Transformation of metals and metal ions by hydrogenases from phototrophic bacteria

The ability of hydrogenases isolated from Thiocapsa roseopersicina and Lamprobacter modestohalophilus to reduce metal ions and oxidize metals has been studied. Hydrogenases from both phototrophic bacteria oxidized metallic Fe, Cd, Zn and Ni into their ionic forms with simultaneous evolution of molecular hydrogen. The metal oxidation rate decreased in the series Zn>Fe>Cd>Ni and depended on the pH. The presence of methyl viologen in the reaction system accelerated this process. T. roseopersicina and L. modestohalophilus cells and their hydrogenases reduced Ni(II), Pt(IV), Pd(II) or Ru(III) to their metallic forms under H₂ atmosphere. These results suggest that metals or metal ions can serve as electron donors or acceptors for hydrogenases from phototrophic bacteria.     

Dual organism design cycle reveals small subunit substitutions that improve [NiFe] hydrogenase hydrogen evolution

Background

Photosynthetic microorganisms that directly channel solar energy to the production of molecular hydrogen are a potential future biofuel system. Building such a system requires installation of a hydrogenase in the photosynthetic organism that is both tolerant to oxygen and capable of hydrogen production. Toward this end, we have identified the [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii “Deep ecotype” that is able to be heterologously expressed in cyanobacteria and has tolerance to partial oxygen. The A. macleodii enzyme shares sequence similarity with the uptake hydrogenases that favor hydrogen uptake activity over hydrogen evolution. To improve hydrogen evolution from the A. macleodii hydrogenase, we examined the three Fe-S clusters found in the small subunit of many [NiFe] uptake hydrogenases that presumably act as a molecular wire to guide electrons to or from the active site of the enzyme. Studies by others altering the medial cluster of a Desulfovibrio fructosovorans hydrogenase from 3Fe-4S to 4Fe-4S resulted in two-fold improved hydrogen evolution activity.

Results

We adopted a strategy of screening for improved hydrogenase constructs using an Escherichia coli expression system before testing in slower growing cyanobacteria. From the A. macleodii enzyme, we created a mutation in the gene encoding the hydrogenase small subunit that in other systems is known to convert the 3Fe-4S medial cluster to 4Fe-4S. The medial cluster substitution did not improve the hydrogen evolution activity of our hydrogenase. However, modifying both the medial cluster and the ligation of the distal Fe-S cluster improved in vitro hydrogen evolution activity relative to the wild type hydrogenase by three- to four-fold. Other properties of the enzyme including thermostability and tolerance to partial oxygen did not appear to be affected by the substitutions.

Conclusions

Our results show that substitution of amino acids altering the ligation of Fe-S clusters in the A. macleodii [NiFe] uptake hydrogenase resulted in increased hydrogen evolution activity. This activity can be recapitulated in multiple host systems and with purified protein. These results validate the approach of using an E. coli-cyanobacteria shuttle system for enzyme expression and improvement.

     

Photoproduction of hydrogen from water in hydrogenase-containing algae.

Scenedesmus obliquus and Chlorella vulgaris cells had active hydrogenase after dark anaerobic adaptation. Illumination of these algae with visible light led to an initial production of small quantities of hydrogen gas which soon ceased owing to production of oxygen by photolysis of water. The presence of oxygen-absorbing systems in a separate chamber, not in contact with the algae, gave only a slight stimulation of hydrogen production. Addition of sodium dithionite directly to the algae led to an extensive light-dependent production of hydrogen. This stimulation was due to oxygen removal by dithionite and not to its serving as an electron donor. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea, an inhibitor of photosystem II, abolished all hydrogen photoproduction. Hydrogen evolution was not accompanied by CO₂ production and little difference was noted between autotrophically and heterotrophically grown cells. Hydrogen was not produced in a photosystem II mutant of Scenedesmus even in the presence of dithionite, establishing that water was the source of hydrogen via photosystems II and I. Hydrogen production was stimulated by the presence of glucose and glucose oxidase as an oxygen-absorbing system. Oxygen inhibited hydrogen photoproduction, even if oxygen was undetectable in the gas phase, if the algal solution did not contain an oxygen absorber. It was demonstrated that under these conditions hydrogenase was still active and the inability to produce hydrogen was probably due to oxidation of the coupling electron carrier.     

