. The primary process in these reaction centers, following excitation, was found to occur in approximately 25 fsec; the long-wavelength band of the primary electron donor (P) was largely homogeneously broadened. In accordance with our previous explanation of hole-burning and photon-echo measurements on Rb. sphaeroides [Meech, S. R., Hoff, A. J. & Wiersma, D. A. (1985) Chem. Phys. Lett. 121, 287-292], we interpret this as resulting from a dephasing of the excitation in P into a background of strongly coupled charge-transfer states. The previously reported picosecond lifetime of the excited P state is assigned to decay of these strongly mixed states. Further, a coupling between P and an adjacent bacteriochlorophyll was observed. The extent of this coupling and the role of charge-transfer states in the functioning of reaction centers is discussed.

Studies on the mechanism of photoinhibition in higher plants. II. Inactivation by high light of photosystem II reaction center function in isolated spinach thylakoids and O2 evolving particles

Studies on the mechanism of photoinhibition in higher plants. II. Inactivation by high light of photosystem II reaction center function in isolated spinach thylakoids and O2 evolving particles

Anacystis nidulans Demonstrates a Photosystem II Cation Requirement Satisfied Only by Ca2+ or Na+

Anacystis nidulans exhibits a total loss of photosystem II (PSII) activity upon incubation in a nutrient medium deficient in Ca(2+) and Na(+) and containing a divalent cation chelator. This loss of activity is light-dependent, which corresponds to an energy requirement. Likewise, Ca(2+) efflux takes place only in cells incubated in light. The loss of PSII activity is reversible by addition of submillimolar amounts of either Ca(2+) or Na(+) to the external medium but not by the addition of any other cation. Restoration of lost PSII activity also requires light. Light saturation curves for partially depleted cells demonstrate both lower maximum O(2) evolution rates and decreased relative quantum yields when compared to control cells. Partial electron transport reactions isolate the site of the Ca(2+)/Na(+) effect to the reaction center itself or immediately on its oxidizing side and exclude the water-splitting complex. O(2) flash yields decline during cation depletion, indicating a decrease in the number of functional PSII reaction centers, but the maximum turnover rate for still functional reaction centers does not decline. Thus, PSII of A. nidulans exhibits an all-or-none cation requirement, satisfied only by Ca(2+) or Na(+).