My research primarily focuses on biomimetics of the active sites of the [FeFe]- and [Fe]-hydrogenases (H2ase) and is classified into three parts. Part A: The one-electron oxidation of asymmetrically disubstituted FeIFeI models of the active site of the [FeFe]-H2ase, (mu-pdt)[Fe(CO)2PMe3][Fe(CO)2NHC] (pdt = 1,3- propanedithiolate, NHC = N-heterocyclic carbene) generates mixed valent FeIIFeI models of the Hox state of [FeFe]-hydrogenase. The spectroscopic properties, structures, reactivities and relative stabilities of the one-electron oxidized mixed valent complexes, (mu-pdt)(mu-CO)[FeII(CO)2PMe3][FeI(CO)NHC]+ are discussed in the context of experimental and theoretical data and biological relevance. Part B: DFT computations find the Fe-Fe bond in the FeIFeI diiron models ((mu- pdt)[Fe(CO)2L][Fe(CO)2L'] ( L, L' = CO, PPh3, or PMe3) is thermodynamically favored to produce the mu-oxo or oxidative addition product, FeII-O-FeII, nevertheless the sulfurbased HOMO-1 accounts for the experimentally observed mono- and bis-O-atom adducts at sulfur. The FeII(mu-H)FeII diiron model, (mu-pdt)(mu-H)[Fe(CO)2PMe3]2 (IV-5), for which the HOMO is largely of sulfur character, exclusively yields S-oxygenation. Deoxygenation with reclamation of the mu-pdt parent complexes occurs in a proton/electron coupled process. The possible biological relevance of oxygenation and deoxygenation studies is discussed. Comprehensive investigations of intramolecular CO site change and intermolecular CO/L (L = PMe3 or CN-) exchange of (mu-pst)[Fe(CO)3]2 (IV-1-O), (mu-pdt)[Fe(CO)3]2 (V-1), and their mono-CN-/PMe3 substituted derivatives indicated that the factors influencing the rate of the CO/L exchange reaction of such diiron carbonyls are intramolecular structural rearrangement (or fluxionality) and nucleophilic attack by the incoming ligand. Part C: X-ray diffraction and spectroscopic studies of a series of mono- and disubstituted complexes, FeI2(CO)xL4-x, x = 2 or 3, showed them to be rudimentary structural models of the [Fe]-H2ase active site in native (FeII(CO)2) or CO-inhibited (FeII(CO)3) states. Full characterization of the advanced model complexes ((NS)FeI(CO)2P, NS = 2-amidophenothiolate; P = phosphine) including x-ray diffraction, DFT computations, and Mossbauer studies revealed the interesting "noninnocent" character of these complexes due to the NS ligand. Ligand-based protonation with a strong acid, HBF4Et2O, interrupted the pi-delocalization over Fe and ligand of complex VII-1 and switched on CO uptake (1 bar) and 12CO/ 13CO exchange of VII-1. The intermediate, VII-1-H+, capable of CO uptake, was defined by DFT calculations.