Oxygen tolerance of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha H16 is based on limited access of oxygen to the active site

Hydrogenases, abundant proteins in the microbial world, catalyze cleavage of H2 into protons and electrons or the evolution of H2 by proton reduction. Hydrogen metabolism predominantly occurs in anoxic environments mediated by hydrogenases, which are sensitive to inhibition by oxygen. Those microorganisms, which thrive in oxic habitats, contain hydrogenases that operate in the presence of oxygen. We have selected the H2-sensing regulatory [NiFe] hydrogenase of Ralstonia eutropha H16 to investigate the molecular background of its oxygen tolerance. Evidence is presented that the shape and size of the intramolecular hydrophobic cavities leading to the [NiFe] active site of the regulatory hydrogenase are crucial for oxygen insensitivity. Expansion of the putative gas channel by site-directed mutagenesis yielded mutant derivatives that are sensitive to inhibition by oxygen, presumably because the active site has become accessible for oxygen. The mutant proteins revealed characteristics typical of standard [NiFe] hydrogenases as described for Desulfovibrio gigas and Allochromatium vinosum. The data offer a new strategy how to engineer oxygen-tolerant hydrogenases for biotechnological application.     

Photobiological hydrogen production: Recent advances and state of the art

Photobiological hydrogen production has advanced significantly in recent years, and on the way to becoming a mature technology. A variety of photosynthetic and non-photosynthetic microorganisms, including unicellular green algae, cyanobacteria, anoxygenic photosynthetic bacteria, obligate anaerobic, and nitrogen-fixing bacteria are endowed with genes and proteins for H₂-production. Enzymes, mechanisms, and the underlying biochemistry may vary among these systems; however, they are all promising catalysts in hydrogen production. Integration of hydrogen production among these organisms and enzymatic systems is a recent concept and a rather interesting development in the field, as it may minimize feedstock utilization and lower the associated costs, while improving yields of hydrogen production. Photobioreactor development and genetic manipulation of the hydrogen-producing microorganisms is also outlined in this review, as these contribute to improvement in the yield of the respective processes.     

Engineering a non-native hydrogen production pathway into Escherichia coli via a cyanobacterial [NiFe] hydrogenase

Biotechnology is a promising approach for the generation of hydrogen, but is not yet commercially viable. Metabolic engineering is a potential solution, but has largely been limited to native pathway optimisation. To widen opportunities for use of non-native [NiFe] hydrogenases for improved hydrogen production, we introduced a cyanobacterial hydrogen production pathway and associated maturation factors into Escherichia coli. Hydrogen production is observed in vivo in a hydrogenase null host, demonstrating coupling to host electron transfer systems. Hydrogenase activity is also detected in vitro. Hydrogen output is increased when formate production is abolished, showing that the new pathway is distinct from the native formate dependent pathway and supporting the conclusion that it couples cellular NADH and NADPH pools to molecular hydrogen. This work demonstrates non-native hydrogen production in E. coli, showing the wide portability of [NiFe] hydrogenase pathways and the potential for metabolic engineering to improve hydrogen yields.     

Improved photobiological H2 production in engineered green algal cells

Oxygenic photosynthetic organisms use solar energy to split water (H2O) into protons (H+), electrons (e-), and oxygen. A select group of photosynthetic microorganisms, including the green alga Chlamydomonas reinhardtii, has evolved the additional ability to redirect the derived H+ and e- to drive hydrogen (H2) production via the chloroplast hydrogenases HydA1 and A2 (H2 ase). This process occurs under anaerobic conditions and provides a biological basis for solar-driven H2 production. However, its relatively poor yield is a major limitation for the economic viability of this process. To improve H2 production in Chlamydomonas, we have developed a new approach to increase H+ and e- supply to the hydrogenases. In a first step, mutants blocked in the state 1 transition were selected. These mutants are inhibited in cyclic e- transfer around photosystem I, eliminating possible competition for e- with H2ase. Selected strains were further screened for increased H2 production rates, leading to the isolation of Stm6. This strain has a modified respiratory metabolism, providing it with two additional important properties as follows: large starch reserves (i.e. enhanced substrate availability), and a low dissolved O2 concentration (40% of the wild type (WT)), resulting in reduced inhibition of H2ase activation. The H2 production rates of Stm6 were 5-13 times that of the control WT strain over a range of conditions (light intensity, culture time, +/- uncoupler). Typically, approximately 540 ml of H2 liter(-1) culture (up to 98% pure) were produced over a 10-14-day period at a maximal rate of 4 ml h(-1) (efficiency = approximately 5 times the WT). Stm6 therefore represents an important step toward the development of future solar-powered H2 production systems.     

