Functional LH1 antenna complexes influence electron transfer in bacterial photosynthetic reaction centers

The effect of the light harvesting 1 (LH1) antenna complex on the driving force for light-driven electron transfer in the Rhodobacter sphaeroides reaction center has been examined. Equilibrium redox titrations show that the presence of the LH1 antenna complex influences the free energy change for the primary electron transfer reaction through an effect on the reduction potential of the primary donor. A lowering of the redox potential of the primary donor due to the presence of the core antenna is consistently observed in a series of reaction center mutants in which the reduction potential of the primary donor was varied over a 130 mV range. Estimates of the magnitude of the change in driving force for charge separation from time-resolved delayed fluorescence measurements in the mutant reaction centers suggest that the mutations exert their effect on the driving force largely through an influence on the redox properties of the primary donor. The results demonstrate that the energetics of light-driven electron transfer in reaction centers are sensitive to the environment of the complex, and provide indirect evidence that the kinetics of electron transfer are modulated by the presence of the LH1 antenna complexes that surround the reaction center in the natural membrane.     

Manganese oxidation by modified reaction centers from Rhodobacter sphaeroides

The transfer of an electron from exogenous manganese (II) ions to the bacteriochlorophyll dimer, P, of bacterial reaction centers was characterized for a series of mutants that have P/P(+) midpoint potentials ranging from 585 to 765 mV compared to 505 mV for wild type. Light-induced changes in optical and EPR spectra of the mutants were measured to monitor the disappearance of the oxidized dimer upon electron donation by manganese in the presence of bicarbonate. The extent of electron transfer was strongly dependent upon the P/P(+) midpoint potential. The midpoint potential of the Mn(2+)/Mn(3+) couple was calculated to decrease linearly from 751 to 623 mV as the pH was raised from 8 to 10, indicating the involvement of a proton. The electron donation had a second order rate constant of approximately 9 x 10(4) M(-1) s(-1), determined from the linear increase in rate for Mn(2+) concentrations up to 200 microM. Weak dissociation constants of 100-200 microM were found. Quantitative EPR analysis of the six-line free Mn(2+) signal revealed that up to seven manganese ions were associated with the reaction centers at a 1 mM concentration of manganese. The association and the electron transfer between manganese and the reaction centers could be inhibited by Ca(2+) and Na(+) ions. The ability of reaction centers with high potentials to oxidize manganese suggests that manganese oxidation could have preceded water oxidation in the evolutionary development of photosystem II.     

Redox potential changes during ATP‐dependent corrinoid reduction determined by redox titrations with europium(II)–DTPA

Corrinoids are essential cofactors of enzymes involved in the C₁ metabolism of anaerobes. The active, super‐reduced [Co^I] state of the corrinoid cofactor is highly sensitive to autoxidation. In O‐demethylases, the oxidation to inactive [Co^II] is reversed by an ATP‐dependent electron transfer catalyzed by the activating enzyme (AE). The redox potential changes of the corrinoid cofactor, which occur during this reaction, were studied by potentiometric titration coupled to UV/visible spectroscopy. By applying europium(II)–diethylenetriaminepentaacetic acid (DTPA) as a reductant, we were able to determine the midpoint potential of the [Co^II]/[Co^I] couple of the protein‐bound corrinoid cofactor in the absence and presence of AE and/or ATP. The data revealed that the transfer of electrons from a physiological donor to the corrinoid as the electron‐accepting site is achieved by increasing the potential of the corrinoid cofactor from ?530 ± 15 mV to ?250 ± 10 mV (E_SHE, pH 7.5). The first 50 to 100 mV of the shift of the redox potential seem to be caused by the interaction of nucleotide‐bound AE with the corrinoid protein or its cofactor. The remaining 150–200 mV had to be overcome by the chemical energy of ATP hydrolysis. The experiments revealed that Eu(II)–DTPA, which was already known as a powerful reducing agent, is a suitable electron donor for titration experiments of low‐potential redox centers. Furthermore, the results of this study will contribute to the understanding of thermodynamically unfavorable electron transfer processes driven by the power of ATP hydrolysis.     

