绿藻[FeFe]氢化酶

该文介绍了绿藻[FeFe]氢化酶的研究现状,包括酶的结构、催化中心、金属簇的性质,以及对氧的敏感性和可能的解决办法。并且对已报道的绿藻[FeFe]氢化酶基因及其调控等问题作了介绍。     

Isolation and first EPR characterization of the [FeFe]-hydrogenases from green algae

Hydrogenase expression in Chlamydomonas reinhardtii can be artificially induced by anaerobic adaptation or is naturally established under sulphur deprivation. In comparison to anaerobic adaptation, sulphur-deprived algal cultures show considerably higher expression rates of the [FeFe]-hydrogenase (HydA1) and develop a 25-fold higher in vitro hydrogenase activity. Based on this efficient induction principle we have established a novel purification protocol for the isolation of HydA1 that can also be used for other green algae. From an eight liter C. reinhardtii culture 0.52 mg HydA1 with a specific activity of 741 micromol H2 min(-1) mg(-1) was isolated. Similar amounts were also purified from Chlorococcum submarinum and Chlamydomonas moewusii. The extraordinarily large yields of protein allowed a spectroscopic characterization of the active site of these smallest [FeFe]-hydrogenases for the first time. An initial analysis by EPR spectroscopy shows characteristic axial EPR signals of the CO inhibited forms that are typical for the Hox-CO state of the active site from [FeFe]-hydrogenases. However, deviations in the g-tensor components have been observed that indicate distinct differences in the electronic structure between the various hydrogenases. At cryogenic temperatures, light-induced changes in the EPR spectra were observed and are interpreted as a photodissociation of the inhibiting CO ligand.     

异源表达【FeFe】氢酶的基因工程大肠杆菌的构建

摘要:【目的】探讨一种构建异源表达【FeFe】氢酶的重组大肠杆菌的新方法。【方法】通过同源重组,依次将来源于丙酮丁醇梭菌中促进【FeFe】氢酶成熟的3 个辅助基因hydE、hydF 和hydG 分别整合到大肠杆菌BW2513-10(缺失氢酶基因) 的丙酮酸甲酸脱氢酶(ybiW)、乳酸脱氢酶(ldh) 和乙醇脱氢酶(adhE) 编码基因位点上。在此基础上进一步将含有来源于丁酸梭菌的氢酶基因的表达载体转化上述重组菌,并对转化子的氢酶活性进行分析。【结果】PCR 和RT-PCR 的检测结果表明,3 个辅助基因都     

Complete activity profile of Clostridium acetobutylicum [FeFe]-hydrogenase and kinetic parameters for endogenous redox partners

In Clostridium acetobutylicum, [FeFe]-hydrogenase is involved in hydrogen production in vivo by transferring electrons from physiological electron donors, ferredoxin and flavodoxin, to protons. In this report, by modifications of the purification procedure, the specific activity of the enzyme has been improved and its complete catalytic profile in hydrogen evolution, hydrogen uptake, proton/deuterium exchange and para-H2/ortho-H2 conversion has been determined. The major ferredoxin expressed in the solvent-producing C. acetobutylicum cells was purified and identified as encoded by ORF CAC0303. Clostridium acetobutylicum recombinant holoflavodoxin CAC0587 was also purified. The kinetic parameters of C. acetobutylicum [FeFe]-hydrogenase for both physiological partners, ferredoxin CAC0303 and flavodoxin CAC0587, are reported for hydrogen uptake and hydrogen evolution activities.     