Cyanobacterial hydrogen production

With the global attention and research now being focussed on looking for an alternative to fossil fuel, hydrogen is the hope of future. Cyanobacteria are highly promising microorganisms for biological photohydrogen production. The review highlights the advancement in the biology of cyanobacterial hydrogen production in recent years. It discusses the enzymes involved in hydrogen production, viz. hydrogenases and nitrogenases, various strategies developed by cyanobacteria to limit nitrogenase inactivation by atmospheric and photosynthetic O₂, different biochemical and physicochemical parameters influencing the commercial cyanobacterial hydrogen production and the methods opted by different researchers for eliminating them to obtain maximum and sustained hydrogen production. Integrating the existing knowledge, techniques and expertise available, much future improvement and progress can be made in the field. This revised version was published online in November 2006 with corrections to the Cover Date.     

Application of immobilized hydrogenase for the detritiation of water

Detritiation of contaminated water is an essential part of nuclear power production. Most promising methods used for this process are based on catalyzed hydrogen isotope exchange reactions. It is proposed herein to replace the platinum catalysts which are currently used in industry with immobilized hydrogenase. Whole bacterial cells of Alcaligenes eutrophus immobilized in calcium alginate or κ-carrageenan gels were found to be efficient catalysts of the reaction of hydrogen–tritium (H–T) exchange in both a batch tank reactor and in a column. The dependence of the reaction rate on the amount of immobilized cells in the system, and on the concentration of the cells in the matrix, indicate that enzymatic H–T exchange is not controlled by diffusion. Immobilized A. eutrophus cells are enzymatically active over a wide range of pH, with a broad maximum from pH 6.0 to 8.0, and are quite resistant to inhibitors of hydrogenases such as O₂ and CO. Upon increasing the temperature from 4 to 37°C, the rate of hydrogenase-catalyzed H–T exchange increases by a factor of 5. From the standpoint of catalytic efficiency, 1 g of PtO₂ is approximately equivalent to 10 g of cells (wet weight). In contrast of platinum-based catalysts, bacterial hydrogenases (1) are potentially inexpensive; (2) can be readily available in bulk quantities; (3) are maximally active in liquid water.     

Hydrogenase: Properties and applications

Hydrogenase is an enzyme which reversibly activates molecular hydrogen and has potential applications in the production of hydrogen by solar energy. This review describes methods employed for assay of the enzyme, the biological role of hydrogenase in normal cellular metabolism and growth, and the properties of the enzyme. Although hydrogenases isolated from different organisms differ in molecular weight, subunit composition, iron and sulphur content, thermal and oxygen stability, they are all iron-sulphur proteins which cleave hydrogen heterolytically to form a hydride and a proton. The review also describes various systems being developed for the biophotolysis of water to produce hydrogen, and the role of hydrogenase in these systems.     

Hydrogenesis in hyperthermophilic microorganisms: implications for biofuels

Hydrothermal microbiotopes are characterized by the consumption and production of molecular hydrogen. Heterotrophic hyperthermophilic microorganisms (growth T(opt)> or =80 degrees C) actively participate in the production of H(2) in these environments through the fermentation of peptides and carbohydrates. Hyperthermophiles have been shown to approach the theoretical (Thauer) limit of 4 mol of H(2) produced per mole of glucose equivalent consumed, albeit at lower volumetric productivities than observed for mesophilic bacteria, especially enterics and clostridia. Potential advantages for biohydrogen production at elevated temperatures include fewer metabolic byproducts formed, absence of catabolic repression for growth on heterogeneous biomass substrates, and reduced loss of H(2) through conversion to H(2)S and CH(4) by mesophilic consortia containing sulfate reducers and methanogens. To fully exploit the use of these novel microorganisms and their constituent hydrogenases for biohydrogen production, development of versatile genetic systems and improvements in current understanding of electron flux from fermentable substrates to H(2) in hyperthermophiles are needed.     