Photoactivation and conformational gating for manganese binding and oxidation in bacterial reaction centers

The influence of illumination history of native bacterial reaction centers (BRCs) on the ability of binding and photo-induced oxidation of manganous ions was investigated in the pH range between 8.0 and 9.4. Binding of manganous ions to a buried site required 6 to 11-fold longer incubation periods, depending on the pH, in dark-adapted BRCs than in BRCs that were previously illuminated prior to manganese binding. The intrinsic electron transfer from the bound manganese ion to the photo-oxidized primary electron donor was found to be limited by a 2 to 5-fold slower precursor conformational step in the dark-adapted samples for the same pH range. The conformational gating could be eliminated by photoactivation, namely if the BRCs were illuminated prior to binding. Unlike in Photosystem II, photoactivation in BRCs did not involve cluster assembly. Photoactivation with manganese already bound was only possible at elevated detergent concentration. In addition, also exclusively in dark-adapted BRCs, a marked breaking point in the Arrhenius-plot was discovered around 15 °C at pH 9.4 indicating a change in the reaction mechanism, most likely caused by the change of orientation of the 2-acetyl group of the inactive bacteriochlorophyll monomer located near the manganese binding site.     

The photosynthetic apparatus and photoinduced electron transfer in the aerobic phototrophic bacteria <Emphasis Type="Italic">Roseicyclus mahoneyensis</Emphasis> and <Emphasis Type="Italic">Porphyrobacter meromictius</Emphasis>

Photosynthetic electron transfer has been examined in whole cells, isolated membranes and in partially purified reaction centers (RCs) of Roseicyclus mahoneyensis, strain ML6 and Porphyrobacter meromictius, strain ML31, two species of obligate aerobic anoxygenic phototrophic bacteria. Photochemical activity in strain ML31 was observed aerobically, but the photosynthetic apparatus was not functional under anaerobic conditions. In strain ML6 low levels of photochemistry were measured anaerobically, possibly due to incomplete reduction of the primary electron acceptor (Q_A) prior to light excitation, however, electron transfer occurred optimally under low oxygen conditions. Photoinduced electron transfer involves a soluble cytochrome c in both strains, and an additional reaction center (RC)-bound cytochrome c in ML6. The redox properties of the primary electron donor (P) and Q_A of ML31 are similar to those previously determined for other aerobic phototrophs, with midpoint redox potentials of +463 mV and −25 mV, respectively. Strain ML6 showed a very narrow range of ambient redox potentials appropriate for photosynthesis, with midpoint redox potentials of +415 mV for P and +94 mV for Q_A. Cytoplasm soluble and photosynthetic complex bound cytochromes were characterized in terms of apparent molecular mass. Fluorescence excitation spectra revealed that abundant carotenoids not intimately associated with the RC are not involved in photosynthetic energy conservation.     

On the electronic structure of the primary electron donor in bacterial photosynthesis – the bacteriochlorophyll dimer as viewed by EPR/ENDOR methods

The primary electron donor (D) plays a prominent role in electron transfer reactions in the primary processes of photosynthesis. In purple photosynthetic bacteria D is a dimer of bacteriochlorophyll molecules. Investigations on its electronic structure using EPR and ENDOR (electron nuclear double resonance) methods are summarized, focussing on results obtained in the last six years. These encompass studies on the cation radical (D+·) of mutants in which the immediate environment of D is modified through mutagenesis, particularly hydrogen bond and heterodimer mutants. Models using these results to describe the electronic interaction of the dimer halves are discussed. Also, High-Field (95 GHz) EPR to obtain the G-tensor of D+· is addressed. Furthermore, ENDOR on the photoexcited triplet state of D (DT), which in some aspects could serve as a model for the excited singlet state, are discussed. Different approaches towards correlating the electronic structure with function, in particular with the rates of electron transfer reactions, are described.     