Hydrogen oxidation by Azospirillum brasilense Sp7 was studied in N(,2)-fixing and NH(,4)('+)-grown batch cultures. The K(,m) for H(,2) of O(,2)-dependent H('3)H oxidation in whole cells was 9 uM. The rates of H('3)H and H(,2) oxidation were very similar, indicating that the initial H(,2) activation step in the overall H(,2) oxidation reaction was not rate-limiting and that H('3)H oxidation was a valid measure of H(,2)-oxidation activity. Hydrogen-oxidation activity was inhibited irreversibly by air. In N-free cultures the O(,2) optima for O(,2)-dependent H(,2) oxidation, ranging from 0.5-1.25% O(,2) depending on the phase of growth, were significantly higher than those of C(,2)H(,2) reduction, 0.15-0.35%, suggesting that the H(,2)-oxidation system may have a limited ability to aid in the protection of nitrogenase against inactivation by O(,2). Oxygen-dependent H(,2) oxidation was inhibited by NO(,2)('-), NO, CO, and C(,2)H(,2) with apparent K(,i) values of 20, 0.4, 28, and 88 uM, respectively. These inhibitors also affected methylene blue-dependent H(,2) oxidation, presumably by acting on the hydrogenase directly. The CO inhibition was easily reversible; the NO(,2)('-) and NO inhibitions were irreversible; and the C(,2)H(,2) inhibition was not readily reversible. Hydrogen-oxidation activity was 50 to 100 times higher in denitrifying cultures when the terminal electron acceptor for growth was N(,2)O rather than NO(,3)('-), possibly due to the irreversible inhibition of hydrogenase by NO(,2)('-) and NO in NO(,3)('-)-grown cultures. THe expression of the H(,2)-oxidation system was independent of nitrogenase expression, did not require added H(,2) (and probably not endogenous H(,2)), was not affected by low concentrations of carbon substrates (less than 30 mM malate), and required low O(,2) concentrations (microaerobic or anaerobic conditions).

Universiẗat, Diss., 2004--München.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1958. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Bibliography: leaves 50-54.

Low concentrations of the group IIA metals Mg²⁺, Ca²⁺, and Ba²⁺ stimulated energy-independent transhydrogenase activity. High concentrations of Mg²⁺ inhibited this activity. Transhydrogenase requires Mg²⁺-complexed NADP(H) rather than free NADP(H) as its substrate. High concentrations of Mg2+, however, may change the conformation of the enzyme to inhibit its enzymatic reaction by binding directly to the NADP(H) site. Upon transhydrogenation between NADPH and 3-acetylpyridine dinucleotide, E. coli pyridine nucleotide transhydrogenase can establish a proton gradient across the cell membrane. The primary component of the proton gradient for energization of transhydrogenase was found to be the pH gradient and not the membrane potential. A similar conclusion was drawn for the ATP-driven transhydrogenase reactions. In strains of E. coli that harbored plasmids to give the cells elevated levels of transhydrogenase, it was found that uncouplers stimulated the aerobic-driven transhydrogenase reaction. This is a chemiosmotic anomaly and is in contrast to the non-plasmid containing parent strains where uncouplers inhibited the activity. Further investigation revealed that the plasmid strains contained a much lower NADH oxidase activity than the non-plasmid strains and that neither KCN nor QNO can inhibit the aerobic-dependent activity in both types of strains even though they were effective in blocking the respiratory chain. These effects prompted us to inquire whether the anomaly was due to differences in the respiratory chain, but no differences were found between the NADH dehydrogenase activities, quinone and cytochrome contents of the plasmid and non-plasmid strains. The bacterial cells with amplified transhydrogenases induce extra intracellular tubular membrane structures to accomodate the extra proteins (Clark, D.M., Pyridine Nucleotide Transhydrogenase, PhD thesis, University of British Columbia, 1986). Separation of the E. coli membrane vesicles on a shallow sucrose gradient, however, did not reveal any differences between the vesicles of the plasmid and non-plasmid strains. Therefore, it seems unlikely that the anomaly is due to the plasmid strains performing a unique form of energization on these induced structures. Finally, it was established by SDS-PAGE and Western blot using anti-transhydrogenase antisera that the plasmid strains express a much higher level of transhydrogenase enzymes in their cell membranes than do the non-plasmid strains. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

