abstract: There is a critical need for the development of clean and efficient energy sources. Hydrogen is being explored as a viable alternative to fuels in current use, many of which have limited availability and detrimental byproducts. Biological photo-production of H2 could provide a potential energy source directly manufactured from water and sunlight. As a part of the photosynthetic electron transport chain (PETC) of the green algae Chlamydomonas reinhardtii, water is split via Photosystem II (PSII) and the electrons flow through a series of electron transfer cofactors in cytochrome b6f, plastocyanin and Photosystem I (PSI). The terminal electron acceptor of PSI is ferredoxin, from which electrons may be used to reduce NADP+ for metabolic purposes. Concomitant production of a H+ gradient allows production of energy for the cell. Under certain conditions and using the endogenous hydrogenase, excess protons and electrons from ferredoxin may be converted to molecular hydrogen. In this work it is demonstrated both that certain mutations near the quinone electron transfer cofactor in PSI can speed up electron transfer through the PETC, and also that a native [FeFe]-hydrogenase can be expressed in the C. reinhardtii chloroplast. Taken together, these research findings form the foundation for the design of a PSI-hydrogenase fusion for the direct and continuous photo-production of hydrogen in vivo. / Dissertation/Thesis / Ph.D. Biochemistry 2013

Molecular hydrogen has been proposed as an energy store to help meet the world's ever increasing demand for clean energy because the oxidation product (produced by either combustion or in a fuel cell) results in the formation of water. To realize this goal, energy efficient catalysts comprised of earth abundant elements must be used. The work in this dissertation describes investigations of diiron dichalcogen catalysts used for proton reduction. These complexes are inspired by the active site of the [FeFe]-hydrogenase enzyme. Catalysts were extensively studied with cyclic voltammetry in conjunction with photoelectron spectroscopy and density functional theory calculations in order to determine the effects that bridging ligands and 2Fe2E (E = S or Se) core substitutions have on the electronic structure and catalytic ability of these complexes. The complex µ-(pyrazine-2,3-dithiolato)diironhexacarbonyl (pyrazine-cat) was prepared and found to catalyze proton reduction at a -0.49 V overpotential, which represents a 16% decrease over the previously studied complex µ-(benzene-1,2-dithiolato)diironhexacarbonyl (benz-cat). Electrochemical investigations in conjunction with DFT calculations indicated the possibility of two mechanisms for proton reduction, both of the ECEC type. The first mechanism is Fe-based and analogous to the mechanism reported for benz-cat. The second is a nitrogen-based mechanism which occurs at more negative potentials than the Fe-based mechanism. Overall, pyrazine-cat maintained the ability to mediate successive redox states similar to benz-cat and the electron withdrawing nature of the pyrazine caused the initial reduction to occur at a lower potential than benz-cat. Ultimately this results in the decreased overpotential for catalytic proton reduction by pyrazine-cat. Investigations of the electronic structure and catalytic ability of complexes of the type (µ-ECH₂XCH₂E-µ)Fe₂(CO)₆ where E = S or Se and X = CH₂, S or Se were also carried out. All complexes were found to catalyze H₂ production from acetic acid in acetonitrile. DFT calculations indicate that when X = S or Se the HOMO changes character from predominatly metal based (X = CH2) to containing significant chalcogen lone pair character. The presence of the chalcogen lone pair character helps to facilitate a rotated structure in either the oxidized or reduced forms of these complexes. Through computations it was found that oxidation of the X = S or Se complexes results in a CO ligand rotating into a semi-bridging position, which opens a vacant site on one of the Fe-centers. The bridgehead bends toward this vacant site donating electron density greatly stabilizing the cation and more interestingly forming a structure which strongly resembles the active site of the [FeFe]-hydorgenase. Complexes which contain a chalcogen in the bridgehead undergo potential inversion, leading to a two-electron initial reduction. This is in part due to electron-electron repulsion between chalcogen lone pair electrons and the reduced Fe-centers, which leads to the formation of a rotated dianion.Complexes with the general structure (µ-E (CH₂)nE-µ)Fe₂ (CO)₆ where E = S or Se and n = 3, 4, or 5 were investigated using cyclic voltammetry, photoelectron spectroscopy, and DFT calculations. Substitution of Se in the 2Fe2E core for S resulted in a lengthening of the FeFe bond. As the linker length increased from n =3 to 5, one of the apical CO's is pushed down due to a steric interaction creating a more obtuse Fe-Fe-C angle. Larger effects of the linker length were seen in the oxidation and reduction chemistry. CV and UPS show that linker length has little effect on the oxidation potential or onset ionization energy. Computations predict that the oxidized structure is rotated, and as the linker length increases there is an agostic interaction which forms between a methylene proton and the vacant site on the rotated Fe-center. Reduction potentials for these complexes are found to decrease with increasing linker length, which was attributed to the steric interaction between the alkane linker and the apical CO helping to facilitate rotation of the anion. Interestingly catalytic potentials were found to depend almost entirely on chalcogen character in the 2Fe2E core, with S-containing catalysts having a lower catalytic potential than Se-containing catalysts. The long known complex [η⁵-CpFe(CO)SMe]₂ was investigated as both a proton reduction and H₂ oxidation catalyst. Reduction of [η⁵-CpFe(CO)SMe]₂ revealed that the complex undergoes a two electron irreversible reduction and the reduced species precipitates onto the glassy carbon electrode surface. The new species on the electrode surface facilitates proton reduction at a -0.3 V overpotential, which is significantly lower (0.9 V) than the most similar complex Fp₂. Unlike previous catalysts of this type, [η⁵-CpFe(CO)SMe]₂ catalytic current does not decrease as overpotential decreases. [η⁵-CpFe(CO)SMe]₂ was also shown to undergo two one-electron oxidations, and in the presence of H₂ and the dication, appears to oxidize H₂. The ability of [η⁵-CpFe(CO)SMe]₂ to both oxidize H₂ and reduce protons to H₂ addresses a known deficiency for catalysts mimicking the function of the active site of the [FeFe]-hydrogenase.