Anaerobic heterotrophic dark metabolism in the cyanobacterium Oscillatoria limnetica: Sulfur respiration and lactate fermentation

The cyanobacterium Oscillatoria limnetica, capable of anoxygenic photosynthesis in the light with sulfide as electron donor can anaerobically break down its intracellular polyglucose in the dark. In the absence of elemental sulfur, the organism carries out lactate fermentation; in its presence, anaerobic respiration occurs in which sulfur is reduced to sulfide. Induction of anoxygenic photosynthesis or synthesis of new proteins is not necessary for either process. Cells adapted in the dark to sulfur reduction are capable of anoxygenic photosynthesis during a subsequent light period, unless protein synthesis has been inhibited during the dark incubation period.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FCCP Carbonylcyanide p-trifluoromethoxyphenylhydrazone - mgat milligramatom - OD optical density     

Species dependence of the redox potential of the primary electron donor p700 in photosystem I of oxygenic photosynthetic organisms revealed by spectroelectrochemistry

The redox potential of the primary electron donor P700, E(m)(P700/P700(+)), of Photosystem I (PSI) has been determined for 10 oxygenic photosynthesis organisms, ranging from cyanobacteria, red algae, green algae to higher plants, by spectroelectrochemistry with an optically transparent thin-layer electrode (OTTLE) cell to elucidate the scattering by as much as 150 mV in reported values of E(m)(P700/P700(+)). The E(m)(P700/P700(+)) values determined within error ranges of ± 1-4 mV exhibited a significant species dependence, with a span >70 mV, from +398 to +470 mV vs. the standard hydrogen electrode (SHE). The E(m)(P700/P700(+)) value appears to change systematically in going from cyanobacteria and primitive eukaryotic red algae, then to green algae and higher plants. From an evolutionary point of view, this result suggests that the species believed to appear later in evolution of photosynthetic organisms exhibit higher values of E(m)(P700/P700(+)). Further, the species dependence of E(m)(P700/P700(+)) seems to originate in the species-dependent redox potentials of soluble metalloproteins, Cyt c(6) and plastocyanin, which re-reduce the oxidized P700 in the electron transfer chain.     

Tuning the redox potential of the primary electron donor in bacterial reaction centers by manganese binding and light-induced structural changes

The influence of transition metal binding on the charge storage ability of native bacterial reaction centers (BRCs) was investigated. Binding of manganous ions uniquely prevented the light-induced conformational changes that would yield to long lifetimes of the charge separated state and the drop of the redox potential of the primary electron donor (P). The lifetimes of the stable charge pair in the terminal conformations were shortened by 50-fold and 7-fold upon manganous and cupric ion binding, respectively. Nickel and zinc binding had only marginal effects. Binding of manganese not only prevented the drop of the potential of P/P⁺ but also elevated it by up to 117 mV depending on where the metal was binding. With variable conditions, facilitating either manganese binding or light-induced structural changes a controlled tuning of the potential of P/P⁺ in multiple steps was demonstrated in a range of ~200 mV without the need of a mutation or synthesis. Under the selected conditions, manganese binding was achieved without its photochemical oxidation thus, the energized but still native BRCs can be utilized in photochemistry that is not reachable with regular BRCs. A 42 Å long hydrophobic tunnel was identified that became obstructed upon manganese binding and its likely role is to deliver protons from the hydrophobic core to the surface during conformational changes.     