The active site of the [FeFe]-hydrogenase from Desulfovibrio desulfuricans. II. Redox properties, light sensitivity and CO-ligand exchange as observed by infrared spectroscopy

In [FeFe]-hydrogenases, the H cluster (hydrogen-activating cluster) contains a di-iron centre ([2Fe]_H subcluster, a (L)(CO)(CN)Fe(μ-RS₂)(μ-CO)Fe(CysS)(CO)(CN) group) covalently attached to a cubane iron-sulphur cluster ([4Fe-4S]_H subcluster). The Cys-thiol functions as the link between one iron (called Fe1) of the [2Fe]_H subcluster and one iron of the cubane subcluster. The other iron in the [2Fe]_H subcluster is called Fe2. The light sensitivity of the Desulfovibrio desulfuricans enzyme in a variety of states has been studied with infrared (IR) spectroscopy. The aerobic inactive enzyme (H_inact state) and the CO-inhibited active form (H_ox–CO state) were stable in light. Illumination of the H_ox state led to a kind of cannibalization; in some enzyme molecules the H cluster was destroyed and the released CO was captured by the H clusters in other molecules to form the light-stable H_ox–CO state. Illumination of active enzyme under ¹³CO resulted in the complete exchange of the two intrinsic COs bound to Fe2. At cryogenic temperatures, light induced the photodissociation of the extrinsic CO and the bridging CO of the enzyme in the H_ox–CO state. Electrochemical redox titrations showed that the enzyme in the H_inact state converts to the transition state (H_trans) in a reversible one-electron redox step (E _{m, pH 7}=–75 mV). IR spectra demonstrate that the added redox equivalent not only affects the [4Fe-4S]_H subcluster, but also the di-iron centre. Enzyme in the H_trans state reacts with extrinsic CO, which binds to Fe2. The H_trans state converts irreversibly into the H_ox state in a redox-dependent reaction most likely involving two electrons (E _{m, pH 7}=–261 mV). These electrons do not end up on any of the six Fe atoms of the H cluster; the possible destiny of the two redox equivalents is discussed. An additional reversible one-electron redox reaction leads to the H_red state (E _{m, pH 7}=–354 mV), where both Fe atoms of the [2Fe]_H subcluster have the same formal oxidation state. The possible oxidation states of Fe1 and Fe2 in the various enzyme states are discussed. Low redox potentials (below –500 mV) lead to destruction of the [2Fe]_H subcluster.     

FeFe]-Hydrogenase active site models with relatively low reduction potentials: Diiron dithiolate complexes containing rigid bridges

Three diiron dithiolate complexes containing rigid and conjugated bridges, [mu-SC(6)H(4)-2-(CO)S-mu]Fe(2)(CO)(6) (1), [2-mu-SC(5)H(3)N-3-(CO)S-mu]Fe(2)(CO)(6) (2), and the PPh(3)-monosubstituted complex [mu-SC(6)H(4)-2-(CO)S-mu]Fe(2)(CO)(5)(PPh(3)) (1-P), were prepared as biomimetic models for the [FeFe]-hydrogenase active site. The structures of complexes 1 and 2 were determined by single crystal X-ray analysis, which shows that each complex features a rigid coplanar dithiolate bridge with a 2-3 degrees deviation from the bisect plane of the molecule. The influence of the rigid bridge on the reduction potentials of complexes 1, 2 and 1-P was investigated by electrochemistry. The cyclic voltammograms of complexes 1 and 2 display large positive shifts for the primary reduction potentials, that is, 380-480mV in comparison to that of the pdt-bridged (pdt=propane-1,3-dithiolato) complex (mu-pdt)Fe(2)(CO)(6) and 160-260mV to that of the bdt-bridged (bdt=benzene-1,2-dithiolato) analogue (mu-bdt)Fe(2)(CO)(6).     

The active site of the [FeFe]-hydrogenase from Desulfovibrio desulfuricans. I. Light sensitivity and magnetic hyperfine interactions as observed by electron paramagnetic resonance