Les protéines à radical S-adénosyl-L-méthionine (SAM) utilisent un centre [Fe4S4] réduit pour initier le clivage réductive homolytique de la SAM et la formation d'une espèce hautement réactive - le radical 5'-déoxyadénosyl ou 5'-dA•. Dans la quasi-totalité de cas ce radical alkyl va arracher un atome d'hydrogène sur le substrat et déclencher ainsi sa conversion en produit. On trouve ces enzymes au niveau d'étapes clé de la synthèse de certaines vitamines, antibiotiques, précurseurs de l'ADN ou encore cofacteurs protéiques où elles sont souvent impliquées dans le clivage ou la formation des liaisons C-C, C-N, C-S ou encore C-P. Les travaux réalisés au cours de cette thèse ont été focalisés sur l'étude structurale et fonctionnelle de la protéine HydE ; une enzyme à radical SAM, qui intervient dans la biosynthèse du site actif organométallique de l'hydrogénase à [FeFe]. L'objectif principal était d'identifier le substrat de HydE et d'étudier les détails du fonctionnement d'une protéine à radical SAM. Nous avons réussi à identifier un groupe de molécules, dérivées de la cystéine, contentant un cycle thiazolidine avec un ou deux groupements carboxylates, qui ont une très bonne affinité pour le site actif de HydE. Certains de ces ligands se sont montrés d’être des substrats non physiologiques de l’enzyme. Grâce à ces substrats nous avons pu mettre en évidence un nouveau mécanisme d’attaque radicalaire dans les protéines à radical SAM. En effet, dans HydE nous avons observé une attaque directe du radical 5'-dA• sur l’atome soufre du thioéther appartenant au cycle thiazolidine. Cette réaction constitue un exemple pas comme les autres d’une insertion d’un atome de soufre (ou de sélénium) catalysée par une enzyme à radical SAM. Il s'agit également d'une première observation d'une réaction radicalaire dans les cristaux protéiques d'une enzyme à radical SAM et également un premier suivi en temps réel par la RMN du 13C et 1H de l'accumulation d'un des produits de la réaction catalysée par ces enzymes. Les résultats de calculs théoriques basés sur nos structures cristallographiques de haute résolution suggèrent que dans le cas de cette superfamille de protéines le radical 5'-dA• serait plutôt un état de transition et donc pas une espèce intermédiaire isolable. / Radical S-adenosyl-L-methionine (SAM) proteins use a reduced [Fe4S4] cluster to initiate homolytic reductive cleavage of SAM, which leads to the formation of highly reactive 5'-deoxyadenosyl radical species or 5'-dA•. In almost all cases this alkyl radical will abstract a hydrogen atom from the substrate and thus trigger its conversion into product. These enzymes are found in key steps of the synthesis of certain vitamins, antibiotics, DNA precursors or protein cofactors. They are often involved in the cleavage or formation of C-C, C-N, C-S or C-P bonds. The present thesis work has been focused on the structural and functional study of HydE protein; a radical SAM enzyme, involved in the biosynthesis of the organometallic active site of [FeFe]-hydrogenase. The main goal was to identify the substrate of HydE and to study details of how radical SAM proteins control the highly oxidizing 5'-dA• species. We managed to identify a group of molecules, derived from cysteine, containing a thiazolidine ring with one or two carboxylate groups, which have a very good affinity for the active site of HydE. We have demonstrated some of these ligands are non-physiological substrates of the enzyme. With these substrates we could highlight a new radical attack mechanism in radical SAM proteins. Indeed, in HydE we observed a direct attack on the 5'-dA • radical on the sulfur atom of the thioether belonging to the thiazolidine ring. This is an unprecedented reaction that contrasts with sulfur (or selenium) atom insertion reactions catalysed by some radical SAM enzymes. This is also the first observation of a radical reaction in the protein crystal of a radical SAM enzyme and also the first real-time monitoring by 1H- & 13C-NMR spectroscopy of the accumulation of products of the reaction catalysed by these enzymes. Theoretical calculations based on our high-resolution crystal structures suggest that in the case of this protein superfamily the 5'-dA• radical, which triggers the reaction in radical SAM enzymes, is a transition state and therefore not an isolable intermediate species.