Photosystem I (PSI) from plants, algae, and cyanobacteria can mediate H2 evolution in vivo and in vitro. A simple, self-platinization procedure that permits stable PSI-mediated H2 evolution in vitro has been developed. The H2 evolution capabilities of PSI from Thermosynechococcus elongatus have been characterized. This organism utilizes cytochrome c6 (cyt c6) as the e- donor to P700. Using a solution-based, self-organized platinization of the PSI nanoparticles, this study demonstrates a sodium ascorbate-cyt-PSI-Pt-H2 electron transport and proton reduction system that yields light-dependent H2. The system was thermostable with H2 evolution increasing up to 55°C. In addition, stability studies have shown the H2 evolution to be very stable, with no significant decrease over the 80 days investigated. Through simple optimization a H2 production rate of ~5.5 mol H2/h/mg Chl [micro-mole H2 per hour per milligram chlorophyll] was attained. To further optimize the H2 production Asc-cyt-PSI-Pt-H2 system, response surface methodology (RSM) was employed. The process parameter studied included temperature, light intensity and platinum salt concentration. The results showed that experimental data had a good fit to the proposed model (R2=0.99 and p < 0.001). Platinum salt concentration, temperature and the interaction between platinum salt concentration and temperature showed significant effects on the total H2 yield. Light intensity had minimal effect of the total H2 yield within the region studied. The optimum parameters for H2 photoproduction were light intensity of 240 μE/m2/s, [micro-eistien per square meter per second], platinum salt concentration of 636 μM [micro-mol/liter] and temperature of 310C. Finally, studies that will improve the H2 yield by increasing the kinetics of electron transfer were done. A hybrid protein was formed by engineering a gene to express a fusion of the membrane-bound [Ni-Fe] hydrogenase from Ralstonia eutropha H16 and the stromal-exposed subunits PsaE and PsaD of PSI from T. elongatus. A PsaE-free mutant of PSI was simultaneously formed by genetically disrupting the expression of the PsaE subunit of a native PSI; that will allow in vitro reconstitution of the desired PsaE-hydrogenase fusion protein with PsaE-free PSI.

Molecular hydrogen is a potential energy carrier for the future. Nitrogen-fixing cyanobacteria are a group of photosynthetic microorganisms with the inherent ability to produce molecular hydrogen via the enzyme complex nitrogenase. This hydrogen is not released, however, but is recaptured by the bacteria using an uptake hydrogenase. In this thesis, genes involved in cyanobacterial hydrogen metabolism were examined, and the possibility of employing genetically modified cyanobacteria for hydrogen production was investigated. Nostoc punctiforme PCC 73102 (ATCC 29133) is a nitrogen-fixing filamentous cyanobacterium containing an uptake hydrogenase encoded by hupSL. The transcription of hupSL was characterised, and putative regulatory elements in the region upstream of the transcription start site were identified. One of these, a binding motif for the global nitrogen regulator NtcA, was further investigated by mobility shift assays, and it was found that the motif is functional in binding NtcA. Also, a set of genes involved in maturation of hydrogenases was identified in N. punctiforme, the hypFCDEAB operon. These genes were found to be situated upstream of hupSL in the opposite direction, and they were preceded by a previously unknown open reading frame, that was found to be transcribed as part of the same operon. The potential for hydrogen production by filamentous cyanobacteria was investigated by studying mutant strains lacking an uptake hydrogenase. A mutant strain of N. punctiforme was constructed, where hupL was inactivated. It was found that cultures of this strain evolve hydrogen during nitrogen fixation. Gas exchange in the hupL- mutant and in wild type N. punctiforme was measured using a mass spectrometer, and conditions under which hydrogen production from the nitrogenase could be increased at the expense of nitrogen fixation were identified. Growth and hydrogen production in continuous cultures of a Hup- mutant of the related strain Nostoc PCC 7120 were also studied. This thesis advances the knowledge about cyanobacterial hydrogen metabolism and opens possibilities for further development of a process for hydrogen production using filamentous cyanobacteria.

