Modelling of the electron transfer reactions in Photosystem I by electron tunnelling theory: The phylloquinones bound to the PsaA and the PsaB reaction centre subunits of PS I are almost isoenergetic to the iron-sulfur cluster FX

Photosystem I is a large macromolecular complex located in the thylakoid membranes of chloroplasts and in cyanobacteria that catalyses the light driven reduction of ferredoxin and oxidation of plastocyanin. Due to the very negative redox potential of the primary electron transfer cofactors accepting electrons, direct estimation by redox titration of the energetics of the system is hampered. However, the rates of electron transfer reactions are related to the thermodynamic properties of the system. Hence, several spectroscopic and biochemical techniques have been employed, in combination with the classical Marcus theory for electron transfer tunnelling, in order to access these parameters. Nevertheless, the values which have been presented are very variable. In particular, for the case of the tightly bound phylloquinone molecule A₁, the values of the redox potentials reported in the literature vary over a range of about 350 mV. Previous models of Photosystem I have assumed a unidirectional electron transfer model. In the present study, experimental evidence obtained by means of time resolved absorption, photovoltage, and electron paramagnetic resonance measurements are reviewed and analysed in terms of a bi-directional kinetic model for electron transfer reactions. This model takes into consideration the thermodynamic equilibrium between the iron-sulfur centre F_X and the phylloquinone bound to either the PsaA (A_1A) or the PsaB (A_1B) subunit of the reaction centre and the equilibrium between the iron-sulfur centres F_A and F_B. The experimentally determined decay lifetimes in the range of sub-picosecond to the microsecond time domains can be satisfactorily simulated, taking into consideration the edge-to-edge distances between redox cofactors and driving forces reported in the literature. The only exception to this general behaviour is the case of phylloquinone (A₁) reoxidation. In order to describe the reported rates of the biphasic decay, of about 20 and 200 ns, associated with this electron transfer step, the redox potentials of the quinones are estimated to be almost isoenergetic with that of the iron sulfur centre F_X. A driving force in the range of 5 to 15 meV is estimated for these reactions, being slightly exergonic in the case of the A_1B quinone and slightly endergonic, in the case of the A_1A quinone. The simulation presented in this analysis not only describes the kinetic data obtained for the wild type samples at room temperature and is consistent with estimates of activation energy by the analysis of temperature dependence, but can also explain the effect of the mutations around the PsaB quinone binding pocket. A model of the overall energetics of the system is derived, which suggests that the only substantially irreversible electron transfer reactions are the reoxidation of A₀ on both electron transfer branches and the reduction of F_A by F_X.     

Spectral and kinetic characterization of electron acceptor A1 in a Photosystem I core devoid of iron-sulfur centers FX,FB and FA

The kinetic and spectroscopic properties of the secondary electron acceptor A₁ were determined by flash absorption spectroscopy at room and cryogenic temperatures in a Photosystem I (PS I) core devoid of the iron-sulfur clusters F_X, F_B and F_A. It was shown earlier (Warren, P.V., Golbeck, J.H. and Warden, J.T. (1993) Biochemistry 32: 849–857) that the majority of the flash-induced absorbance increase at 820 nm, reflecting formation of P700⁺, decays with a t_1/2 of 10 s due to charge recombination between P700⁺ and A₁ ^–. Following A₁ ^– directly around 380 nm, where absorbance changes due to the formation of P700⁺ are negligible, two major decay components were resolved in this study with t_1/2 of 10 s and 110 s at an amplitude ratio of 2.5:1. The difference spectra between 340 and 490 nm of the two kinetic phases are highly similar, showing absorbance increases from 340 to 400 nm characteristic of the one-electron reduction of the phylloquinone A₁. When measured at 10 K, the flash-induced absorbance changes around 380 nm can be fitted with two decay phases of t_1/2 15 s and 150 s at an amplitude ratio 1:1. The difference spectra of both kinetic phases from 340 to 400 nm are similar to those determined at 298 K and are therefore attributed to charge recombination in the pair P700⁺A₁ ^–. These results indicate that the backreaction between P700⁺ and A₁ ^– is multiphasic when F_X, F_B and F_A are removed, and only slightly temperature dependent in the range of 298 K to 10 K.Abbreviations Chl chlorophyll - D pathlength for the measuring light through the sample - DPIP 2,6-dichlorophenolindophenol - EPR electron paramagnetic resonance - IR infrared - PS I Photosystem I - Tris Tris(hydroxymethyl)aminomethane - UV ultraviolet Published as Journal Series #10890 of the University of Nebraska Agricultural Research Division and supported by a grant from the National Science Foundation (MCB-9205756).     

