Uphill energy transfer in photosystem I from Chlamydomonas reinhardtii. Time-resolved fluorescence measurements at 77 K.

Energetic properties of chlorophylls in photosynthetic complexes are strongly modulated by their interaction with the protein matrix and by inter-pigment coupling. This spectral tuning is especially striking in photosystem I (PSI) complexes that contain low-energy chlorophylls emitting above 700 nm. Such low-energy chlorophylls have been observed in cyanobacterial PSI, algal and plant PSI-LHCI complexes, and individual light-harvesting complex I (LHCI) proteins. However, there has been no direct evidence of their presence in algal PSI core complexes lacking LHCI. In order to determine the lowest-energy states of chlorophylls and their dynamics in algal PSI antenna systems, we performed time-resolved fluorescence measurements at 77 K for PSI core and PSI-LHCI complexes isolated from the green alga Chlamydomonas reinhardtii. The pool of low-energy chlorophylls observed in PSI cores is generally smaller and less red-shifted than that observed in PSI-LHCI complexes. Excitation energy equilibration between bulk and low-energy chlorophylls in the PSI-LHCI complexes at 77 K leads to population of excited states that are less red-shifted (by ~ 12 nm) than at room temperature. On the other hand, analysis of the detection wavelength dependence of the effective trapping time of bulk excitations in the PSI core at 77 K provided evidence for an energy threshold at ~ 675 nm, above which trapping slows down. Based on these observations, we postulate that excitation energy transfer from bulk to low-energy chlorophylls and from bulk to reaction center chlorophylls are thermally activated uphill processes that likely occur via higher excitonic states of energy accepting chlorophylls.

Species-dependent alteration of electron transfer in the early stages of charge stabilization in Photosystem I.

Electron transfer (ET) in Photosystem I (PS I) is bidirectional, occurring in two pseudosymmetric branches of cofactors. The relative use of two branches in the green alga Chlamydomonas reinhardtii and the cyanobacterium Synechocystis sp. PCC 6803 has been studied by changing the Met axial ligands of the chlorophyll a acceptor molecules, A0A and A0B, to His. The nature of the effect on the ET is found to be species dependent. In C. reinhardtii, transient absorption and transient EPR data show that in the M688HPsaA variant, forward ET from A0A to the quinone, A1A, is blocked in 100% of the PS I complexes. In contrast, in Synechocystis sp. PCC 6803, forward ET from A0A to A1A is blocked in only 50% of the PS I complexes, but in those PS I complexes in which electrons reach A1A, further transfer to the iron-sulfur cluster FX is blocked. Similar species differences are found for the corresponding B-branch variants. One possible explanation of this behavior is that it is the result of two conformers in which an H-bond between the His side chain and the O1 carbonyl group of A1 is either present or absent. The spectroscopic data suggest that the two conformers are present in nearly equal amounts in the Synechocystis sp. PCC 6803 variants, while only the conformer without the H-bond is present in the same variants of C. reinhardtii.

Purification and photobiochemical profile of photosystem 1 from a high-salt tolerant, oleaginous Chlorella (Trebouxiophycaea, Chlorophyta).

The eukaryotic green alga Chlamydomonas reinhardtii has been studied extensively within the biofuel industry as a model organism, as researchers look towards algae to provide chemical feedstocks (i.e., lipids) for the production of liquid transportation fuels. C. reinhardtii, however, is unsuitable for high-level production of such precursors due to its relatively poor lipid accumulation and fresh-water demand. In this study we offer insight into the primary light harvesting and electron transfer reactions that occur during phototropic growth in a high-salt tolerant strain of Chlorella (a novel strain introduced here as NE1401), a single-celled eukaryotic algae also in the phylum Chlorophyta. Under nutrient starvation many eukaryotic algae increase dramatically the amount of lipids stored in lipid bodies within their cell interiors. Microscopy and lipid analyses indicate that Chlorella sp. NE1401 may become a superior candidate for algal biofuels production. We have purified highly active Photosystem 1 (PS1) complexes to study in vitro, so that we may understand further the photobiochemisty of this promising biofuel producer and how its characteristics compare and contrast with that of the better understood C. reinhardtii. Our findings suggest that the PS1 complex from Chlorella sp. NE1401 demonstrates similar characteristics to that of C. reinhardtii with respect to light-harvesting and electron transfer reactions. We also illustrate that the relative extent of the light state transition performed by Chlorella sp. NE1401 is smaller compared to C. reinhardtii, although they are triggered by the same dynamic light stresses.