Tuning the redox properties of a [4Fe-4S] center to modulate the activity of Mo-bisPGD periplasmic nitrate reductase

Molybdoenzymes are ubiquitous in living organisms and catalyze, for most of them, oxidation-reduction reactions using a large range of substrates. Periplasmic nitrate reductase (NapAB) from Rhodobacter sphaeroides catalyzes the 2-electron reduction of nitrate into nitrite. Its active site is a Mo bis-(pyranopterin guanine dinucleotide), or Mo-bisPGD, found in most prokaryotic molybdoenzymes. A [4Fe-4S] cluster and two c-type hemes form an intramolecular electron transfer chain that deliver electrons to the active site. Lysine 56 is a highly conserved amino acid which connects, through hydrogen-bonds, the [4Fe-4S] center to one of the pyranopterin ligands of the Mo-cofactor. This residue was proposed to be involved in the intramolecular electron transfer, either defining an electron transfer pathway between the two redox cofactors, and/or modulating their redox properties.In this work, we investigated the role of this lysine by combining site-directed mutagenesis, activity assays, redox titrations, EPR and HYSCORE spectroscopies. Removal of a positively-charged residue at position 56 strongly decreased the redox potential of the [4Fe-4S] cluster at pH?8 by 230?mV to 400?mV in the K56H and K56M mutants, respectively, thus affecting the kinetics of electron transfer from the hemes to the [4Fe-4S] center up to 5 orders of magnitude. This effect was partly reversed at acidic pH in the K56H mutant likely due to protonation of the imidazole ring of the histidine. Overall, our study demonstrates the critical role of a charged residue from the second coordination sphere in tuning the reduction potential of the [4Fe-4S] cluster in RsNapAB and related molybdoenzymes.     

Redox study of electron donation to P-680 in Photosystem II

Flash-induced absorption changes at 820 nm were studied as a function of redox potential in Tris-extracted Photosystem II oxygen-evolving particles and Triton subchloroplast fraction II particles. The rereduction kinetics of P-680+ in both preparations showed biphasic recovery phases with half-times of 42 and 625 microseconds at pH 4.5. The magnitude of the 42 microseconds phase of P-680+ rereduction was strongly dependent on the redox potential of the medium. This absorption transient, attributed to electron donation from D1 (the secondary electron donor in oxygen-inhibited chloroplasts), titrated as a single redox component with a midpoint potential of +240 +/- 35 mV. The experimentally determined midpoint potential was found to be independent of pH over the tested range 4.5-6.0. In contrast, the magnitude of the 625 microseconds phase of P-680+ rereduction was independent of redox potential between +350 and +100 mV. These results are interpreted in terms of a model in which an alternate electron donor with Em approximately equal to 240 mV, termed D0, serves as a rapid donor (t 1/2 less than or equal to 2 microseconds) to P-680+ in Tris-extracted and Triton-treated Photosystem-II preparations. According to this model, the slower electron donor, D1, is functional only when D0 becomes oxidized.     

Selective incorporation of 5-hydroxytryptophan blocks long range electron transfer in oxalate decarboxylase

Oxalate decarboxylase from Bacillus subtilis is a binuclear Mn-dependent acid stress response enzyme that converts the mono-anion of oxalic acid into formate and carbon dioxide in a redox neutral unimolecular disproportionation reaction. A π-stacked tryptophan dimer, W96 and W274, at the interface between two monomer subunits facilitates long-range electron transfer between the two Mn ions and plays an important role in the catalytic mechanism. Substitution of W96 with the unnatural amino acid 5-hydroxytryptophan leads to a persistent EPR signal which can be traced back to the neutral radical of 5-hydroxytryptophan with its hydroxyl proton removed. 5-Hydroxytryptophan acts as a hole sink preventing the formation of Mn(III) at the N-terminal active site and strongly suppresses enzymatic activity. The lower boundary of the standard reduction potential for the active site Mn(II)/Mn(III) couple can therefore be estimated as 740 mV against the normal hydrogen electrode at pH 4, the pH of maximum catalytic efficiency. Our results support the catalytic importance of long-range electron transfer in oxalate decarboxylase while at the same time highlighting the utility of unnatural amino acid incorporation and specifically the use of 5-hydroxytryptophan as an energetic sink for hole hopping to probe electron transfer in redox proteins.     