Metabolic regulation of citrate and iron by aconitases: role of iron–sulfur cluster biogenesis

Iron and citrate are essential for the metabolism of most organisms, and regulation of iron and citrate biology at both the cellular and systemic levels is critical for normal physiology and survival. Mitochondrial and cytosolic aconitases catalyze the interconversion of citrate and isocitrate, and aconitase activities are affected by iron levels, oxidative stress and by the status of the Fe–S cluster biogenesis apparatus. Assembly and disassembly of Fe–S clusters is a key process not only in regulating the enzymatic activity of mitochondrial aconitase in the citric acid cycle, but also in controlling the iron sensing and RNA binding activities of cytosolic aconitase (also known as iron regulatory protein IRP1). This review discusses the central role of aconitases in intermediary metabolism and explores how iron homeostasis and Fe–S cluster biogenesis regulate the Fe–S cluster switch and modulate intracellular citrate flux.     

Reconstitution of iron-sulfur center FB results in complete restoration of NADP+ photoreduction in Hg-treated Photosystem I complexes from Synechococcus sp. PCC 6301

The F_B iron-sulfur cluster is destroyed preferentially by treating Photosystem I complexes with HgCl₂(Kojima Y, Niinomi Y, Tsuboi S, Hiyama T and Sakurai H (1987) Bot Mag 100: 243–53). When F_B is 95% depleted but F_Ais quantitatively retained in cyanobacterial PS I complexes, the reduction potential of F_A remains highly electronegative (E_m=–530 mV, n=1), the EPR spectral and spin relaxation properties of F_A and F_Xremain unchanged, but NADP⁺ photoreduction rates decline from 552 to 72 mol mg Chl^–1 h^–1.When F_B is reconstituted with FeCl₃, Na₂S and -mercaptoethanol, NADP⁺photoreduction rates recover to 528 mol mg Chl^–1 h^–1. The correlation between the presence of F_Band NADP⁺ photoreduction provides direct experimental evidence that this iron-sulfur cluster is required for electron throughput from cytochromec ₆ to flavodoxin or ferredoxin in Photosystem I.Abbreviations Chl chlorophyll - DPIP dichlorophenolindophenol - PS I Photosystem I Published as Journal Series #11091 of the University of Nebraska Agricultural Research Division. This paper is dedicated to the memory of the late Professor Daniel Arnon, who is remembered for his gracious and generous encouragement of the senior author's early career.     

GABA-mediated effects of some taurine derivatives injected i.c.v. on rabbit rectal temperature and gross motor behavior

Summary. Some synthetic taurine analogues, namely ethanolamine-O-sulphate (EOS), N,N-dimethyltaurine (DMT), N,N,N-trimethyltaurine (TMT) and 2-aminoethylphosphonic acid (AEP) were shown to interact with rabbit brain GABA_A- or GABA_B-receptors, while (±)piperidine-3-sulfonic acid (PSA) inhibited the activity of rabbit brain 4-aminobutyrate transaminase. This suggests that they behave like direct/indirect GABA agonists or GABA antagonists and affect thermoregulation and gross motor behaviour (GMB) which are under GABA control. In the present study micromole (1.2–48) amounts of these compounds were i.c.v. injected in conscious, restrained rabbits while monitoring rectal temperature (RT), ear skin temperature (EST) and GMB. AEP, EOS, DMT and TMT induced a dose-related hyperthermia, ear vasoconstriction and excitation of GMB, while PSA induced a dose-related hypothermia, ear vasodilation and inhibition of GMB. EOS antagonized in a dose-related fashion hypothermia induced by 60 nmol THIP, a GABA_A agonist, while AEP, DMT and TMT counteracted that induced by 8 nmol R(-)Baclofen, a GABA_B agonist. In conclusion, EOS and AEP, DMT, TMT seem to act as GABA_A and GABA_B antagonists, respectively, while PSA behaves like an indirect GABA agonist, all affecting the central mechanisms which drive rabbit thermoregulation.     