The hydrogen-activating cluster (H cluster) in [FeFe]-hydrogenases consists of two moieties. The [2Fe]_H subcluster is a (L)(CO)(CN)Fe(μ-RS₂)(μ-CO)Fe(CysS)(CO)(CN) centre. The Cys-bound Fe is called Fe1, the other iron Fe2. The Cys-thiol forms a bridge to a [4Fe–4S] cluster, the [4Fe–4S]_H subcluster. We report that electron paramagnetic resonance (EPR) spectra of the ⁵⁷Fe-enriched enzyme from Desulfovibrio desulfuricans in the H_ox–CO state are consistent with a magnetic hyperfine interaction of the unpaired spin with all six Fe atoms of the H cluster. In contrast to the inactive aerobic enzyme, the active enzyme is easily destroyed by light. The [2Fe]_H subcluster in some enzyme molecules loses CO by photolysis, whereupon other molecules firmly bind the released CO to form the H_ox–CO state giving rise to the so-called axial 2.06 EPR signal. Though not destroyed by light, the H_ox–CO state is affected by it. As demonstrated in the accompanying paper [49] two of the intrinsic COs, both bound to Fe2, can be exchanged by extrinsic ¹³CO during illumination at 2 °C. We found that only one of the three ¹³COs, the one at the extrinsic position, gives an EPR-detectable isotropic superhyperfine interaction of 0.6 mT. At 30 K both the inhibiting extrinsic CO bound to Fe2 and one more CO can be photolysed. EPR spectra of the photolysed products are consistent with a 3d ⁷ system of Fe with the formal oxidation state +1. The damaged enzyme shows a light-sensitive g=5 signal which is ascribed to an S=3/2 form of the [2Fe]_H subcluster. The light sensitivity of the enzyme explains the occurrence of the g=5 signal and the axial 2.06 signal in published EPR spectra of nearly all preparations studied thus far.     

Genetic diversity and amplification of different clostridial [FeFe] hydrogenases by group-specific degenerate primers

Aims: The aim of this study was to explore and characterize the genetic diversity of [FeFe] hydrogenases in a representative set of strains from Clostridium sp. and to reveal the existence of neither yet detected nor characterized [FeFe] hydrogenases in hydrogen‐producing strains. Methods and Results: The genomes of 57 Clostridium strains (34 different genotypic species), representing six phylogenetic clusters based on their 16S rRNA sequence analysis (cluster I, III, XIa, XIb, XIV and XVIII), were screened for different [FeFe] hydrogenases. Based on the obtained alignments, ten pairs of [FeFe] hydrogenase cluster‐specific degenerate primers were newly designed. Ten Clostridium strains were screened by PCRs to assess the specificity of the primers designed and to examine the genetic diversity of [FeFe] hydrogenases. Using this approach, a diversity of hydrogenase genes was discovered in several species previously shown to produce hydrogen in bioreactors: Clostridium sartagoforme, Clostridium felsineum, Clostridium roseum and Clostridium pasteurianum. Conclusions: The newly designed [FeFe] hydrogenase cluster‐specific primers, targeting the cluster‐conserved regions, allow for a direct amplification of a specific hydrogenase gene from the species of interest. Significance and Impact of the Study: Using this strategy for a screening of different Clostridium ssp. will provide new insights into the diversity of hydrogenase genes and should be a first step to study a complex hydrogen metabolism of this genus.     

Atypical effect of temperature tuning on the insertion of the catalytic iron−sulfur center in a recombinant [FeFe]‐hydrogenase

The expression of recombinant [FeFe]‐hydrogenases is an important step for the production of large amount of these enzymes for their exploitation in biotechnology and for the characterization of the protein‐metal cofactor interactions. The correct assembly of the organometallic catalytic site, named H‐cluster, requires a dedicated set of maturases that must be coexpressed in the microbial hosts or used for in vitro assembly of the active enzymes. In this work, the effect of the post‐induction temperature on the recombinant expression of CaHydA [FeFe]‐hydrogenase in E. coli is investigated. The results show a peculiar behavior: the enzyme expression is maximum at lower temperatures (20°C), while the specific activity of the purified CaHydA is higher at higher temperature (30°C), as a consequence of improved protein folding and active site incorporation.     

Properties of the [NiFe]-hydrogenase maturation protein HypD

A mutational screen of amino acid residues of hydrogenase maturation protein HypD from Escherichia coli disclosed that seven conserved cysteine residues located in three different motifs in HypD are essential. Evidence is presented for potential functions of these motifs in the maturation process.     