Carbon dioxide assimilation in oxygenic and anoxygenic photosynthesis

This article represents a summary of our contemporary understanding of carbon dioxide assimilation in photosynthesis, including both the oxygen-evolving (oxygenic) type characteristic of cyanobacteria, algae and higher plants, and the non-oxygen-evolving (anoxygenic) type characteristic of other bacteria. Mechanisms functional in the regulation of the reductive pentose phosphate cycle of oxygenic photosynthesis are emphasized, as is the reductive carboxylic acid cycle-the photosynthetic carbon pathway functional in anoxygenic green sulfur bacteria. Thioredoxins, an ubiquitous group of low molecular weight proteins with catalytically active thiols, are also described in some detail, notably their role in regulating the reductive pentose phosphate cycle of oxygenic photosynthesis and their potential use as markers to trace the evolutionary development of photosynthesis.Abbreviations NADP-GAPDH-NADP glyceraldehyde 3-phosphate dehydrogenase - FBPase fructose 1,6-bisphosphatase - FTR ferredoxin-thioredoxin reductase - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - SBPase sedoheptulos 1,7-bisphosphatase - PRK phosphoribulokinase - NADP-MDH-NADP malate dehydrogenase - CF1-ATPase chloroplast coupling factor - G6PDH glucose 6-phosphate dehydrogenase Most of the references cited in this article are reviews. For references to specific material, readers should consult the appropriate review.     

Ferredoxin-Dependent Photosynthetic Electron Transport and Coupled Processes of Energy Storage in Chloroplasts of Wheat Plants Grown under Different Levels of Nitrogen Supply

The rates of electron transfer in the presence of natural cofactors, ferredoxin and NADP, which were added in the amounts catalyzing noncyclic or cyclic electron transfer, were studied in thylakoids isolated from 17-day-old wheat seedlings. Upon excitation of both photosystems (PS) of photosynthesis, the potential rate of NADP reduction in thylakoids isolated from plants grown on nitrogen-free nutrient solution did not differ from that in thylakoids from the control plants. However, the P/2e ratio was significantly lower in thylakoids isolated from nitrogen-deficient plants. On the contrary, in the presence of DCMU, the rate of PSI-driven electron transfer from an artificial donor to NADP was considerably higher in these than in the control thylakoids. In the presence of ferredoxin under anaerobic conditions, the rate of phosphorylation coupled to cyclic electron transport was also significantly higher in thylakoids isolated from nitrogen-deficient plants, than in thylakoids isolated from control plants. Our data show that PSI-driven electron transport and cyclic photophosphorylation are activated in nitrogen-starved wheat plants, at least at the initial stages of starvation.     

Structural and spectropotentiometric analysis of Blastochloris viridis heterodimer mutant reaction center

Heterodimer mutant reaction centers (RCs) of Blastochloris viridis were crystallized using microfluidic technology. In this mutant, a leucine residue replaced the histidine residue which had acted as a fifth ligand to the bacteriochlorophyll (BChl) of the primary electron donor dimer M site (HisM200). With the loss of the histidine-coordinated Mg, one bacteriochlorophyll of the special pair was converted into a bacteriopheophytin (BPhe), and the primary donor became a heterodimer supermolecule. The crystals had dimensions 400 × 100 × 100 μm, belonged to space group P4₃2₁2, and were isomorphous to the ones reported earlier for the wild type (WT) strain. The structure was solved to a 2.5 Å resolution limit. Electron-density maps confirmed the replacement of the histidine residue and the absence of Mg. Structural changes in the heterodimer mutant RC relative to the WT included the absence of the water molecule that is typically positioned between the M side of the primary donor and the accessory BChl, a slight shift in the position of amino acids surrounding the site of the mutation, and the rotation of the M194 phenylalanine. The cytochrome subunit was anchored similarly as in the WT and had no detectable changes in its overall position. The highly conserved tyrosine L162, located between the primary donor and the highest potential heme C₃₈₀, revealed only a minor deviation of its hydroxyl group. Concomitantly to modification of the BChl molecule, the redox potential of the heterodimer primary donor increased relative to that of the WT organism (772 mV vs. 517 mV). The availability of this heterodimer mutant and its crystal structure provides opportunities for investigating changes in light-induced electron transfer that reflect differences in redox cascades.     