Excitation dynamics in Photosystem I from Chlamydomonas reinhardtii. Comparative studies of isolated complexes and whole cells.

Identical time-resolved fluorescence measurements with ~3.5-ps resolution were performed for three types of PSI preparations from the green alga, Chlamydomonas reinhardtii: isolated PSI cores, isolated PSI-LHCI complexes and PSI-LHCI complexes in whole living cells. Fluorescence decay in these types of PSI preparations has been previously investigated but never under the same experimental conditions. As a result we present consistent picture of excitation dynamics in algal PSI. Temporal evolution of fluorescence spectra can be generally described by three decay components with similar lifetimes in all samples (6-8ps, 25-30ps, 166-314ps). In the PSI cores, the fluorescence decay is dominated by the two fastest components (~90%), which can be assigned to excitation energy trapping in the reaction center by reversible primary charge separation. Excitation dynamics in the PSI-LHCI preparations is more complex because of the energy transfer between the LHCI antenna system and the core. The average trapping time of excitations created in the well coupled LHCI antenna system is about 12-15ps longer than excitations formed in the PSI core antenna. Excitation dynamics in PSI-LHCI complexes in whole living cells is very similar to that observed in isolated complexes. Our data support the view that chlorophylls responsible for the long-wavelength emission are located mostly in LHCI. We also compared in detail our results with the literature data obtained for plant PSI.

Expression and characterization of cytochrome c553 from Heliobacterium modesticaldum.

Cytochrome c553 of Heliobacterium modesticaldum is the donor to P800 (+), the primary electron donor of the heliobacterial reaction center (HbRC). It is a membrane-anchored 14-kDa cytochrome that accomplishes electron transfer from the cytochrome bc complex to the HbRC. The petJ gene encoding cyt c 553 was cloned and expressed in Escherichia coli with a hexahistidine tag replacing the lipid attachment site to create a soluble donor that could be made in a preparative scale. The recombinant cytochrome had spectral characteristics typical of a c-type cytochrome, including an asymmetric α-band, and a slightly red-shifted Soret band when reduced. The EPR spectrum of the oxidized protein was characteristic of a low-spin cytochrome. The midpoint potential of the recombinant cytochrome was +217 ± 10 mV. The interaction between soluble recombinant cytochrome c 553 and the HbRC was also studied. Re-reduction of photooxidized P800 (+) was accelerated by addition of reduced cytochrome c 553. The kinetics were characteristic of a bimolecular reaction with a second order rate of 1.53 × 10(4) M(-1) s(-1) at room temperature. The rate manifested a steep temperature dependence, with a calculated activation energy of 91 kJ mol(-1), similar to that of the native protein in Heliobacillus gestii cells. These data demonstrate that the recombinant soluble cytochrome is comparable to the native protein, and likely lacks a discrete electrostatic binding site on the HbRC.

Introduction of a hydrogen bond between phylloquinone PhQ(A) and a threonine side-chain OH group in photosystem I.

The phylloquinone acceptor PhQ(A) in photosystem I binds to the protein through a single H-bond to the backbone nitrogen of PsaA-L722. Here, we investigate the effect of this H-bond on the electron transfer (ET) kinetics by substituting threonine for PsaA-L722. Room temperature spin-polarized transient EPR measurements show that in the PsaA-L722T mutant, the rate of PhQ(A)(-) to F(X) ET increases and the hyperfine coupling to the 2-methyl group of PhQ(A) is much larger than in the wild type. Molecular dynamics simulations and ONIOM type electronic structure calculations indicate that it is possible for the OH group of the Thr side chain to form an H-bond to the carbonyl oxygen atom, O(4) of the phylloquinone, and that this results in an increase in the 2-methyl hyperfine couplings as observed in the transient EPR data. The Arrhenius plot of the PhQ(A)(-) to F(X) ET in the PsaA-L722T mutant suggests that the increased rate is probably the result of a slight change in the electronic coupling between PhQ(A)(-) and F(X). The strong deviation from Arrhenius behavior observed at ∼200 K can be reproduced using a semiclassical model, which takes the zero-point energy of the mode coupled to the ET into account. However, since the change in slope of the Arrhenius plot occurs at the protein glass transition temperature, it is argued that it could be the result of a change in the protein relaxation dynamics at this temperature rather than quantum mechanical effects.

