Differential electron flow around photosystem I by two C(4)-photosynthetic-cell-specific ferredoxins

In the C(4) plant maize (Zea mays L.), two ferredoxin isoproteins, Fd I and Fd II, are expressed specifically in mesophyll and bundle-sheath cells, respectively. cDNAs for these ferredoxins were introduced separately into the cyanobacterium Plectonema boryanum with a disrupted endogenous ferredoxin gene, yielding TM202 and KM2-9 strains expressing Fd I and Fd II. The growth of TM202 was retarded under high light (130 micromol/m(2)/s), whereas KM2-9 grew at a normal rate but exhibited a nitrogen-deficient phenotype. Measurement of photosynthetic O(2) evolution revealed that the reducing power was not efficiently partitioned into nitrogen assimilation in KM2-9. After starvation of the cells in darkness, the P700 oxidation level under far-red illumination increased significantly in TM202. However, it remained low in KM2-9, indicating an active cyclic electron flow. In accordance with this, the cellular ratio of ATP/ADP increased and that of NADPH/NADP(+) decreased in KM2-9 as compared with TM202. These results demonstrated that the two cell type-specific ferredoxins differentially modulate electron flow around photosystem I.     

Molecular structure of an apolipoprotein determined at 2.5-A resolution

The three-dimensional structure of an apolipoprotein isolated from the African migratory locust Locusta migratoria has been determined by X-ray analysis to a resolution of 2.5 A. The overall molecular architecture of this protein consists of five long alpha-helices connected by short loops. As predicted from amino acid sequence analyses, these helices are distinctly amphiphilic with the hydrophobic residues pointing in toward the interior of the protein and the hydrophilic side chains facing outward. The molecule falls into the general category of up-and-down alpha-helical bundles as previously observed, for example, in cytochrome c'. Although the structure shows the presence of five long amphiphilic alpha-helices, the alpha-helical moment and hydrophobicity of the entire molecule fall into the range found for normal globular proteins. Thus, in order for the amphiphilic helices to play a role in the binding of the protein to a lipid surface, there must be a structural reorganization of the protein which exposes the hydrophobic interior to the lipid surface. The three-dimensional motif of this apolipoprotein is compatible with a model in which the molecule binds to the lipid surface via a relatively nonpolar end and then spreads on the surface in such a way as to cause the hydrophobic side chains of the helices to come in contact with the lipid surface, the charged and polar residues to remain in contact with water, and the overall helical motif of the protein to be maintained.     

Structure of actinidin: details of the polypeptide chain conformation and active site from an electron density map at 2-8 A resolution

A 2·8 Å resolution electron density map of the sulphydryl protease, actinidin, has been calculated. Two isomorphous heavy-atom derivatives, prepared with uranyl acetate and dichloroethylenediamineplatinum(II), were used to calculate phases by the method of isomorphous replacement, giving an overall figure of merit of 0·81. The polypeptide chain is well-defined in the present map and many side-chains can be identified from their appearance. The molecule consists of a single chain of 220 residues, the last two of which appear disordered in the map and contains at least two, and probably three, disulphide bridges. The conformation of the polypeptide chain is remarkably similar to that of papain. It is folded into two domains, domain I consisting of residues 19–115 and 214–218, and domain II residues 1–18 and 116–213. There are three significant stretches of α-helix, involving residues 25–42, 69–81 and 120–129, together with several shorter pieces, while much of domain II consists of a twisted β-sheet structure. When compared with papain, actinidin has two additional residues inserted between 59 and 60, one inserted between 78 and 79, and four between 168 and 169 (papain numbering) while one residue (194) has been deleted from the papain structure. All these changes are in external parts of the molecule and have little effect on the conformation. The positions and orientations of the catalytically-important side-chains in the active site are virtually identical with those in papain, but some of the side-chains lining the non-polar binding pocket are clearly different.     

Electrochemical and spectro-kinetic evidence for an intermediate electron acceptor in photosystem I

Absorption changes accompanying light-induced P-700 formation and its decay in the dark at 15 K in Photosystem-I particles poised at various redox potentials have been examined. In unpoised samples, the light-induced absorption change is practically irreversible. At increasingly negative potentials, an increasing fraction of the absorption change, proportional to the fraction of bound iron-sulfur protein chemically reduced, becomes reversible, and the titration curve has a midpoint potential of --530 mV (vs. normal hydrogen electrode). At --66 mV, the P-700 absorption change is 97% reversible. The total P-700-signal amplitude decreases over the same potential span and levels off at about 43% (to slightly over 50% at a substantially higher excitation intensity). These results provide additional support to previous suggestions of an existence of an intermediate electron acceptor located between the primary donor, P-700, and the more stable primary electron acceptor (P-430 or bound iron-sulfur protein).     