Light-induced charge separation in ruthenium based triads - New variations on an old theme

Success with artificial photosynthesis requires control of the photoinduced electron transfer reactions leading to charge-separated states. In this review, some new ideas to optimize such charge-separated states in ruthenium(II) polypyridyl based three-component systems with respect to: (1) long lifetimes and (2) ability to store sufficient energy for catalytic water splitting, are presented. To form long-lived charge-separated states, a manganese complex as electron donor and potential catalyst for water oxidation has been used. The recombination reaction is unusually slow because it occurs deep down in the Marcus normal region as a consequence of the large bond reorganization following the manganese oxidation. For the creation of high energy charge-separated states, a strategy using bichromophoric systems is presented. By consecutive excitations of the two chromophores, the formation of charge-separated states that lie higher in energy than either of the two excited states could in theory be achieved, the first results of which will be discussed in this review.     

Sulfide and pH effects on variable fluorescence of photosystem II in two strains of the cyanobacterium Oscillatoria amphigranulata

Changes in fluorescence of photosystem II (PS II) chlorophyll were used to monitor the in vivo effects of sulfide and pH on photosynthesis by the cyanobacterium Oscillatoria amphigranulata. O. amphigranulata is capable of both oxygenic photosynthesis and sulfide dependent anoxygenic photosynthesis. A genetic variant of O. amphigranulata which photosynthesizes oxygenically at normal rates, but is incapable of anoxygenic photosynthesis and cannot tolerate sulfide, was also used to explore the mode of action of sulfide. In vivo fluorescence responses of PS II chlorophyll in the first few seconds of exposure to light (Kautsky transients) reflected the electrochemical states of PS II and associated electron donors and acceptors. Kautsky transients showed a distinct difference between PS II of the wild type and the variant, but sulfide lowered fluorescence in both. Kautsky transients with sulfide were similar to transients with addition of NH₂OH, NH₄ ⁺ or HCN, indicating sulfide interacts with a protein on the donor side of PS II. The fluorescence steady-state (after 2 min) was measured in the presence of sulfide, cyanide and ammonium with pH ranging from 7.2–8.7. Sulfide and cyanide had the most impact at pH 7.2, ammonium at pH 8.7. This suggests that the uncharged forms (HCN, NH₃ and H₂S) had the strongest effect on PS II, possibly because of increased membrane permeability.Abbreviations DCMU 3(3,4-dichlorophenyl)-1,1-dimethyl-urea - Oa-wt Oscillatoria amphigranulata (wild type) - Oa-2 Oscillatoria amphigranulata (genetic variant)     

Comparative study of reaction centers from purple photosynthetic bacteria: Isolation and optical spectroscopy