Today, mankind faces a new challenge in energetic terms: a new Industrial Revolution is imperative, already called by some as an Energetic Revolution. This corresponds to a conversion to clean, environmentally friendly and renewable energy sources. In this context, hydrogen arises as a valid alternative, since its combustion produces a considerable amount of energy and releases solely water as a by-product. In the present thesis, two model cyanobacteria, namely Synechocystis sp. strain PCC 6803 and Anabaena/Nostoc sp. strain PCC 7120, were used to examine the hydrogen metabolism. The efforts were focused on to understand the transcription regulation of the hox genes, encoding the structural elements of the bidirectional hydrogenase enzyme. Here, it is shown that such regulation is operated in a very distinct and intricate way, with different factors contributing to its delicate tuning. While in Synechocystis sp. strain PCC 6803 the hox genes were shown to be transcribed as a single operon, in Anabaena/Nostoc sp. strain PCC 7120 they were shown to be transcribed as two independent operons (possibly three). Two transcription factors, LexA and AbrB-like protein, were identified and further characterized in relation to the hydrogen metabolism. Furthermore, different environmental conditions were demonstrated to operate changes on the transcription of the bidirectional hydrogenase genes. In addition, functional studies of three open reading frames found within the hox operon of Synechocystis sp. strain PCC 6803 suggest that this may be a stress responsive operon. However, based on the gained knowledge, it is still not possible to connect the signal transduction pathways, from the environmental signal, through the response regulator, to the final regulation of the hox genes. Nevertheless, the crucial importance of studying the transcription regulation of the different players involved in the hydrogen metabolism is now established and a new era seems to be rising.

[FeFe]-Hydrogenases (H2ases) are metalloenzymes that can catalyze the reversible reduction of protons to molecular hydrogen as part of the metabolism of certain cyanobacteria and green algae. Due to the low availability of the enzyme, synthetic complexes that mimic the natural active site in structure, function and activity are highly sought after. In this thesis, a number of [FeFe]-H2ases active site model complexes were synthesized to answer open questions of the active site and to develop unprecedented bio-inspired proton reduction catalysts. The first part describes the synthesis and the protonation properties of a [Fe2(μ-adt)(CO)4(PMe3)2] (adt = azadithiolate) complex which contains two basic sites that are similar to those found in the enzyme active site. Unusual kinetic factors give rise to four discrete protonation states. The twofold protonated state is the first model complex that simultaneously carries a proton at the azadithiolate nitrogen and a bridging hydride at the Fe-Fe bond. In the second part, a model complex with an unprecedented amine ligand was synthesized and studied. In analogy to the enzyme active site, the labile amine ligand is expelled after electrochemical reduction. The third part describes a series of model complexes with electronically different aromatic dithiolate ligands. It is demonstrated in one case that the tuning of the ligand by electron-withdrawing substituents results in proton reduction catalysis at an overpotential that is lower than that required by the non-substituted parent compound. The design and the synthetic work towards a new ruthenium-diiron dyad for light-driven hydrogen production are presented in the fourth part. In the final part, differently isotope-labelled mixed valent Fe(I)-Fe(II) model complexes were synthesized, in particular the unprecedented 15N labelled analogue, with the aim to provide EPR-spectroscopic references that will allow the elucidation of the nature of the central atom in the dithiolate bridge of the [FeFe] hydrogenase active site.

Biofuels that are renewable and environmentally benign constitute an important area of research, as the supply of fossil fuels decreases and the amount of green house gases in the atmosphere increases. Biohydrogen is not as well explored as other biofuels, but its properties render it a promising complement, as it is clean and can be used directly in fuel cells to generate electricity, the only waste products being water and heat. Hydrogenproducing microorganisms have the potential to be used to recycle industrial waste, such as carbohydrates from food manufacturing. Hence the cost of waste disposal could be reduced whilst biofuel is being produced through microbial processes. Escherichia coli is a well-known microorganism that produces hydrogen under fermentative conditions, through the conversion of formate to hydrogen gas and carbon dioxide, via an enzyme complex called formate hydrogenlyase (FHL). The complex is anchored to the inner cell membrane and consists of seven subunits: a formate dehydrogenase, a [Ni-Fe] hydrogenase, three electron carrier proteins, which together make up a large ‘hydrophilic domain’, and two integral membrane proteins (the ‘membrane domain’). Even though the entire bacterial genome is known, the FHL complex remains little understood and has proven difficult to isolate and characterise. During this project, a genetically modified strain producing only the hydrophilic domain of FHL was constructed, and the resultant sub-complex was purified. It was hoped that, if a stable and homogenous core complex could be isolated, it might be subjected to further analysis, such as elucidating the subunit stoichiometry and solving the structure. Furthermore, FHL is notoriously oxygen labile, which hampers its study and technological development. However, oxygen tolerance is a natural feature found in some other [Ni-Fe] hydrogenases, and recent research shows that this property is likely dependent on the presence of extra cysteine residues near an important metal cluster in the enzyme. These cysteines are not present in FHL and a complex that could be active in both aerobic and anaerobic conditions may be a useful tool in optimising microbial biohydrogen processes. Thus, three strains that each expressed a modified FHL variant carrying single Cysteine-for-Glycine substitutions were constructed. The modified FHL complexes proved to remain active in vivo, and can serve as the basis of genetically engineering oxygen tolerance into this important enzyme.