Stage- and tissue-specific expression of rice OsIsu1 gene encoding a scaffold protein for mitochondrial iron–sulfur-cluster biogenesis

Isu is a scaffold protein involved in mitochondrial iron–sulfur-cluster biogenesis, which affects redox and iron homeostasis in human and yeast cells. A BLASTP search identified two putative Isu genes in rice, and we designated one of them as OsIsu1. When expressed in onion epidermal cells, OsIsu1::GFP was localized to the mitochondria. Northern analysis showed that OsIsu1 was down-regulated in iron-deficient rice root. OsIsu1 promoter-GUS was introduced into Arabidopsis thaliana and histochemical GUS-staining showed that OsIsu1 expression was regulated in a stage- and tissue-specific manner. OsIsu1 was expressed ectopically in Arabidopsis under the control of the CaMV35S promoter, which increased weight of plants. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Iron–sulfur cluster biosynthesis: characterization of IscU–IscS complex formation and a structural model for sulfide delivery to the [2Fe–2S] assembly site

Recent work on the bacterial iron–sulfur cluster (isc) family of gene products, and eukaryotic homologs, has advanced the molecular understanding of cellular mechanisms of iron–sulfur cluster biosynthesis. Members of the IscS family are pyridoxyl-5′-phosophate dependent proteins that deliver inorganic sulfide during assembly of the [2Fe–2S] cluster on the IscU scaffold protein. Herein it is demonstrated through calorimetry, fluorescence, and protein stability measurements that Thermotoga maritima IscS forms a 1:1 complex with IscU in a concentration-dependent manner (K _D varying from 6 to 34 μM, over an IscS concentration range of approximately 2–50 μM). Docking simulations of representative IscU and IscS proteins reveal critical contact surfaces at the N-terminal helix of IscU and a C-terminal loop comprising a chaperone binding domain. Consistent with the isothermal titration calorimetry results described here, an overall dominant contribution of charged surfaces with a change in the molar heat capacity of binding, ΔC _p ~ 199.8 kcal K⁻¹ mol⁻¹, is observed that accounts for approximately 10% of the total accessible surface area at the binding interface. Both apo and holo IscUs and homologs were found to bind to IscS in an enthalpically driven reaction with comparable K _D values. Both helix and loop regions are highly conserved among phylogenetically diverse organisms from a pool of archael, bacterial, fungal, and mammalian representatives.     

Structure of a [2Fe–2S] ferredoxin from Rhodobacter capsulatus likely involved in Fe–S cluster biogenesis and conformational changes observed upon reduction

FdVI from Rhodobacter capsulatus is structurally related to a group of [2Fe–2S] ferredoxins involved in iron–sulfur cluster biosynthesis. Comparative genomics suggested that FdVI and orthologs found in α-Proteobacteria are involved in this process. Here, the crystal structure of FdVI has been determined for both the oxidized and the reduced protein. The [2Fe–2S] cluster lies 6 Å below the protein surface in a hydrophobic pocket without access to the solvent. This particular cluster environment might explain why the FdVI midpoint redox potential (?306 mV at pH 8.0) did not show temperature or ionic strength dependence. Besides the four cysteines that bind the cluster, FdVI features an extra cysteine which is located close to the S1 atom of the cluster and is oriented in a position such that its thiol group points towards the solvent. Upon reduction, the general fold of the polypeptide chain was almost unchanged. The [2Fe–2S] cluster underwent a conformational change from a planar to a distorted lozenge. In the vicinity of the cluster, the side chain of Met24 was rotated by 180°, bringing its S atom within hydrogen-bonding distance of the S2 atom of the cluster. The reduced molecule also featured a higher content of bound water molecules, and more extensive hydrogen-bonding networks compared with the oxidized molecule. The unique conformational changes observed in FdVI upon reduction are discussed in the light of structural studies performed on related ferredoxins.     

Expression, purification and characterization of a high potential iron–sulfur protein from Acidithiobacillus ferrooxidans

The high potential iron–sulfur protein (HiPIP) is involved in the iron respiratory electron transport chain of Acidithiobacillus ferrooxidans but its exact role is unclear. The gene of HiPIP from A. ferrooxidans ATCC 23270 was cloned and expressed in Escherichia coli, and the protein then purified by one-step affinity chromatography to homogeneity. The molecular mass of the HiPIP monomer was 7250.43 Da by MALDI-TOF MS, indicating the presence of the [Fe₄S₄] cluster. The optical and EPR spectra results of the recombinant protein confirmed that the iron–sulfur cluster was correctly inserted into the active site of the protein. Site-directed mutagenesis results revealed that Cys25, Cys28, Cys37 and Cys50 were involved in ligating to the iron–sulfur cluster.     