Double reduction of plastoquinone to plastoquinol in photosystem 1.

In Photosystem 1 (PS1), phylloquinone (PhQ) acts as a secondary electron acceptor from chlorophyll ec(3) and also as an electron donor to the iron-sulfur cluster F(X). PS1 possesses two virtually equivalent branches of electron transfer (ET) cofactors from P(700) to F(X), and the lifetime of the semiquinone intermediate displays biphasic kinetics, reflecting ET along the two different branches. PhQ in PS1 serves only as an intermediate in ET and is not normally fully reduced to the quinol form. This is in contrast to PS2, in which plastoquinone (PQ) is doubly reduced to plastoquinol (PQH(2)) as the terminal electron acceptor. We purified PS1 particles from the menD1 mutant of Chlamydomonas reinhardtii that cannot synthesize PhQ, resulting in replacement of PhQ by PQ in the quinone-binding pocket. The magnitude of the stable flash-induced P(700)(+) signal of menD1 PS1, but not wild-type PS1, decreased during a train of laser flashes, as it was replaced by a ~30 ns back-reaction from the preceding radical pair (P(700)(+)A(0)(-)). We show that this process of photoinactivation is due to double reduction of PQ in the menD1 PS1 and have characterized the process. It is accelerated at lower pH, consistent with a rate-limiting protonation step. Moreover, a point mutation (PsaA-L722T) in the PhQ(A) site that accelerates ET to F(X) ~2-fold, likely by weakening the sole H-bond to PhQ(A), also accelerates the photoinactivation process. The addition of exogenous PhQ can restore activity to photoinactivated PS1 and confer resistance to further photoinactivation. This process also occurs with PS1 purified from the menB PhQ biosynthesis mutant of Synechocystis PCC 6803, demonstrating that it is a general phenomenon in both prokaryotic and eukaryotic PS1.

Physiologically focused triage criteria improve utilization of pediatric surgeon-directed trauma teams and reduce costs.

INTRODUCTION: Pediatric surgeon-directed trauma teams (STTs) provide lifesaving treatment but at a high cost. We used physiologically based criteria to improve STT utilization. METHODS: We reviewed 152 consecutive STT activations at one center, comparing standard and physiologically focused criteria and 24-hour hospital costs/charges for overtriaged patients vs level 2 (emergency department managed) blunt trauma patients matched for age, Injury Severity Score (ISS), and necessity for operation. RESULTS: Our cohort (73.0% male; 86.8% blunt; median age, 8.0 [interquartile range, 4.0-14.0] years) had 10 deaths (6.6%) and 18 (11.8%) emergent operations. Twenty-nine patients met neither standard nor physiologic criteria (group 1), 25 met standard but not physiologic criteria (overtriaged, group 2), and 98 met physiologic criteria (group 3). Group 3 had higher median ISS (19.0 [10.0-33.0] vs 10.0 [4.0-17.0] and 5.5 [5.0-16.75] for groups 1 and 2, P = .001), more intensive care unit admissions (67.2% vs 31.0% and 52.0%, P = .001), longer hospitalization (5.0 [3.0-9.25] days vs 3.0 [1.0-5.0] and 4.0 [2.0-5.0] days, P = .002), and all patients who died or required emergent operation (P < .001). Physiologic criteria maintained 100% sensitivity but improved specificity (49.2% vs 23.0%). Overtriaged patients (n = 18) had 78.2% higher charges ($4700; 95% confidence interval, 13.3%-180.1%; P = .013) and 53.4% higher costs ($800; 95% confidence interval, 1.8%-131.2%; P = .041) than level 2 patients (n = 259) after adjusting for age, ISS, and need for operation, largely because of computed tomography and emergency department charges (66% of overtriaged charges). CONCLUSIONS: Physiologic STT activation criteria would have saved 25 activations, $20,000 in costs, and $120,000 in charges annually without compromising patient safety.