Reaction centers from two species of purple bacteria, Rhodospirillum rubrum and Rhodospirillum centenum, have been characterized and compared to reaction centers from Rhodobacter sphaeroides and Rhodobacter capsulatus. The reaction centers purified from these four species can be divided into two classes according to the spectral characteristics of the primary donor. Reaction centers from one class have a donor optical band at a longer wavelength, 865 nm compared to 850 nm, and an optical absorption band associated with the oxidized donor at 1250 nm that has a larger oscillator strength than reaction centers from the second class. Under normal buffering conditions, reaction centers isolated from Rb. sphaeroides and Rs. rubrum exhibit characteristics of the first class while those from Rb. capsulatus and Rs. centenum exhibit characteristics of the second class. However, the reaction centers can be converted between the two groups by the addition of charged detergents. Thus, the observed spectral differences are not due to intrinsic differences between reaction centers but represent changes in the electronic structure of the donor due to interactions with the detergents as has been confirmed by recent ENDOR measurements (Rautter J, Lendzian F, Lubitz W, Wang S and Allen JP (1994) Biochemistry 33: 12077–12084). The oxidation midpoint potential for the donor has values of 445 mV, 475 mV, 480 mV and 495 mV for Rs. rubrum, Rs. centenum, Rb. capsulatus, and Rb. sphaeroides, respectively. Despite this range of values for the midpoint potential, the decay rates of the stimulated emission are all fast with values of 4.1 ps, 4.5 ps. 5.5 ps and 6.1 ps for quinone-reduced RCs from Rs. rubrum, Rb. capsulatus, Rs. centenum, and Rb. sphaeroides, respectively. The general spectral features of the initial charge separated state are essentially the same for the four species, except for differences in the wavelengths of the absorption changes due to the different donor band positions. The pH dependence of the charge recombination rates from the primary and secondary quinones differ for reaction centers from the four species indicating different interactions between the quinones and ionizable residues. A different mechanism for charge recombination from the secondary quinone, that probably is direct recombination, is proposed for RCs from Rs. centenum.Abbreviations RC reaction center - P bacteriochlorophyll dimer - H bacteriopheophytin - Q quinone - Rb Rhodobacter - Rs Rhodospirillum - Rps Rhodopseudomonas - EDTA (ethylenediamine)tetraaceticacid - LDAO N,N-dimethyl-dodecylamine-N-oxide - CTAB cetyltrimethylammonium bromide - DOC deoxycholate - Tris Tris-(hydroxymethyl)aminomethane - ns nanosecond - ps picosecond - fs femtosecond     

Energetics for oxidation of a bound manganese cofactor in modified bacterial reaction centers

The energetics of a Mn cofactor bound to modified reaction centers were determined, including the oxidation/reduction midpoint potential and free energy differences for electron transfer. To determine these properties, a series of mutants of Rhodobacter sphaeroides were designed that have a metal-ion binding site that binds Mn2+ with a dissociation constant of 1 μM at pH 9.0 (Thielges et al. (2005) Biochemistry 44, 7389-7394). In addition to the Mn binding site, each mutant had changes near the bacteriochlorophyll dimer, P, that resulted in altered P/P+ oxidation/reduction midpoint potentials, which ranged from 480 mV to above 800 mV compared to 505 mV for wild type. The bound Mn2+ is redox active and after light excitation can rapidly reduce the oxidized primary electron donor, P+. The extent of P+ reduction was found to systematically range from a full reduction in the mutants with high P/P+ midpoint potentials to no reduction in the mutant with a potential comparable to wild type. This dependence of the extent of Mn2+ oxidation on the P/P+ midpoint potential can be understood using an equilibrium model and the Nernst equation, yielding a Mn2+/Mn3+ oxidation/reduction midpoint potential of 625 mV at pH 9. In the presence of bicarbonate, the Mn2+/Mn3+ potential was found to be 90 mV lower with a value of 535 mV suggesting that the bicarbonate serves as a ligand to the bound Mn. Measurement of the electron transfer rates yielded rate constants for Mn2+ oxidation ranging from 30 to 120 s(-1) as the P/P+ midpoint potentials increased from 670 mV to approximately 805 mV in the absence of bicarbonate. In the presence of bicarbonate, the rates increased for each mutant with values ranging from 65 to 165 s(-1), reflecting an increase in the free energy difference due to the lower Mn2+/Mn3+ midpoint potential. This dependence of the rate constant on the P/P+ midpoint potential can be understood using a Marcus relationship that yielded limits of at least 150 s(-1) and 290 meV for the maximal rate constant and reorganization energy, respectively. The implications of these results are discussed in terms of the energetics of proteins with redox active Mn cofactors, in particular, the Mn4Ca cofactor of photosystem II.     

光合作用氧释放机理研究进展

植物在光合作用过程中不仅为同化CO2提供能量和还原力,同时裂解水放出氧气。放氧反应主要由光系统Ⅱ(PSⅡ)氧化侧的4个锰原子组成的锰簇催化完成的。因此,锰簇在光合放氧过程中起看至关重要的作用。文章概述了对锰簇及其微环境的结构和功能的研究进展。