Chemical rescue of a site-modified ligand to a [4Fe-4S] cluster in PsaC, a bacterial-like dicluster ferredoxin bound to Photosystem I

Chemical rescue of site-modified amino acids using externally supplied organic molecules represents a powerful method to investigate structure-function relationships in proteins. Here we provide definitive evidence that aryl and alkyl thiolates, reagents typically used for in vitro iron-sulfur cluster reconstitutions, serve as rescue ligands to a site-specifically modified [4Fe-4S]^1+,2+ cluster in PsaC, a bacterial dicluster ferredoxin-like subunit of Photosystem I. PsaC binds two low-potential [4Fe-4S]^1+,2+ clusters termed F_A and F_B. In the C13G/C33S variant of PsaC, glycine has replaced cysteine at position 13 creating a protein that is missing one of the ligating amino acids to iron-sulfur cluster F_B. Using a variety of analytical techniques, including non-heme iron and acid-labile sulfur assays, and EPR, resonance Raman, and Mössbauer spectroscopies, we showed that the C13G/C33S variant of PsaC binds two [4Fe-4S]^1+,2+ clusters, despite the absence of one of the biological ligands. ¹⁹F NMR spectroscopy indicated that the external thiolate replaces cysteine 13 as a substitute ligand to the F_B cluster. The finding that site-modified [4Fe-4S]^1+,2+ clusters can be chemically rescued with external thiolates opens new opportunities for modulating their properties in proteins. In particular, it provides a mechanism to attach an additional electron transfer cofactor to the protein via a bound, external ligand.     

Effects of ultraviolet radiation on photosynthesis and related enzyme reactions of marine macroalgae

 Changes in physiological parameters related to photosynthesis were studied in five macroalgal species from Spitsbergen (Monostroma arcticum, Laminaria solidungula, Alaria esculenta, Palmaria palmata, Phycodrys rubens) during a 72-h exposure to UV radiation. Maximal quantum yield of photochemistry (Fv/Fm) and maximal electron transport rate (ETRmax) were measured with a pulse-amplitude-modulated fluorometer; the activity of the Calvin cycle enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were estimated using a photometric test. Proteins of crude extracts were separated by SDS gel electrophoresis and changes in cellular concentrations of Rubisco were determined. Moreover, the concentration of chlorophyll a (Chl a), and protein content, were measured photometrically. In all species, Chl a content, maximal quantum yield as well as ETRmax decreased during the UV treatment. Changes in ETRmax were related to the changes in the overall activity of Rubisco. Analysis of SDS gels showed that in P. rubens, L. solidungula, M. arcticum and A. esculenta decreasing Rubisco activity partly resulted from a degradation of the enzyme. However, in A. esculenta, the formation of a high-molecular-weight polypeptide was observed. In all species, the activity of Rubisco was more strongly impaired than that of G3PDH. Exposure to UV resulted in loss of total protein only in the deepwater species L. solidungula and P. rubens. The different sensitivities to UV exposure of the species tested reflect their zonation pattern in the field. Received: 4 October 1999 / Accepted: 15 February 2000     

Mechanisms of iron–sulfur cluster assembly: the SUF machinery

Biosynthesis of iron-sulfur clusters is a cellular process which depends on complex protein machineries. Escherichia coli contains two such biosynthetic systems, ISC and SUF. In this review article we specifically make a presentation of the various Suf proteins and discuss the molecular mechanisms by which these proteins work together to assemble Fe and S atoms within a scaffold and to transfer the resulting cluster to target proteins. An erratum to this article can be found at     

Replacement of the methionine axial ligand to the primary electron acceptor A0 slows the A0 reoxidation dynamics in Photosystem I

The recent crystal structure of photosystem I (PSI) from Thermosynechococcus elongatus shows two nearly symmetric branches of electron transfer cofactors including the primary electron donor, P₇₀₀, and a sequence of electron acceptors, A, A₀ and A₁, bound to the PsaA and PsaB heterodimer. The central magnesium atoms of each of the putative primary electron acceptor chlorophylls, A₀, are unusually coordinated by the sulfur atom of methionine 688 of PsaA and 668 of PsaB, respectively. We [Ramesh et al. (2004a) Biochemistry 43:1369-1375] have shown that the replacement of either methionine with histidine in the PSI of the unicellular green alga Chlamydomonas reinhardtii resulted in accumulation of A₀⁻ (in 300-ps time scale), suggesting that both the PsaA and PsaB branches are active. This is in contrast to cyanobacterial PSI where studies with methionine-to-leucine mutants show that electron transfer occurs predominantly along the PsaA branch. In this contribution we report that the change of methionine to either leucine or serine leads to a similar accumulation of A₀⁻ on both the PsaA and the PsaB branch of PSI from C. reinhardtii, as we reported earlier for histidine mutants. More importantly, we further demonstrate that for all the mutants under study, accumulation of A₀⁻ is transient, and that reoxidation of A₀⁻ occurs within 1-2 ns, two orders of magnitude slower than in wild type PSI, most likely via slow electron transfer to A₁. This illustrates an indispensable role of methionine as an axial ligand to the primary acceptor A₀ in optimizing the rate of charge stabilization in PSI. A simple energetic model for this reaction is proposed. Our findings support the model of equivalent electron transfer along both cofactor branches in Photosystem I.     