This dissertation describes computational and experimental studies of synthetic complexes that model the active site of the iron-iron hydrogenase [FeFe]H2ase enzyme. Simple dinuclear iron dithiolate complexes act as functional models of the ironiron hydrogenase enzyme by catalyzing isotopic exchange in D2/H2O mixtures. Density Functional Theory (DFT) calculations and new experiments have been performed that suggest reasonable mechanistic explanations for this reactivity. Evidence for the existence of an acetone derivative of the di-iron complex, as suggested by theory, is presented. Bis-phosphine substituted dinuclear iron dithiolate complexes react with the electrophilic species, H+ and Et+ (Et+ = CH3CH2 +) with differing regioselectivity; H+ reacts to form a 3c-2eâ Fe-H-Fe bond, while Et+ reacts to form a new C-S bond. The instability of a bridging ethyl complex is attributed to the inability of the ethyl group, in contrast to a hydride, to form a stable 3c-2eâ bond with the two iron centers. Gas-phase density functional theory calculations are used to predict the solutionphase infrared spectra for a series of CO and CN-containing dinuclear iron complexes dithiolate. It is shown that simple linear scaling of the computed C-O and C-N stretching frequencies yields accurate predictions of the experimentally determined ν(CO) and ν(CN) values. An N-heterocyclic carbene containing [FeFe]H2ase model complex, whose X-ray structure displays an apical carbene, is shown to undergo an unexpected simultaneous two-electron reduction. DFT shows, in addition to a one-electron Fe-Fe reduction, that the aryl-substituted N-heterocyclic carbene can accept a second electron more readily than the Fe-Fe manifold. The juxtaposition of these two one-electron reductions resembles the [FeFe]H2ase active site with an FeFe di-iron unit joined to the electroactive 4Fe4S cluster. Simple synthetic di-iron dithiolate complexes synthesized to date fail to reproduce the precise orientation of the diatomic ligands about the iron centers that is observed in the molecular structure of the reduced form of the enzyme active site. Herein, DFT computations are used for the rational design of synthetic complexes as accurate structural models of the reduced form of the enzyme active site.

The electronic structures and reaction mechanisms of transition-metal complexes can be calculated accurately by density functional theory (DFT) in cooperation with the continuum solvation model. The palladium catalyzed Heck reaction, iron-model complexes for cytochrome P450 and superoxide reductase (SOR), and tetrairon hexathiolate hydrogenase model were investigated. The DFT calculations on the catalytic Heck reaction (between phenyl-bromide and ethylene to form the styrene product), catalyzed by palladium diphosphine indicate a four-step mechanism: oxidative addition of C6H5Br, migratory insertion of C6H5 to C2H4, b-hydride transfer/olefin elimination of styrene product, and catalyst regeneration by removal of HBr. For the oxidative addition, the rate-determining step, the reaction through monophosphinopalladium complex is more favorable than that through either the diphosphinopalladium or ethylene-bound monophosphinopalladium. In further study, for a steric phosphine, PtBu3, the oxidative-addition barrier is lower on monopalladium monophosphine than dipalladium diphosphine whereas for a small phosphine, PMe3, the oxidative addition proceeds more easily via dipalladium diphosphine. Of the phosphine-free palladium complexes examined: free-Pd, PdBr-, and Pd(h2-C2H4), the olefin-coordinated intermediate has the lowest barrier for the oxidativeaddition. P450 and SOR have the same first-coordination-sphere, Fe[N4S], at their active sites but proceed through different reaction paths. The different ground spin states of the intermediate FeIII(OOH)(SCH3)(L) model {L = porphyrin for P450 and four imidazoles for SOR} produce geometric and electronic structures that assist i) the protonation on distal oxygen for P450, which leads to O-O bond cleavage and formation of (FeIV=O)(SCH3)(L) H2O, and ii) the protonation on proximal oxygen for SOR, which leads to (FeIII-HOOH)(SCH3)(L) formation before the Fe-O bond cleavage and H2O2 production. The hydrogen bonding from explicit waters also stabilizes FeIII-HOOH over FeIV=O H2O products in SOR. The electrochemical hydrogen production by Fe4[MeC(CH2S)3]2(CO)8 (1) with 2,6-dimethylpyridinium (LutH ) were studied by the DFT calculations of proton-transfer free energies relative to LutH and reduction potentials (vs. Fc/Fc ) of possible intermediates. In hydrogen production by 1, the second, more highly reductive, applied potential (-1.58 V) has the advantage over the first applied potential (-1.22 V) in that the more highly reduced intermediates can more easily add protons to produce H2.

