The growing global energy demand coupled with the need for a low-carbon economy requires innovative solutions. Microalgal oxygenic photosynthesis provides a sustainable platform for efficient capture of sunlight and storage of some of the energy in the form of reduced carbon derivatives. Under certain conditions, the photosynthetic reductant can be shunted to molecular hydrogen production, yet the efficiency and longevity of such processes are insufficient. In this work, re-engineering of the heterodimeric type I reaction center, also known as photosystem I (PSI), in the green microalga Chlamydomonas reinhardtii was shown to dramatically change algal metabolism and improve photobiological hydrogen production in vivo. First, an internal fusion of the small PsaC subunit of PSI harboring the terminal photosynthetic electron transport chain cofactors with the endogenous algal hydrogenase 2 (HydA2) was demonstrated to assemble on the PSI core in vivo, albeit at ~15% the level of normal PSI accumulation, and make molecular hydrogen from water oxidation. Second, the more physiologically active algal endogenous hydrogenase 1 (HydA1) was fused to PsaC in a similar fashion, resulting in improved levels of accumulation (~75%). Both algal hydrogenases chimeras remained extremely oxygen sensitive and benefited from oxygen removal methods. On the example of PSI-HydA1 chimera, it was demonstrated that the active site of hydrogenase can be reactivated in vivo after complete inactivation by oxygen without the need for new polypeptide synthesis. Third, the hydrogenase domain of Megasphaera elsdenii bacterial hydrogenase (MeHydA) was also fused with psaC, resulting in expression of a PSI-hydrogenase chimera at ~25% the normal level. The heterologous hydrogenase chimera could be activated with the algal maturation system, despite only 32 % sequence identity (43 % similarity). All constructs demonstrated diminished ability to reduce PSI electron acceptors (ferredoxin and flavodoxin) in vitro and indirect evidence indicated that this was true in vivo as well. Finally, chimeric design considerations are discussed in light of the models generated by Alphafold2 and how could they be used to further optimize stability of the PSI-hydrogenase chimeric complexes.

Alkylphosphine- and alkylpyridine-substituted 2,6-bis(imino)pyridines (pyridine diimines, PDI) have recently been used as polydentate, redox non-innocent ligands that support the development of highly active catalysts. The alkyl phosphine-substituted ligand, Ph2PPrPDI, was added to (Ph3P)3CoCl and subsequent reduction using excess sodium amalgam yielded (Ph2PPrPDI)Co. Electronic structure analysis revealed a cobalt(I) complex that features a singly reduced PDI chelate. Additionally, low valent Ph2PPrPDI complexes of Fe and Ni were synthesized and structurally characterized. Furthermore, a series of Ph2PPrPDI Mn, Fe, Co, and Ni complexes were investigated to evaluate ligand denticity and redox activity. Finally, the catalytic hydrosilylation of carbonyls was investigated to compare the activity of the series and determine whether electron count plays a role in catalysis. An analogous ligand system featuring alkylpyridine substituents, PyEtPDI, was added to CoCl2, affording a cobalt dichloride complex with the formula [(PyEtPDI)CoCl][Cl]. Single crystal X-ray diffraction revealed a high-spin cobalt(II) center that possesses an octahedral geometry and an outer-sphere chloride ion. Further treatment using 2 equivalents of NaEt3BH resulted in the formation of (κ4-N,N,N,N-PyEtIPCHMeNEtPy)Co, which has been verified by multinuclear NMR spectroscopy and single crystal X-ray diffraction. (κ4-N,N,N,N-PyEtIPCHMeNEtPy)Co was then used in the catalytic hydroboration of nitriles at ambient conditions to yield the corresponding N,N-diborylamines, which were used as precursors for amide formation.

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.

I present for the first time a broad-scale assessment of dissolved organic matter in the continental hot springs of Yellowstone National Park. The concentration of dissolved organic carbon in hot springs is highly variable, but demonstrates distinct trends with the geochemical composition of springs. The dissolved organic carbon concentrations are lowest in the hottest, most deeply sourced hot springs. Mixing of hydrothermal fluids with surface waters or reaction with buried sedimentary organic matter is typically indicated by increased dissolved organic carbon concentrations. I assessed the bulk composition of organic matter through fluorescence analysis that demonstrated different fluorescent components associated with terrestrial organic matter, microbial organic matter, and several novel fluorescent signatures unique to hot springs. One novel fluorescence signature is observed exclusively in acidic hot springs, and it is likely an end product of thermally-altered sedimentary organic matter. This acid-spring component precipitates out of solution under neutral or alkaline conditions and characterization of the precipitate revealed evidence for a highly condensed aromatic structure. This acid-spring component serves as a reliable tracer of acidic, hot water that has cycled through the subsurface. Overall, dissolved organic carbon concentrations and fluorescent features correlate with the inorganic indicators traditionally used to infer spring fluid mixing in the subsurface. Further, the fluorescence information reveals subtle differences in mixing between fluid phases that are not distinguishable through classic inorganic indicator species. My work assessing dissolved organic carbon in the Yellowstone National Park hot springs reveals that the organic matter in hydrothermal systems is different from that found in surface waters, and that the concentration and composition of hot spring dissolved organic matter reflects the subsurface geochemical and hydrological environment.