A thermostable hybrid cluster protein from Pyrococcus furiosus: effects of the loss of a three helix bundle subdomain

Pyrococcus furiosus hybrid cluster protein (HCP) was expressed in Escherichia coli, purified, and characterized. This is the first archaeal and thermostable HCP to be isolated. Compared with the protein sequences of previously characterized HCPs from mesophiles, the protein sequence of P. furiosus HCP exhibits a deletion of approximately 13 kDa as a single amino acid stretch just after the N-terminal cysteine motif, characteristic for class-III HCPs from (hyper)thermophilic archaea and bacteria. The protein was expressed as a thermostable, soluble homodimeric protein. Hydroxylamine reductase activity of P. furiosus HCP showed a K _m value of 0.40 mM and a k _cat value of 3.8 s⁻¹ at 70 °C and pH 9.0. Electron paramagnetic resonance spectroscopy showed evidence for the presence of a spin-admixed, S = 3/2 [4Fe–4S]⁺ cubane cluster and of the hybrid cluster. The cubane cluster of P. furiosus HCP is presumably coordinated by a CXXC–X₇–C–X₅–C motif close to the N-terminus, which is similar to the CXXC–X₈–C–X₅–C motif of the Desulfovibrio desulfuricans and Desulfovibrio vulgaris HCPs. Amino acid sequence alignment and homology modeling of P. furiosus HCP reveal that the deletion results in a loss of one of the two three-helix bundles of domain 1. Clearly the loss of one of the three-helix bundles of domain 1 does not diminish the hydroxylamine reduction activity and the incorporation of the iron–sulfur clusters.     

The structure of genetically modified iron-sulfur cluster Fx in photosystem I as determined by X-ray absorption spectroscopy

Photosystem I (PS I) mediates light-induced electron transfer from P700 through a chlorophyll a, a quinone and a [4Fe-4S] iron-sulfur cluster F_X, located on the core subunits PsaA/B to iron-sulfur clusters F_A/B on subunit PsaC. Structure function relations in the native and in the mutant (psaB-C565S/D566E) of the cysteine ligand of F_X cluster were studied by X-ray absorption spectroscopy (EXAFS) and transient spectroscopy. The structure of F_X was determined in PS I lacking clusters F_A/B by interruption of the psaC2 gene of PS I in the cyanobacterium Synechocystis sp PCC 6803. PsaC-deficient mutant cells assembled the core subunits of PS I which mediated electron transfer mostly to the phylloquinone. EXAFS analysis of the iron resolved a [4Fe-4S] cluster in the native PsaC-deficient PS I. Each iron had 4 sulfur and 3 iron atoms in the first and second shells with average Fe-S and Fe-Fe distances of 2.27 Å and 2.69 Å, respectively. In the C565S/D566E serine mutant, one of the irons of the cluster was ligated to three oxygen atoms with Fe-O distance of 1.81 Å. The possibility that the structural changes induced an increase in the reorganization energy that consequently decreased the rate of electron transfer from the phylloquinone to F_X is discussed.     

Pyrococcus furiosus 4Fe-ferredoxin, chemisorbed on gold, exhibits gated reduction and ionic strength dependent dimerization