Application of hyperthermophiles and their enzymes

Enzymes from hyperthermophiles display extreme (thermo)stability and a wide range of enzymes have been examined to explore their potential for various biotechnological processes. In addition, recent years have witnessed the development of genetic systems in a number of hyperthermophilic archaea. This has provided the means to initiate cell engineering studies in these organisms. Biofuel production is now an important topic in microbial biotechnology, and the hydrogen producing capabilities of (hyper)thermophiles, as well as their thermostable hydrogenases, are now attracting much attention.     

Hydrogenase-independent uptake and metabolism of electrons by the archaeon Methanococcus maripaludis

Direct, shuttle-free uptake of extracellular, cathode-derived electrons has been postulated as a novel mechanism of electron metabolism in some prokaryotes that may also be involved in syntrophic electron transport between two microorganisms. Experimental proof for direct uptake of cathodic electrons has been mostly indirect and has been based on the absence of detectable concentrations of molecular hydrogen. However, hydrogen can be formed as a transient intermediate abiotically at low cathodic potentials (<−414 mV) under conditions of electromethanogenesis. Here we provide genetic evidence for hydrogen-independent uptake of extracellular electrons. Methane formation from cathodic electrons was observed in a wild-type strain of the methanogenic archaeon Methanococcus maripaludis as well as in a hydrogenase-deletion mutant lacking all catabolic hydrogenases, indicating the presence of a hydrogenase-independent mechanism of electron catabolism. In addition, we discovered a new route for hydrogen or formate production from cathodic electrons: Upon chemical inhibition of methanogenesis with 2-bromo-ethane sulfonate, hydrogen or formate accumulated in the bioelectrochemical cells instead of methane. These results have implications for our understanding on the diversity of microbial electron uptake and metabolism.     

Development of an in vitro compartmentalization screen for high-throughput directed evolution of [FeFe] hydrogenases

Background

[FeFe] hydrogenase enzymes catalyze the formation and dissociation of molecular hydrogen with the help of a complex prosthetic group composed of common elements. The development of energy conversion technologies based on these renewable catalysts has been hindered by their extreme oxygen sensitivity. Attempts to improve the enzymes by directed evolution have failed for want of a screening platform capable of throughputs high enough to adequately sample heavily mutated DNA libraries. In vitro compartmentalization (IVC) is a powerful method capable of screening for multiple-turnover enzymatic activity at very high throughputs. Recent advances have allowed [FeFe] hydrogenases to be expressed and activated in the cell-free protein synthesis reactions on which IVC is based; however, IVC is a demanding technique with which many enzymes have proven incompatible.

Methodology/Principal Findings

Here we describe an extremely high-throughput IVC screen for oxygen-tolerant [FeFe] hydrogenases. We demonstrate that the [FeFe] hydrogenase CpI can be expressed and activated within emulsion droplets, and identify a fluorogenic substrate that links activity after oxygen exposure to the generation of a fluorescent signal. We present a screening protocol in which attachment of mutant genes and the proteins they encode to the surfaces of microbeads is followed by three separate emulsion steps for amplification, expression, and evaluation of hydrogenase mutants. We show that beads displaying active hydrogenase can be isolated by fluorescence-activated cell-sorting, and we use the method to enrich such beads from a mock library.

Conclusions/Significance

[FeFe] hydrogenases are the most complex enzymes to be produced by cell-free protein synthesis, and the most challenging targets to which IVC has yet been applied. The technique described here is an enabling step towards the development of biocatalysts for a biological hydrogen economy.     

Effect of redox potential on activity of hydrogenase 1 and hydrogenase 2 in Escherichia coli

This report elucidates the distinctions of redox properties between two uptake hydrogenases in Escherichia coli. Hydrogen uptake in the presence of mediators with different redox potential was studied in cell-free extracts of E. coli mutants HDK103 and HDK203 synthesizing hydrogenase 2 or hydrogenase 1, respectively. Both hydrogenases mediated H(2) uptake in the presence of high-potential acceptors (ferricyanide and phenazine methosulfate). H(2) uptake in the presence of low-potential acceptors (methyl and benzyl viologen) was mediated mainly by hydrogenase 2. To explore the dependence of hydrogen consumption on redox potential of media in cell-free extracts, a chamber with hydrogen and redox ( E(h)) electrodes was used. The mutants HDK103 and HDK203 exhibited significant distinctions in their redox behavior. During the redox titration, maximal hydrogenase 2 activity was observed at the E(h) below -80 mV. Hydrogenase 1 had maximum activity in the E(h) range from +30 mV to +110 mV. Unlike hydrogenase 2, the activated hydrogenase 1 retained activity after a fast shift of redox potential up to +500 mV by ferricyanide titration and was more tolerant to O(2). Thus, two hydrogenases in E. coli are complementary in their redox properties, hydrogenase 1 functioning at higher redox potentials and/or at higher O(2) concentrations than hydrogenase 2.     