A synthetic protein selected for ligand binding affinity mediates ATP hydrolysis.

How primitive enzymes emerged from a primordial pool remains a fundamental unanswered question with important practical implications in synthetic biology. Here we show that a de novo evolved ATP binding protein, selected solely on the basis of its ability to bind ATP, mediates the regiospecific hydrolysis of ATP to ADP when crystallized with 1 equiv of ATP. Structural insights into this reaction were obtained by growing protein crystals under saturating ATP conditions. The resulting crystal structure refined to 1.8 A resolution reveals that this man-made protein binds ATP in an unusual bent conformation that is metal-independent and held in place by a key bridging water molecule. Removal of this interaction using a null mutant results in a variant that binds ATP in a normal linear geometry and is incapable of ATP hydrolysis. Biochemical analysis, including high-resolution mass spectrometry performed on dissolved protein crystals, confirms that the reaction is accelerated in the crystalline environment. This observation suggests that proteins with weak chemical reactivity can emerge from high affinity ligand binding sites and that constrained ligand-binding geometries could have helped to facilitate the emergence of early protein enzymes.

Functional heterogeneity of photosystem II in domain specific regions of the thylakoid membrane of spinach (Spinacia oleracea L.).

A mild sonication and phase fractionation method has been used to isolate five regions of the thylakoid membrane in order to characterize the functional lateral heterogeneity of photosynthetic reaction centers and light harvesting complexes. Low-temperature fluorescence and absorbance spectra, absorbance cross-section measurements, and picosecond time-resolved fluorescence decay kinetics were used to determine the relative amounts of photosystem II (PSII) and photosystem I (PSI), to determine the relative PSII antenna size, and to characterize the excited-state dynamics of PSI and PSII in each fraction. Marked progressive increases in the proportion of PSI complexes were observed in the following sequence: grana core (BS), whole grana (B3), margins (MA), stroma lamellae (T3), and purified stromal fraction (Y100). PSII antenna size was drastically reduced in the margins of the grana stack and stroma lamellae fractions as compared to the grana. Picosecond time-resolved fluorescence decay kinetics of PSII were characterized by three exponential decay components in the grana fractions, and were found to have only two decay components with slower lifetimes in the stroma. Results are discussed in the framework of existing models of chloroplast thylakoid membrane lateral heterogeneity and the PSII repair cycle. Kinetic modeling of the PSII fluorescence decay kinetics revealed that PSII populations in the stroma and grana margin fractions possess much slower primary charge separation rates and decreased photosynthetic efficiency when compared to PSII populations in the grana stack.

Regulation of the distribution of chlorophyll and phycobilin-absorbed excitation energy in cyanobacteria. A structure-based model for the light state transition.

The light state transition regulates the distribution of absorbed excitation energy between the two photosystems (PSs) of photosynthesis under varying environmental conditions and/or metabolic demands. In cyanobacteria, there is evidence for the redistribution of energy absorbed by both chlorophyll (Chl) and by phycobilin pigments, and proposed mechanisms differ in the relative involvement of the two pigment types. We assayed changes in the distribution of excitation energy with 77K fluorescence emission spectroscopy determined for excitation of Chl and phycobilin pigments, in both wild-type and state transition-impaired mutant strains of Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803. Action spectra for the redistribution of both Chl and phycobilin pigments were very similar in both wild-type cyanobacteria. Both state transition-impaired mutants showed no redistribution of phycobilin-absorbed excitation energy, but retained changes in Chl-absorbed excitation. Action spectra for the Chl-absorbed changes in excitation in the two mutants were similar to each other and to those observed in the two wild types. Our data show that the redistribution of excitation energy absorbed by Chl is independent of the redistribution of excitation energy absorbed by phycobilin pigments and that both changes are triggered by the same environmental light conditions. We present a model for the state transition in cyanobacteria based on the x-ray structures of PSII, PSI, and allophycocyanin consistent with these results.