Charge recombination in photosystem I at low temperatures kinetics of electron tunneling

Absorption changes accompanying light-induced P-700 oxidation and the decay of P-700+ in the dark were measured in the temperature range 294-5 K over a broad time scale (three to four orders of magnitude). Two qualitatively different types of kinetics for the dark decay of P-700⁺ were observed. In the 294-240K region, a usual exponential kinetics is observed with the rate constant κ = 1 · 10¹⁰ · exp(-16 000/RT) s^?1, with R in cal/mol per degree. Below 220 K, a rather unusual logarithmic or near-logarithmic kinetics are observed. These kinetics can be explained quantitatively if one assumes for the various (P-700+ ··· X^-) pairs a broad rectangular or near-rectangular distribution over the values of the rate constant. The following kinetic equation corresponding to this model was obtained: n_t/n_o = [In(κ_max/κ_min)]-1 - [In(1/κ_min)? In t] where no and nt are respectively the initial concentration of P-700⁺ and its concentration at time t, and kmax and kmin the maximum and minimum values of the rate constant, respectively. The decay processes observed can be ascribed to electron tunneling. Distribution over the values of k can be accounted for by different environments or different mutual orientations of P-700⁺ and X^?, or by different distances between them in the various reacting pairs.The corresponding distribution function was reconstructed from the experimentally measured P-700+-decay curves. The rate of tunneling was found to be temperature dependent. In the 160-80-K region, the temperature dependence corresponds to an activation energy of 2.9 kcal/mol. Below 80 K, new modes of P-700+ decay with lower activation energy become operative. The tunneling distance for the majority of the (P-700+ ··· X^?) pairs was estimated from the EPR linewidth of P-700+ to exceed 13.2 A.     

The difficulty of estimating the electron transport rate at photosystem I

It is still a controversial issue how the electron transport reaction is carried out around photosystem I (PSI) in the photosynthetic electron transport chain. The measurable component in PSI is the oxidized P700, the reaction center chlorophyll in PSI, as the absorbance changes at 820–830 nm. Previously, the quantum yield at PSI [Y(I)] has been estimated as the existence probability of the photo-oxidizable P700 by applying the saturated-pulse illumination (SP; 10,000–20,000 µmol photons m^?2 s^?1). The electron transport rate (ETR) at PSI has been estimated from the Y(I) value, which was larger than the reaction rate at PSII, evaluated as the quantum yield of PSII, especially under stress-conditions such as CO₂-limited and high light intensity conditions. Therefore, it has been considered that the extra electron flow at PSI was enhanced at the stress condition and played an important role in dealing with the excessive light energy. However, some pieces of evidence were reported that the excessive electron flow at PSI would be ignorable from other aspects. In the present research, we confirmed that the Y(I) value estimated by the SP method could be easily misestimated by the limitation of the electron donation to PSI. Moreover, we estimated the quantitative turnover rate of P700⁺ by the light-to-dark transition. However, the turnover rate of P700 was much slower than the ETR at PSII. It is still hard to quantitatively estimate the ETR at PSI by the current techniques.

     

The three-dimensional structure of bovine platelet factor 4 at 3.0-A resolution

Platelet factor 4 (PF4), which is released by platelets during coagulation, binds very tightly to negatively charged oligosaccharides such as heparin. To date, six other proteins are known that are homologous in sequence with PF4 but have quite different functions. The structure of a tetramer of bovine PF4 complexed with one Ni(CN)4(2-) molecule has been determined at 3.0 A resolution and refined to an R factor of 0.28. The current model contains residues 24-85, no solvent, and one overall temperature factor. Residues 1-13, which carried an oligosaccharide chain, were removed with elastase to induce crystallization; residues 14-23 and presumably 86-88 are disordered in the electron density map. Because no heavy atom derivative was isomorphous with the native crystals, the complex of PF4 with one Ni(CN)4(2-) molecule was solved using a single, highly isomorphous Pt(CN)4(2-) derivative and the iterative, single isomorphous replacement method. The secondary structure of the PF4 subunit, from amino- to carboxyl-terminal end, consists of an extended loop, three strands of antiparallel beta-sheet arranged in a Greek key, and one alpha-helix. The tetramer contains two extended, six-stranded beta-sheets, each formed by two subunits, which are arranged back-to-back to form a "beta-bilayer" structure with two buried salt bridges sandwiched in the middle. The carboxyl-terminal alpha-helices, which contain lysine residues that are thought to be intimately involved in binding heparin, are arranged as antiparallel pairs on the surface of each extended beta-sheet.     