Ruthenium (II) thiacrown complexes as hydrogen-transfer reduction catalysts

An investigation into the potential of a series of Ruthenium (II) thiacrown complexes with general formula [Ru([9]aneS₃)Cl₂(L)] and [Ru([n]aneS₄)Cl(L)]⁺, where L = DMSO or Ph₃P, n = 12, 14, or 16, as hydrogen transfer reduction catalysts is reported. As part of these studies two new complexes incorporating [Ru([12]aneS₄)] and [Ru([16]aneS₄)] metal centres have been synthesised and fully characterised. The X-ray structure of one of these complexes is also reported. The UV/Vis spectra of these complexes are dominated by π → π* transitions, with weaker d → d transitions also being apparent. Using these eight structurally related complexes, studies on the transfer hydrogenation of acetophenone using propan-2-ol as the hydride source were carried out. This work revealed that the complexes displayed catalytic activity in the presence of a base promoter. Although the activity of these complexes were considerably lower than that of structurally related mixed-donor ligand systems, there was some evidence that the flexibility of the ligand did have an effect on initial catalytic activity.     

The Three-Dimensional Structure of [NiFeSe] Hydrogenase from Desulfovibrio vulgaris Hildenborough: A Hydrogenase without a Bridging Ligand in the Active Site in Its Oxidised, “as-Isolated” State

Hydrogen is a good energy vector, and its production from renewable sources is a requirement for its widespread use. [NiFeSe] hydrogenases (Hases) are attractive candidates for the biological production of hydrogen because they are capable of high production rates even in the presence of moderate amounts of O₂, lessening the requirements for anaerobic conditions. The three-dimensional structure of the [NiFeSe] Hase from Desulfovibrio vulgaris Hildenborough has been determined in its oxidised “as-isolated” form at 2.04-Å resolution. Remarkably, this is the first structure of an oxidised Hase of the [NiFe] family that does not contain an oxide bridging ligand at the active site. Instead, an extra sulfur atom is observed binding Ni and Se, leading to a SeCys conformation that shields the NiFe site from contact with oxygen. This structure provides several insights that may explain the fast activation and O₂ tolerance of these enzymes.     

Photoinduced reduction of water by tin(IV) and ruthenium(II) porphyrins

In the paper are presented new photoredox systems for the reduction of water in which water- soluble Sn(IV) and Ru(II) porphyrins have been used as photosensitizers It has been found that during the photolysis of water Sn(IV) porphyrin underwent photoreduction whereas Ru(II) porphyrin underwent photooxidation. The successive photo- products of Sn(IV) porphyrin in the reaction from EDTA were, first, Sn(IV) chlorin and, second, Sn(IV) bacteriochlorin. In the experiments on the photo- generation of hydrogen, a correlation between the rates of hydrogen evolution and the reduction potentials of the electron carriers has been observed. The highest rate of hydrogen generation by means of Sn(IV) and Ru(II) porphyrins has been found for those electron carriers whose values of reduction potentials were tau; 0.55 and tau; 0.45 V. In the case of Ru(II) porphyrin, the rate of hydrogen evolution additionally depended on the molecular structure of the electron carrier. It has been found that during the water photolysis, viologens show a tendency to form their respective complexes with Ru(II) porphyrin, but only when they occur in a one-electron reduced form in the solution.     

Engineering model proteins for Photosystem II function

Our knowledge of Photosystem II and the molecular mechanism of oxygen production are rapidly advancing. The time is now ripe to exploit this knowledge and use it as a blueprint for the development of light-driven catalysts, ultimately for the splitting of water into O₂ and H₂. In this article, we outline the background and our approach to this technological application through the reverse engineering of Photosystem II into model proteins.