The synthesis of diiron thiolate complexes was carried out using two ligands that were expected to furnish improved catalytic activity, solubility in water, and stability to the metal complexes. The water-soluble phosphine 1,3,5-triaza-7- phosphaadamantane, PTA, coordinates to the Fe centers forming the disubstituted complex (m-pdt)[Fe(CO)2PTA]2, which presents one PTA in each iron in a transoid arrangement. Substitution of one CO ligand in the (m-pdt)[Fe(CO)3]2 parent complex forms the asymmetric (m-pdt)[Fe(CO)3][Fe(CO)2PTA]. Enhanced water solubility was achieved through reactions with electrophiles, H+ and CH3 +, which reacted with the N on the PTA ligand forming the protonated and methylated derivatives, respectively. The 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene), IMes, was reacted with (m-pdt)[Fe(CO)3]2 yielding the asymmetric (m-pdt)[Fe(CO)3][Fe(CO)2IMes], an electron rich, air stable complex that does not show reactivity with H+. Electrocatalytic production of hydrogen was studied for the all-CO, bis-PMe3, mono- and di-PTA FeIFeI complexes, as well as the PTA-protonated and -methylated derivatives. The all-CO species produce H2, in the presence of the weak HOAc, at their second reduction event, FeIFe0 ?? Fe0Fe0, that occurs at ca. ??1.9 V, through an EECC mechanism. The mono- and di-substituted phosphine complexes present electrocatalytic production of H2 from the Fe0FeI redox state; this reduction takes place at ??1.54 V for (m-pdt)[Fe(CO)3][Fe(CO)2PTA], and at ca. ??1.8 for the disubstituted PMe3 and PTA derivatives. A positive charge on the starting complex does not have an effect on the production of H2. It was found that the protonated and methylated derivatives are not the catalytic species for H2 production. At their first reduction event the neutral precursor forms, and catalysis occurs from the FeIFeI complex in all cases. The possibility of enhanced catalytic activity in the presence of H2 O was explored by conducting electrochemical experiments in the mixed CH3CN:H2O solvent system for the PTA-substituted complexes. The reduction potential of the catalytic peak is shifted to more positive values by the presence of H2 O. The cyclic voltammogram of {(m-pdt)[Fe(CO)2(PTA?? H)]2}2+ in CH3CN:H2O 3:1 shows the reduction of a more easily reduced species in the return scan. This curve-crossing event provides evidence for the (h2-H2)FeII intermediate proposed in the ECCE mechanism.

TRANSCRIPTIONAL ANALYSIS OF HYDROGENASE GENES IN RHODOBACTER SPHAEROIDES O.U.001 In photosynthetic non-sulphur bacteria, hydrogen production is catalyzed by nitrogenases and hydrogenases. Hydrogenases are metalloenzymes that are basically classified into: the Fe hydrogenases, the Ni-Fe hydrogenases and metal-free hydrogenases. Two distinct Ni-Fe hydrogenases are described as uptake hydrogenases and bidirectional hydrogenases. The uptake hydrogenases are membrane bound dimeric enzymes consisting of small (hupS) and large (hupL) subunits, and are involved in uptake and the recycling of hydrogen, providing energy for nitrogen fixation and other metabolic processes. In this study the presence of the uptake hydrogenase genes was shown in Rhodobacter sphaeroides O.U.001 strain for the first time and hupS gene sequence was determined. The sequence shows 93% of homology with the uptake hydrogenase hupS of R.sphaeroides R.V. There was no significant change in growth of the bacteria at different concentrations of metal ions (nickel, molybdenum and iron in growth media). The effect of metal ions on hydrogen production of the organism was also studied. The maximum hydrogen gas production was achieved in 8.4&micro / M of nickel and 0.1 mM of iron containing media. The expression of uptake hydrogenase genes were examined by RT-PCR. Increasing the concentration of Ni++ up to 8.4&micro / M increased the expression of uptake hydrogenase genes (hupS). At varied concentrations of Fe-citrate (0.01 mM-0.1 mM) expression of hupS was not detected until hydrogen production stopped. These results will be significant for the improvement strategies of Rhodobacter sphaeroides O.U.001 to increase hydrogen production efficiency. In order to examine the presence of hupL genes, different primers were designed. However, the products could not be observed by PCR.

Techn. University, Diss., 2001--Berlin.