Extraction and physico-chemical characterization of a versatile biodegradable polysaccharide obtained from green algae

During the last years, considerable attention has been given to different marine organisms, like algae, as potential sources of valuable materials. The continuous demand for novel materials and technologies is high and research on the underexploited marine green algae, including its polysaccharidic part—ulvan, has increased accordingly. In this research work, a novel method for extraction of ulvan from green algae is proposed and demonstrated successfully. Different characterization techniques were employed to characterize the isolated algal polysaccharide, namely, on what concerns its thermal trace and crystallinity. Upon heating, ulvan behaves as a non-meltable polysaccharide that is thermally stable before degradation at 220 °C. Ulvan is semi-crystalline in nature and possesses high hygroscopic features, as revealed in this research work. Due to its properties, ulvan can be considered, pure or modified, as a versatile biodegradable polymer for different applications, including tissue engineering and regenerative medicine.     

Roles of the F-domain in [FeFe] hydrogenase

The role of accessory Fe-S clusters of the F-domain in the catalytic activity of M3-type [FeFe] hydrogenase and the contribution of each of the two Fe-S surface clusters in the intermolecular electron transfer from ferredoxin are both poorly understood. We designed, constructed, produced and spectroscopically, electrochemically and biochemically characterized three mutants of Clostridium acetobutylicum CaHydA hydrogenase with modified Fe-S clusters: two site-directed mutants, HydA_C100A and HydA_C48A missing the FS4C and the FS2 surface Fe-S clusters, respectively, and a HydA_ΔDA mutant that completely lacks the F-domain. Analysis of the mutant enzyme activities clearly demonstrated the importance of accessory clusters in retaining full enzyme activity at potentials around and higher than the equilibrium 2H⁺/H₂ potential but not at the lowest potentials, where all enzymes have a similar turnover rate. Moreover, our results, combined with molecular modelling approaches, indicated that the FS2 cluster is the main gate for electron transfer from reduced ferredoxin.     

Importance of the protein framework for catalytic activity of [FeFe]-hydrogenases

The active center (H-cluster) of [FeFe]-hydrogenases is embedded into a hydrophobic pocket within the protein. We analyzed several amino acids, located in the vicinity of this niche, by site-directed mutagenesis of the [FeFe]-hydrogenases from Clostridium pasteurianum (CpI) and Chlamydomonas reinhardtii (CrHydA1). These amino acids are highly conserved and predicted to be involved in H-cluster coordination. Characterization of two hydrogenase variants confirmed this hypothesis. The exchange of residues CrHydA1Met(415) and CrHydA1Lys(228) resulted in inactive proteins, which, according to EPR and FTIR analyses, contain no intact H-cluster. However, [FeFe]-hydrogenases in which CpIMet(353) (CrHydA1Met(223)) and CpICys(299) (CrHydA1Cys(169)) were exchanged to leucine and serine, respectively, showed a structurally intact H-cluster with catalytic activity either absent (CpIC299S) or strongly diminished (CpIM353L). In the case of CrHydA1C169S, the H-cluster was trapped in an inactive state exhibiting g values and vibrational frequencies that resembled the H(trans) state of DdH from Desulfovibrio desulfuricans. This cysteine residue, interacting with the bridge head nitrogen of the di(methyl)amine ligand, seems therefore to represent an essential contribution of the immediate protein environment to the reaction mechanism. Exchanging methionine CpIM(353) (CrHydA1M(223)) to leucine led to a strong decrease in turnover without affecting the K(m) value of the electron donor. We suggest that this methionine constitutes a "fine-tuning" element of hydrogenase activity.     