abstract: Oxidoreductases catalyze transformations important in both bioenergetics and microbial technologies. Nonetheless, questions remain about how to tune them to modulate properties such as preference for catalysis in the oxidative or reductive direction, the potential range of activity, or coupling of multiple reactions. Using protein film electrochemistry, the features that control these properties are defined by comparing the activities of five [FeFe]-hydrogenases and two thiosulfate reductases. Although [FeFe]-hydrogenases are largely described as hydrogen evolution catalysts, the catalytic bias of [FeFe]-hydrogenases, i.e. the ratio of maximal reductive to oxidative activities, spans more than six orders of magnitude. At one extreme, two [FeFe]-hdyrogenases, Clostridium pasteuriaunum HydAII and Clostridium symbiosum HydY, are far more active for hydrogen oxidation than hydrogen evolution. On the other extreme, Clostridium pasteurianum HydAI and Clostridium acetobutylicum HydA1 have a neutral bias, in which both proton reduction and hydrogen oxidation are efficient. By investigating a collection of site-directed mutants, it is shown that the catalytic bias of [FeFe]-hydrogenases is not trivially correlated with the identities of residues in the primary or secondary coordination sphere. On the other hand, the catalytic bias of Clostridium acetobutylicum HydAI can be modulated via mutation of an amino acid residue coordinating the terminal [FeS] cluster. Simulations suggest that this change in catalytic bias may be linked to the reduction potential of the cluster. Two of the enzymes examined in this work, Clostridium pasteurianum HydAIII and Clostridium symbiosum HydY, display novel catalytic properties. HydY is exclusively a hydrogen oxidizing catalyst, and it couples this activity to peroxide reduction activity at a rubrerythrin center in the same enzyme. On the other hand, CpIII operates only in a narrow potential window, inactivating at oxidizing potentials. This suggests it plays a novel physiological role that has not yet been identified. Finally, the electrocatalytic properties of Pyrobaculum aerophilum thiosulfate reductase with either Mo or W in the active site are compared. In both cases, the onset of catalysis corresponds to reduction of the active site. Overall, the Mo enzyme is more active, and reduces thiosulfate with less overpotential. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2020

C. acetobutylicum is an anaerobic bacterium that produces acetic and butyric acids, hydrogen gas, and carbon dioxide during the exponential phase of growth. When the culture pH is allowed to remain near 4.5, the metabolism switches to the production of the neutral compounds (solvents) - acetone, n-butanol, and ethanol. The two metabolic phases are known as the acidogenic and solventogenic phases. The enzyme hydrogenase plays an important role in this bacterium because it converts excess reducing power into hydrogen gas to maintain a balance in the oxidation-reduction state in the cell. During solventogenesis, additional reducing power is used in the production of n-butanol and ethanol, which leaves excess reducing power to be vented as hydrogen gas. There are conflicting reports about the level of hydrogenase in acidogenic and solventogenic cells. There is also evidence that hydrogenase may consume too much reducing power during solventogenensis that it actually decreases the cell's capacity to produce solvents. The purpose of this study was to examine the level of hydrogenase in acidogenic and solventogenic cells and to search for clues that may indicate the presence of multiple forms of hydrogenase in C. acetobutylicum. Both the hydrogen-oxidation (uptake) and the hydrogen-production (evolution) activities were measured in this study. The level of hydrogenase was found higher in acidogenic cells than in solventogenic cells, but there was no difference in the molecular weight of hydrogenase from these two types of cells. A significant increase in the ratio of the hydrogen-uptake over the hydrogen-evolution activity was observed in oxygen or heat-treated cell extracts and in hydrogenase partially purified on a DEAE-cellulose column. The results suggest the presence of more than one type of hydrogenase in this species or hydrogenase activities in the two directions may be differentially altered. These possibilities will be investigated in a future study. / Master of Science

No description available.