Tertiary structure of oxidized flavodoxin from an eukaryotic red alga Chondrus crispus at 2.35-A resolution. Localization of charged residues and implication for interaction with electron transfer partners

The crystal structure of the oxidized form of a flavodoxin from an eukaryotic red alga, Chondrus crispus, has been determined by multiple isomorphous replacement and anomalous scattering methods. A model of the 173 residues and flavin mononucleotide (FMN) has been refined by a restrained least squares method to a crystallographic R-factor of 22.6% using 6236 reflections between 6.0 and 2.35 A with F greater than 3 sigma F. This molecule has a sheet consisting of five parallel beta-strands with two alpha-helices on one side of the sheet and three on the other side, and has a (beta alpha)5 structure. The molecule incorporates a substantial insertion in beta 5, as in Anacystis nidulans flavodoxin, which distinguishes these flavodoxins from the short-chain type. The isoalloxazine ring of FMN is sandwiched between the side chains of Trp-56 and Tyr-98, with its C-7 and C-8 methyl groups being exposed to solvent. The phosphate group of FMN is located at the N-terminal end of alpha 1, and forms extensive hydrogen bonds with the loop (T8-T13) between beta 1 and alpha 1 of the protein. Six of the total 11 lysine residues are clustered at the opposing face to the FMN-binding site, while about two-thirds of the total 35 acidic residues are located in the half of the molecule which includes the FMN-binding site. Such localization of charged residues produces a dipole within the molecule, which may be important in its recognition of the other proteins participating in electron transfer reactions.     

Projection map of tubulin in zinc-induced sheets at 4 A resolution.

Tubulin polymerizes into two-dimensional, crystalline sheets in the presence of zinc ions. These sheets are well suited to structural studies by electron crystallography. We have developed conditions for forming sheets which are large and well ordered enough to provide both electron diffraction and image data to better than 4 A resolution. In projection maps calculated from this data, the alpha and beta monomers can be identified within the protofilaments. These results indicate that we should be able to determine the structure of tubulin in these sheets at atomic resolution.     

Light harvesting in photosystem I: modeling based on the 2.5-A structure of photosystem I from Synechococcus elongatus

The structure of photosystem I from the thermophilic cyanobacterium Synechococcus elongatus has been recently resolved by x-ray crystallography to 2.5-A resolution. Besides the reaction center, photosystem I consists also of a core antenna containing 90 chlorophyll and 22 carotenoid molecules. It is their function to harvest solar energy and to transfer this energy to the reaction center (RC) where the excitation energy is converted into a charge separated state. Methods of steady-state optical spectroscopy such as absorption, linear, and circular dichroism have been applied to obtain information on the spectral properties of the complex, whereas transient absorption and fluorescence studies reported in the literature provide information on the dynamics of the excitation energy transfer. On the basis of the structure, the spectral properties and the energy transfer kinetics are simultaneously modeled by application of excitonic coupling theory to reveal relationships between structure and function. A spectral assignment of the 96 chlorophylls is suggested that allows us to reproduce both optical spectra and transfer and emission spectra and lifetimes of the photosystem I complex from S. elongatus. The model calculation allowed to study the influence of the following parameters on the excited state dynamics: the orientation factor, the heterogeneous site energies, the modifications arising from excitonic coupling (redistribution of oscillator strength, energetic splitting, reorientation of transition dipoles), and presence or absence of the linker cluster chlorophylls between antenna and reaction center. For the F?rster radius and the intrinsic primary charge separation rate, the following values have been obtained: R(0) = 7.8 nm and k(CS) = 0.9 ps(-1). Variations of these parameters indicate that the excited state dynamics is neither pure trap limited, nor pure transfer (to-the-trap) limited but seems to be rather balanced.     

Spectroscopic resolution of the picosecond reduction kinetics of the secondary electron acceptor A1 in photosystem I

Forward electron transfer in photosystem I from Synechocystis sp. PCC 6803 has been studied in the picosecond time range with transient absorption spectroscopy in the blue and near-UV spectral regions. From the direct measurement, at 380-390 nm, of the reduction kinetics of the phylloquinone secondary acceptor A1 and from the absence of spectral evolution between 100 ps and 2 ns, we conclude that electron transfer, from the chlorophyll a primary acceptor A0, to A1 occurs directly and completely with a time constant of about 30 ps.     

Reversible inhibition and reactivation of electron transfer in photosystem I

Photosynthesis Research - In photosystem I (PSI) complexes at room temperature electron transfer from A1– to FX is an order of magnitude faster on the B-branch compared to the A-branch. One...     

Crystal and molecular structure of human plasminogen kringle 4 refined at 1.9-A resolution.