Tempo-spatial distribution and species diversity of green algae micro-propagules in the Yellow Sea during the large-scale green tide development

From 2008 to 2013, vast green tides mainly composed of Ulva prolifera consecutively invaded the coast of Qingdao (36°06′N, 120°25′E, PR China) in June and July. Previous studies have shown that the early green tides initially formed in the Porphyra yezoensis aquaculture area of the Subei Shoal, southern Yellow Sea. To date, multiple studies have demonstrated that green algae micro-propagules play an important role in the formation of green tides. In this study, we aimed to assess the temporal and spatial distribution of green algae micro-propagules in an extensive area of the Yellow Sea and to determine the species diversity of propagules during the development of the large-scale green tide. We found that the quantity of micro-propagules increased with the free-floating biomass from the initial generation to the development phase of the green tide in mid May. From late May to mid June, the micro-propagule density decreased sharply despite a continuous increase of the floating macroalgae biomass. In addition, our data indicate that the coastal area of the Subei Shoal has always been the distribution center of the micro-propagules, even prior to the large-scale green tide formation. Furthermore, diverse green algae species, including Ulva prolifera, Ulva linza, Ulva flexuosa, Ulva compressa, Ulva pertusa and Blidingia sp., were identified among the micro-propagules in the survey sea area. Finally, we determined that the distribution of U. prolifera micro-propagules is closely related to the floating algal mats and attached macroalgae on Porphyra aquaculture rafts.     

Maturation of [NiFe]-hydrogenases in Escherichia coli: the HypC cycle

Carbamoyl phosphate (CP) has been implicated as an educt for the synthesis of the CO and CN ligands of the metal centre of [NiFe]-hydrogenases in Escherichia coli, since CP synthetase mutants (carAB) are unable to generate active hydrogenases due to a block in enzyme maturation. Citrulline, when added to the growth medium in high concentrations, compensated for the phenotype of the mutants. It is now shown that overexpression of the argI gene lowered the effective concentration of citrulline, thus proving that the amino acid serves as a source for CP. The DeltaCarAB mutant accumulated a complex consisting of the hydrogenase maturation proteins HypC and HypD. This complex was resolved upon citrulline addition and followed-up by the appearance of a complex between HypC and the precursor of the large subunit of hydrogenase 3, preHycE. In the absence of the hycE gene, the HypC-HypD complex did not disappear upon addition of citrulline but developed into a form migrating slower in a non-denaturing polyacrylamide gel, providing strong evidence for the notion that the HypC-HypD complex is the intermediate in hydrogenase maturation where CP or its products are added to the iron atom of the metal centre. This step precedes nickel insertion, since extracts of carAB cells that had been cultivated in the absence of citrulline are unable to process preHycE after the addition of nickel. Complex formation between HypC and HypD, and between HypC and preHycE display dependence on identical primary structure elements of HypC. On the basis of the results, a cycle of HypC activity is proposed whose function is to transfer the iron atom that has been liganded at the HypC-HypD complex to the precursor of the large hydrogenase subunit.     

Diversity and geographic distribution of desmids and other coccoid green algae

Taxonomic diversity of desmids and other coccoid green algae is discussed in relation to different species concepts. For want of unambiguous criteria about species delimitation, no reliable estimations of global species richness can be given. Application of the biological species concept is seriously hampered by lack of sexual reproduction in many species. Molecular analyses demonstrated cases of close affiliation between morphologically highly different taxa and, contrary, examples of little relationship between morphologically similar taxa. Despite the fact that desmids and chlorococcal algae, because of their microbial nature, can be readily distributed, cosmopolitan species are relatively scarce. The geographic distribution of some well-recognizable morphospecies is discussed in detail. Of some species a recent extension of their area could be established, e.g., in the desmids Micrasterias americana and Euastrum germanicum, and in the chlorococcaleans Desmodesmus perforatus and Pediastrum simplex. Special Issue: Protist diversity and geographic distribution. Guest editor: W. Foissner.     