Pyrococcus furiosus ferredoxin is a small metalloprotein that shuttles electrons between redox enzymes. In its native 4Fe-4S form the protein is highly thermostable. In addition to three cluster-ligating cysteines, two surface cysteine residues (C21 and C48) are present. We used the reactivity of these surface thiols to directly immobilize ferredoxin on a bare gold electrode, with an orientation in which the cluster is exposed to solution. Voltammetry, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM) studies established the immobilization of the 4Fe form. Native and recombinant wild-type ferredoxins were compared with the C48S, C21S, and C21S/C48S mutants. The variants with one and two surface cysteines can be directly chemisorbed on bare gold. Cyclic voltammetry demonstrated that the reduction potentials are similar to those in solution. The interfacial electron transfer kinetics revealed that the reduction is gated by the interconversion between two oxidized species. AFM images showed that dimers are chemisorbed at low ionic strength, while monomers are present at high ionic strength. XPS spectra revealed the presence of S, Fe, C, N, and O at the surface, which are assigned to the corresponding atoms in the peptide and the cofactor. Analysis of the sulfur spectrum corroborates that both C21 and C48 form gold-thiolate bonds. Moreover, two inorganic sulfide and two iron species were identified, suggesting an inhomogeneous charge distribution in the 4Fe-4S cluster. In conclusion, P. furiosus ferredoxin can be directly and vectorially chemisorbed on gold with retention of its properties. This may provide a biocompatible electrode surface with docking sites for redox enzymes.     

Cluster N1 of complex I from Yarrowia lipolytica studied by pulsed EPR spectroscopy

After reduction with nicotinamide adenine dinucleotide (NADH), NADH:ubiquinone oxidoreductase (complex I) of the strictly aerobic yeast Yarrowia lipolytica shows clear signals from five different paramagnetic iron-sulfur (FeS) clusters (N1-N5) which can be detected using electron paramagnetic resonance (EPR) spectroscopy. The ligand environment and the assignment of several FeS clusters to specific binding motifs found in several subunits of the complex are still under debate. In order to characterize the hyperfine interaction of the surrounding nuclei with FeS cluster N1, one- and two-dimensional electron spin echo envelope modulation experiments were performed at a temperature of 30 K. At this temperature only cluster N1 contributes to the overall signal in a pulsed EPR experiment. The hyperfine and quadrupole tensors of a nitrogen nucleus and the isotropic and dipolar hyperfine couplings of two sets of protons could be determined by numerical simulation of the one- and two-dimensional spectra. The values obtained are in perfect agreement with a ferredoxin-like binding structure by four cysteine amino acid residues and allow the assignment of the nitrogen couplings to a backbone nitrogen nucleus and the proton couplings to the beta-protons of the bound cysteine residues.     

Impacts of chlorination and heat shocks on growth, pigments and photosynthesis of Phaeodactylum tricornutum (Bacillariophyceae)

The main impacts of cooling water from thermal (nuclear) power plants on aquatic organisms were caused by chlorination and temperature increase. In this study, we investigated the impacts of residual chlorine and short-term heat shocks on growth, pigment contents and photosynthesis of Phaeodactylum tricornutum. Growth of P. tricornutum was completely inhibited; Chlorophyll a and carotenoids contents deceased about 63.3% and 61.4% in 24 h treated with 0.2 mg L^− 1 chlorine. The negative effects of chlorination increased with enhanced concentration and prolonged exposure time. Relative electrode transfer rate (rETR) of P. tricornutum was significantly suppressed when treated with 0.2 mg L^− 1 residual chlorine for 24 h. Furthermore, the effective quantum yield (F_v'/F_m') decreased first but then recovered with prolonged exposure when residual chlorine ranged between 0.1 and 0.2 mg L^− 1. The cells were less sensitive to heat shocks compared with chlorination: the rETR and F_v'/F_m' was suppressed only when the temperature exceeded 35 °C for 1 h. When P. tricornutum was exposed to chlorination combined with heat shocks, the rETR was further inhibited at 35 °C. It indicated that both chlorination and heat shocks had negative impacts on the primary producers living in discharging coastal waters; furthermore, there were synergistic effects of heat shocks on chlorination toxicity.     

In vitro activation of [FeFe] hydrogenase: new insights into hydrogenase maturation

The in vitro activation of the [FeFe] hydrogenase is accomplished by combining Escherichia coli cell extracts containing the heterologously expressed inactive HydA with extracts in which hydrogenase-specific maturation proteins HydE, HydF, and HydG are expressed in concert. Interestingly, the process of HydA activation occurs rapidly and in the absence of potential substrates, which suggests that the hydrogenase accessory proteins synthesize an H-cluster precursor that can be quickly transferred to the hydrogenase enzyme to affect activation. HydA activity is observed to be dependent on the protein fraction containing all three accessory proteins expressed in concert and cannot be accomplished with addition of heat-treated extract or extract filtrate, suggesting that the activation of the hydrogenase structural protein is mediated by interaction with the accessory assembly protein(s). These results represent the first important step in understanding the process of H-cluster assembly and provide significant insights into hydrogenase maturation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.