Exoelectrogenic microorganisms can grow by transferring electrons from their internal metabolism to extracellular substrates in a process known as extracellular electron transfer (EET). This dissertation explores the mechanisms of EET by both chemotrophic and phototrophic organisms and constructs a novel supramolecular structure that can be used as a model for microbial, long-range electron transfer. Geobacter sulfurreducens has been hypothesized to secrete and use riboflavin as a soluble, extracellular redox shuttle in conjunction with multi-heme, outer membrane, c-type cytochromes, but the required proteins and their properties have not been defined. To address the mechanism of extracellular electron transfer by G. sulfurreducens, the first part of this work explores the interaction between an outer membrane, octaheme, c-type cytochrome OmcZs from G. sulfurreducens and riboflavin. Interrogation via multiple physical techniques shows that OmcZs transfers electrons to riboflavin. By analogy to other characterized systems, riboflavin then likely interacts with extracellular acceptors directly. The second part of this work addresses the mechanisms of EET by the model cyanobacterium Synechocystis sp. PCC 6803. It has been hypothesized that Synechocystis employs conductive pili for production of extracellular current. However, the results herein show that a strain that does not have pili produces extracellular photocurrent in a direct electrochemical cell at a level similar to that by wild type cells. Furthermore, conductive atomic force microscopy (AFM) imaging is used to show that pili produced by the wild type organism are not conductive. Thus, an alternative EET mechanism must be operable. In the third part of this work, a supramolecular structure comprised of peptide and cytochromes designed to serve as a model for long-range electron transfer through cytochrome rich environments is described. The c-type cytochromes in this synthetic nanowire retain their redox activity after assembly and have suitable characteristics for long-range electron transfer. Taken together, the results of this dissertation not only inform on natural microbial mechanisms for EET but also provide a starting point to develop novel, synthetic systems.

In my thesis, I characterize multi-nuclear manganese cofactors in modified reaction

centers from the bacterium Rhodobacter sphaeroides. I characterized interactions

between a variety of secondary electron donors and modified reaction centers. In Chapter

1, I provide the research aims, background, and a summary of the chapters in my thesis.

In Chapter 2 and Chapter 3, I present my work with artificial four-helix bundles as

secondary electron donors to modified bacterial reaction centers. In Chapter 2, I

characterize the binding and energetics of the P1 Mn-protein, as a secondary electron

donor to modified reaction centers. In Chapter 3, I present the activity of a suite of four

helix bundles behaving as secondary electron donors to modified reaction centers. In

Chapter 4, I characterize a suite of modified reaction centers designed to bind and oxidize

manganese. I present work that characterizes bound manganese oxides as secondary

electron donors to the oxidized bacteriochlorophyll dimer in modified reaction centers. In

Chapter 5, I present my conclusions with a short description of future work in

characterizing multiple electron transfers from a multi-nuclear manganese cofactor in

modified reaction centers. To conclude, my thesis presents a characterization of a variety

of secondary electron donors to modified reaction centers that establish the feasibility to

characterize multiple turnovers from a multi-nuclear manganese cofactor.

To mimic the membrane environment for the photosynthetic reaction center of the photoheterotrophic Heliobacterium modesticaldum, a proteoliposome system was developed using the lipids found in native membranes, as well as a lipid possessing a Ni(II)-NTA head group. The liposomes were also saturated with menaquinone-9 to provide further native conditions, given that menaquinone is active within the heliobacterial reaction center in some way. Purified heliobacterial reaction center was reconstituted into the liposomes and a recombinant cytochrome c553 was decorated onto the liposome surface. The native lipid-attachment sequence of cytochrome c553 was truncated and replaced with a hexahistidine tag. Thus, the membrane-anchoring observed in vivo was simulated through the histidine tag of the recombinant cytochrome binding to the Ni(II)-NTA lipid's head group. The kinetics of electron transfer in this system was measured and compared to native membranes using transient absorption spectroscopy. The preferential-orientation of reconstituted heliobacterial reaction center was also measured by monitoring the proteoliposome system's ability to reduce a soluble acceptor, flavodoxin, in both whole and detergent-solubilized proteoliposome conditions. These data demonstrate that this proteoliposome system is reliable, biomimetic, and efficient for selectively testing the function of the photosynthetic reaction center of Heliobacterium modesticaldum and its interactions with both donors and acceptors. The recombinant cytochrome c553 performs similarly to native cytochrome c553 in heliobacterial membranes. These data also support the hypothesis that the orientation of the reconstituted reaction center is inherently selective for its bacteriochlorophyll special pair directed to the outer-leaflet of the liposome.