The hydrogenase enzyme is an effective replacement for the expensive platinum catalysts used in hydrogen fuel cells today. However, many enzymes themselves are found in extreme environments and are inactive under standard conditions, but current active site models have a much larger over-potential for H+ reduction than the actual enzyme. Most research today involves the improvement of these synthetic models in an attempt to lower reduction potential, increase reaction kinetics, or improve catalytic activity. Research focuses on the synthesis of active site models with a carbon chain bridgehead linker of varying length. Synthesis of these molecules is achieved by the reaction of a dithiol with triiron dodecacarbonyl under an inert atmosphere to avoid the formation of by-products. Dithiols with four or more carbon atoms must first be converted to cyclic disulfides before the reaction with the iron The hydrogenase enzyme is an effective replacement for the expensive platinum catalysts used in hydrogen fuel cells today. However, many enzymes themselves are found in extreme environments and are inactive under standard conditions, but current active site models have a much larger over-potential for H+ reduction than the actual enzyme. Most research today involves the improvement of these synthetic models in an attempt to lower reduction potential, increase reaction kinetics, or improve catalytic activity. Research focuses on the synthesis of active site models with a carbon chain bridgehead linker of varying length. Synthesis of these molecules is achieved by the reaction of a dithiol with triiron dodecacarbonyl under an inert atmosphere to avoid the formation of by-products. Dithiols with four or more carbon atoms must first be converted to cyclic disulfides before the reaction with the iron dodecacarbonyl. This prevents the formation of an unwanted side product. Both butyl- and pentyldithiolatohexacarbonyldiiron model complexes have been characterized by IR, NMR, and X-ray spectroscopy. Active site models can also feature two unlinked sulfur atoms. These models have two conformational isomers that depend on the spatial location of the R-group bonded to each sulfur atom. This research also focuses on the synthesis of unlinked active site models with a variety of R-groups, and a temperature controlled NMR study of the isomeration reaction to determine the reaction rate. / Review of literature -- Synthesis of [FeFe]-hydrogenase active site mimics with bridged sulfur atoms -- Preliminary kinetics study of [FeFe]-hydrogenase active site mimics. / Department of Chemistry

氫化酶作為一種催化劑，能催化氫分子成為質子及電子的相互轉換。 [鎳鐵]- 氫化酶散播最廣的一種氫化酶，從古菌到細菌都能找到 [鎳鐵]- 氫化酶。完整成熟的 [鎳鐵]-氫化酶需要插入鐵、氰化物、一氧化碳以及鎳到它的催化核心。這複雜的過程需要其它由若干 hyp 基因編譯的輔助蛋白酶的幫助，其中蛋白HypA 與 HypB 負責將鎳運送到[鎳鐵] -氫化酶的催化核心。敲除了 hypA 或hypB 基因的細菌株缺失[鎳鐵] -氫化酶的活性，如在生長介質裡添補鎳可恢復部份[鎳鐵] -氫化酶的活性。當HypB 與鳥嘌呤核苷酸結合時會變成蛋白二聚體。對比HypB 脫輔基蛋白及與HypB 與鳥嘌呤三核苷酸類似物的蛋白複合物的晶體結構可發現，HypB 透過一個保守賴氨酸殘基( Archaeoglobus fulgidus HypB 的殘基 148 )組成分子間鹽橋以構成蛋白二聚體。Escherichia coli 的體內實驗顯示，此保守賴氨酸殘基對活性氫化酶的製造起必要的作用，反映由此殘基所構成的鹽橋對HypB 功能的重要性。此外，本研究展示了A. fulgidusHypA 及 HypB 蛋白之間的相互作用。通過在A. fulgidus HypB 上進行系統性的突變，發現HypB 利用其GTP 酶域上的一段氨基端區域與HypA 相互作用。跟據這個結果，我們進而在E. coli HypB 上發現了兩個保守的非極性殘基與HypA 相互作用。當以丙氨酸取代在HypB 上的這兩個非極性殘基時，HypB 無法激活E. coli 中的氫化酶，導置降低的氫化酶活性，這表明了HypA 和HypB 的相互作用對[鎳鐵] -氫化酶成熟過程的必要性。 / Hydrogenases catalyze the inter-conversion of molecular hydrogen into protons and electrons. [NiFe]-hydrogenase is the most widely distributed hydrogenases, which is found in organisms ranging from archaea to bacteria. Maturation of [NiFe]-hydrogenase requires the insertion of iron, cyanide and carbon monoxide, followed by nickel, to the catalytic core of the enzyme. The maturation process of hydrogenase is a complicated procedure, which requires many accessory proteins encoded by hyp genes. HypA and HypB participate in the nickel delivery step to the catalytic core of hydrogenase, which is supported by the fact that strain deficient in hypA or hypB gene lack hydrogenase activity which can be recovered partially by elevating nickel content in the medium. HypB is capable to form dimer in solution upon guanine nucleotide binding. By comparing the crystal structures of HypB in dimer and monomer form, an important lysine residue (residue 148 in A. fulgidus HypB) which is required to form an intermolecular salt bridge during GTP-dependent dimerization, has been identified. Substitution of this lysine resiue with alanie would break HypB dimer in vitro. In vivo complementation study in E. coli showed that the corresponding lysine residue in E. coli HypB is required for active hydrogenase production indicating the importance of this intermolecular salt bridge to the biological function of HypB. Besides, interaction between A. fulgidus HypA and HypB are demonstrated in this work. By making systematic mutation to A. fulgidus HypB, the N‐terminal region of the GTPase‐domain has been identified to be important for its interaction with HypA. Further mutagenesis study has been done on E. coli HypB and two conserved non‐polar residues responsible for interaction with HypA have been identified. Alanine substitution of these conserved non‐polar residues result in HypB mutants which failed to rescue hydrogenase activity in vivo in E. coli showing that HypA/HypB interaction is required for hydrogenase maturation. / Detailed summary in vernacular field only. / Chan, Kwok Ho. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 88-95). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Chapter Chapter 1 --- Introduction: Hydrogenase biosynthesis requires insertion of nickel facilitated by protein HypA and HypB --- p.1 / Chapter 1.1 --- What is hydrogenase? --- p.1 / Chapter 1.2 --- [NiFe] hydrogenase contains a complex catalytic core composed of metal atoms and diatomic ligands --- p.2 / Chapter 1.3 --- The [NiFe] catalytic core --- p.4 / Chapter 1.4 --- Building the catalytic [NiFe] core --- p.4 / Chapter 1.5 --- Nickel insertion into the hydrogenase precursor involves the proteins HypB, HypA and SlyD --- p.7 / Chapter 1.5.1 --- Protein HypB --- p.7 / Chapter 1.5.2 --- Protein HypA --- p.11 / Chapter 1.5.3 --- Protein SlyD --- p.12 / Chapter 1.6 --- Objectives - How HypB dimerization and HypA/HypB interaction are involved in hydrogenase maturation process? --- p.13 / Chapter Chapter 2 --- A conserved Lys residue is required for GTP-dependent dimerization and hydrogenase maturation --- p.17 / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Materials and Methods --- p.22 / Chapter 2.2.1 --- Recombinant Plasmid Construction --- p.22 / Chapter 2.2.2 --- HypB mutant construction by site-directed Mutagenesis --- p.22 / Chapter 2.2.3 --- Protein Expression and purification --- p.23 / Chapter 2.2.4 --- HypB protein purification --- p.23 / Chapter 2.2.5 --- Analytical gel filtration chromatography coupled with Light Scattering (SEC/LS) --- p.24 / Chapter 2.2.6 --- Nucleotide binding affinity determination --- p.25 / Chapter 2.2.7 --- GTPase activity determination --- p.26 / Chapter 2.2.8 --- Sample preparation for hydrogenase activity assay --- p.26 / Chapter 2.2.9 --- Hydrogenase activity determination --- p.27 / Chapter 2.3 --- Results --- p.29 / Chapter 2.3.1 --- AfHypB undergoes GTP-dependent dimerization --- p.29 / Chapter 2.3.2 --- Analysis of Structural difference between the apo form and GTP S-bound form suggests a mechanism of GTP-dependent dimerization for HypB --- p.30 / Chapter 2.3.3 --- Lys-148 is essential for GTP-dependent dimerization --- p.31 / Chapter 2.3.4 --- Disruption of dimerization by K148 mutation did not affect nucleotide binding and GTP hydrolysis activity significantly --- p.32 / Chapter 2.3.5 --- The conserved lysine residue is required for hydrogenase maturation in E. coli --- p.33 / Chapter 2.4 --- Discussion --- p.45 / Chapter 2.4.1 --- A conserved intermolecular salt‐bridge is required for GTP-dependent dimerization of HypB and hydrogenase maturation --- p.45 / Chapter 2.4.2 --- The extra metal binding site at the dimeric interface of HypB may provide a mechanism of why GTP-dependent dimerization is essential to Ni insertion --- p.46 / Chapter Chapter 3 --- N-terminal region of GTPase‐domain of HypB is required for interaction with HypA --- p.51 / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.2 --- Methods and materials --- p.53 / Chapter 3.2.1 --- Recombinant Plasmid Construction --- p.53 / Chapter 3.2.2 --- HypB variant construction by site‐directed Mutagenesis --- p.53 / Chapter 3.2.3 --- Protein Expression --- p.54 / Chapter 3.2.4 --- Tag‐free AfHypA and AfHypB purification --- p.54 / Chapter 3.2.5 --- Analytical size exclusion chromatography coupled with Light Scattering --- p.54 / Chapter 3.2.6 --- GST pull‐down of GST‐AfHypA and AfHypB --- p.55 / Chapter 3.2.7 --- Tandem affinity pull‐down of GST‐EcHypA and His‐SUMO‐EcHypB --- p.55 / Chapter 3.2.8 --- GST pull‐down of GST‐EcHypA and His‐SUMO‐EcHypB --- p.56 / Chapter 3.2.9 --- Hydrogenase activity determination --- p.57 / Chapter 3.3 --- Results --- p.58 / Chapter 3.3.1 --- HypA and HypB from A. fulgidus form 1:1 heterodimer in solution --- p.58 / Chapter 3.3.2 --- The N‐terminal regions upstream of the first helix of A. fulgidus HypB is required for HypA-HypB interaction --- p.59 / Chapter 3.3.3 --- Two conserved hydrophobic residues on HypB from E. coli are required to interact with HypA --- p.60 / Chapter 3.3.4 --- HypA-HypB interaction is required for hydrogenase maturation in E. coli --- p.62 / Chapter 3.4 --- Discussion --- p.73 / Chapter 3.4.1 --- The N‐terminal region of the GTPase domain is required for interaction with HypA and hydrogenase maturation in E. coli --- p.73 / Chapter 3.4.2 --- Location of interaction site on HypB reveals possible role for HypA/HypB interaction --- p.74 / Chapter 3.4.3 --- Mode of specific interaction with HypA: Interaction via a disordered region implies a coupled folding and binding process --- p.75 / Chapter Chapter 4 --- Conclusion and Future Perspectives --- p.80 / Chapter A1.1 --- Summary of findings in this work --- p.80 / Chapter A1.2 --- Implications in hydrogenase maturation --- p.81 / Chapter A1.3 --- Questions unresolved --- p.82 / Chapter 4.3.1 --- Factors that activate GTPase activity of HypB are still elusive --- p.82 / Chapter 4.3.2 --- How nickel delivery is regulated by HypA/HypB complex is still unclear --- p.83 / References --- p.88 / Chapter Appendix 1 --- Preliminary results of HypA/HypB protein complex structural study --- p.96 / Chapter A1.1 --- Structural study may provide invaluable insights to the role of HypA‐HypB interaction --- p.96 / Chapter A1.2 --- X‐ray crystallography as an approach to determine HypA/HypB complex structure --- p.96 / Chapter A1.3 --- Initial crystal hits were obtained with purified AfHypA/HypB complex --- p.97 / Chapter Appendix 2 --- Publications associated to the thesis --- p.100 / Chapter Appendix 3 --- Constructs and Primers used --- p.101