The crystal structure of human plasminogen kringle 4 (PGK4) has been solved by molecular replacement using the bovine prothrombin kringle 1 (PTK1) structure as a model and refined by restrained least-squares methods to an R factor of 14.2% at 1.9-A resolution. The K4 structure is similar to that of PTK1, and an insertion of one residue at position 59 of the latter has minimal effect on the protein folding. The PGK4 structure is highly stabilized by an internal hydrophobic core and an extensive hydrogen-bonding network. Features new to this kringle include a cis peptide bond at Pro30 and the presence of two alternate, perpendicular, and equally occupied orientations for the Cys75 side chain. The K4 lysine-binding site consists of a hydrophobic trough formed by the Trp62 and Trp72 indole rings, with anionic (Asp55/Asp57) and cationic (Lys35/Arg71) charge pairs at either end. With the adjacent Asp5 and Arg32 residues, these result in triply charged anionic and cationic clusters (pH of crystals at 6.0), which, in addition to the unusually high accessibility of the Trp72 side chain, serve as an obvious marker of the binding site on the K4 surface. A complex intermolecular interaction occurs between the binding sites of symmetry-related molecules involving a highly ordered sulfate anion of solvation in which the Arg32 side chain of a neighboring kringle occupies the binding site.     

EGTA, a calcium chelator, inhibits electron transport in photosystem II of spinach chloroplasts at two different sites

A fifteen minute incubation of spinach chloroplasts with the divalent Ca²⁺ chelator, EGTA, in concentrations 50–250 μM, inhibits electron transport through both photosystems. All photosystem II partial reactions, including indophenol, ferricyanide and the DCMU-insensitive silicomolybdate reduction are inhibited from 70–100%. The photosystem II donor reaction, diphenyl carbazide → indophenol, is also inhibited, indicating that the inhibition site comes after the Mn²⁺ site, and that the first Ca²⁺ effect noted (site II) is not on the water oxidation enzyme, as is commonly assumed, but between the Mn²⁺ site and plastoquinone A pool. The other photosystem II effect of EGTA (Ca²⁺ site I), occurs in the region between plastoquinone A and P700 in the electron transport chain of chloroplasts. About 50% inhibition of the reaction ascorbate + TMPD → methyl viologen is given by incubation with 200 μM EGTA for 15 min. Ca²⁺ site II activity can be restored with 20 mM CaCl₂. Ca²⁺ site I responds to Ca²⁺ and plastocyanin added jointly. More than 90% activity in the ascorbate + TMPD → methylviologen reaction can be restored. Various ways in which Ca²⁺ ions could affect chloroplast structure and function are discussed. Since EGTA is more likely to penetrate chloroplast membranes than EDTA, which is known to remove CF₁, the coupling factor, from chloroplast membranes, and since Mg²⁺ ions are ineffective in restoring activity, it is concluded that Ca²⁺ may function in the electron transport chain of chloroplasts in a hitherto unsuspected manner.     

Localization of polylysine inhibition in a photosystem I subchloroplast particle.

Polylysine was found to increase the efficiency of electron donation from plastocyanin to P700⁺ in highly resolved Photosystem I subchloroplast particles. This increase in efficiency is due to a decrease in the Km for plastocyanin in the presence of polylysine and is similar to results obtained with divalent cations. Cytochrome f photooxidation is observed in the presence of plastocyanin and divalent cations but not in the presence of plastocyanin and polylysine. The results indicate that the binding of polylysine to plastocyanin prevents the reduction of plastocyanin by cytochrome f.     

Contributions of the protein environment to the midpoint potentials of the A1 phylloquinones and the Fx iron-sulfur cluster in photosystem I

Electrostatic calculations have predicted that the partial negative charge associated with D575PsaB plays a significant role in modulating the midpoint potentials of the A1A and A1B phylloquinones in photosystem I. To test this prediction, the side chain of residue 575PsaB was changed from negatively charged (D) to neutral (A) and to positively charged (K). D566PsaB, which is located at a considerable distance from either A1A or A1B, and should affect primarily the midpoint potential of FX, was similarly changed. In the 575PsaB variants, the rate of electron transfer from A1A to FX is observed to decrease slightly according to the sequence D/A/K. In the 566PsaB variants, the opposite effect of a slight increase in the rate is observed according to the same sequence D/A/K. These results are consistent with the expectation that changing these residues will shift the midpoint potentials of nearby cofactors to more positive values and that the magnitude of this shift will depend on the distance between the cofactors and the residues being changed. Thus, the midpoint potentials of A1A and A1B should experience a larger shift than will FX in the 575PsaB variants, while FX should experience a larger shift than will either A1A or A1B in the 566PsaB variants. As a result, the driving energy for electron transfer from A1A and A1B to FX will be decreased in the former and increased in the latter. This rationalization of the changes in kinetics is compared with the results of electrostatic calculations. While the altered amino acids shift the midpoint potentials of A1A, A1B, and FX by different amounts, the difference in the shifts between A1A and FX or between A1B and FX is small so that the overall effect on the electron transfer rate between A1A and FX or between A1B and FX is predicted to be small. These conclusions are borne out by experiment.