Synthesis, characterization and electrocatalysis of diiron propanediselenolate derivatives as the active site models of [FeFe]-hydrogenases

As an extension of our study on the H-cluster model compounds, a series of diiron propanediselenolate (PDS)-type models have been successfully synthesized. Reaction of diselenol HSe(CH₂)₃SeH with Fe₃(CO)₁₂ in THF (tetrahydrofuran) at reflux gave the parent model compound [μ-Se(CH₂)₃Se-μ]Fe₂(CO)₆ (1) in 48% yield. Further reaction of 1 with PPh₃ or PPh₂H in the presence of Me₃NO in MeCN at room temperature afforded the phosphine-monosubstituted model compounds [μ-Se(CH₂)₃Se-μ]Fe₂(CO)₅(L) (2, L = PPh₃; 3, L = PPh₂H) in 76% and 68% yields, respectively. Similarly, the N-heterocyclic carbene I_Mes-monosubstituted model compound [μ-Se(CH₂)₃Se-μ]Fe₂(CO)₅(I_Mes) (4) could be prepared in 46% yield by reaction of imidazolium salt I_Mes · HCl with n-BuLi followed by treatment of the resulting I_Mes ligand with 1 in THF at room temperature. Compounds 1-4 were fully characterized by elemental analysis and various spectroscopic methods. While the structures of 1-4 were further confirmed by X-ray crystallography, the comparative study of 1 and its analog [μ-S(CH₂)₃S-μ]Fe₂(CO)₆ demonstrates that 1 is a better catalyst for TsOH proton reduction to hydrogen under electrochemical conditions.     

Stability enhancement of an O2-tolerant NAD+-reducing [NiFe]-hydrogenase by a combination of immobilisation and chemical modification

The oxygen-tolerant, NAD⁺-reducing soluble hydrogenase (SH) from Ralstonia eutropha H16 is a promising catalyst for cofactor regeneration in enzyme-catalysed reduction processes. The technical use of the isolated enzyme, however, is limited by its relatively low stability under operational conditions such as agitation, elevated temperature or addition of co-solvents. The maximum half-life at a reaction temperature of 35 °C and pH 8.0 was only 5.3 h. In order to enhance the stability of the enzyme, it was immobilised onto the anionic resin Amberlite™ FPA54. At an immobilisation yield of 93.4% for adsorptive and 100% for covalent attachment, corresponding activities of 48.9 and 39.3%, respectively, were obtained. Covalent binding always yielded superior stabilisation. At elevated temperature and under agitation, stabilisation was further increased by modification of the covalently bound SH with methoxy-poly(ethylene) glycol (mPEG). A comparable effect was not achieved when SH modification was performed before immobilisation. In stationary aqueous solution, half-lives of up to 161 h at 25 °C and 32 h at 35 °C were obtained. In presence of the technically relevant co-solvents DMSO, DMF, 2-propanol and [EMIM][EtSO₄] half-lives of 14–29 h can now be achieved.     

Purification and biochemical characterization of a membrane-bound [NiFe]-hydrogenase from a hydrogen-oxidizing, lithotrophic bacterium, Hydrogenophaga sp. AH-24

Membrane-bound [NiFe]-hydrogenase from Hydrogenophaga sp. AH-24 was purified to homogeneity. The molecular weight was estimated as 100±10 kDa, consisting of two different subunits (62 and 37 kDa). The optimal pH values for H₂ oxidation and evolution were 8.0 and 4.0, respectively, and the activity ratio (H₂ oxidation/H₂ evolution) was 1.61 × 10² at pH 7.0. The optimal temperature was 75 °C. The enzyme was quite stable under air atmosphere (the half-life of activity was c . 48 h at 4 °C), which should be important to function in the aerobic habitat of the strain. The enzyme showed high thermal stability under anaerobic conditions, which retained full activity for over 5 h at 50 °C. The activity increased up to 2.5-fold during incubation at 50 °C under H₂. Using methylene blue as an electron acceptor, the kinetic constants of the purified membrane-bound homogenase (MBH) were V _max=336 U mg⁻¹, k _cat=560 s⁻¹, and k _cat/ K _m=2.24 × 10⁷ M⁻¹ s⁻¹. The MBH exhibited prominent electron paramagnetic resonance signals originating from [3Fe–4S]⁺ and [4Fe–4S]⁺ clusters. On the other hand, signals originating from Ni of the active center were very weak, as observed in other oxygen-stable hydrogenases from aerobic H₂-oxidizing bacteria. This is the first report of catalytic and biochemical characterization of the respiratory MBH from Hydrogenophaga .