Biological systems have long been known to utilize two processes for energy conservation: substrate-level phosphorylation and electron transport phosphorylation. Recently, a new bioenergetic process was discovered that increases ATP yields: flavin-based electron bifurcation (FBEB). This process couples an energetically favorable reaction with an energetically unfavorable one to conserve energy in the organism. Currently, the mechanisms of enzymes that perform FBEB are unknown. In this work, NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (Nfn), a FBEB enzyme, is used as a model system to study this phenomenon. Nfn is a heterodimeric enzyme that reversibly couples the exergonic reduction of NADP+ by reduced ferredoxin with the endergonic reduction of NADP+ by NADH. Protein film electrochemistry (PFE) has been utilized to characterize the catalytic properties of three ferredoxins, possible substrates for Nfn enzymes, from organisms that perform FBEB: Pyrococcus furiosus (PfFd), Thermotoga maritima (TmFd), and Caldicellulosiruptor bescii (CbFd). Additionally, PFE is utilized to characterize three Nfn enzymes from two different archaea in the family Thermococcaceae: two from P. furiosus (PfNfnI and PfXfn), and one from Thermococcus sibiricus (TsNfnABC). Key results are as follows. The reduction potentials of the [4Fe4S]2+/1+ couple for all three ferredoxins are pH independent and modestly temperature dependent, and the Marcus reorganization energies of PfFd and TmFd are relatively small, suggesting optimized electron transfer. Electrocatalytic experiments show that PfNfnI is tuned for NADP+ reduction by both fast rates and a low binding constant for NADP+. A PfNfnI variant engineered to have only cysteines as coordinating ligands for its [FeS] clusters has significantly altered rates of electrocatalysis, substrate binding, and FBEB activity. This suggests that the heteroligands in the primary coordination sphere of the [FeS] clusters play a role in controlling catalysis by Nfn. Furthermore, a variant of PfNfnI lacking its small subunit, designed to probe allosteric effects at the bifurcating site, has altered substrate binding at the NADP(H) binding site, i.e. the bifurcation site. PfXfn and TsNfnABC, representing different types of Nfn enzymes, have different electrocatalytic properties than PfNfnI, including slower rates of FBEB. This suggests that Nfn enzymes vary significantly over phylogenetically similar organisms despite relatively high primary sequence homology.

The linear chromosomes ends in eukaryotes are protected by telomeres, a nucleoprotein structure that contains telomeric DNA with repetitive sequence and associated proteins. Telomerase is an RNA-dependent DNA polymerase that adds telomeric DNA repeats to the 3'-ends of chromosomes to offset the loss of terminal DNA repeats during DNA replication. It consists of two core components: a telomerase reverse transcriptase (TERT) and a telomerase RNA (TR). Telomerase uses a short sequence in its integral RNA component as template to add multiple DNA repeats in a processive manner. However, it remains unclear how the telomerase utilizes the short RNA template accurately and efficiently during DNA repeat synthesis. As previously reported human telomerase nucleotide synthesis arrests upon reaching the end of its RNA template by a unique template-embedded pause signal. In this study, I demonstrate pause signal remains active following template regeneration and inhibits the intrinsic processivity and rate of telomerase repeat addition. Furthermore, I have found that the human telomerase catalytic cycle comprises a crucial and slow incorporation of the first nucleotide after template translocation. This slow nucleotide incorporation step drastically limits repeat addition processivity and rate, which is alleviated with elevated concentrations of dGTP. Additionally, molecular mechanism of the disease mutants on telomerase specific motif T, K570N, have been explored. Finally, I studied how telomerase selective inhibitor BIBR 1532 reduce telomerase repeat addition processivity by function assay. Together, these results shed new light on telomerase catalytic cycle and the importance of telomerase for biomedicine.

The evolution of photosynthesis caused the oxygen-rich atmosphere in which we thrive today. Although the reaction centers involved in oxygenic photosynthesis probably evolved from a protein like the reaction centers in modern anoxygenic photosynthesis, modern anoxygenic reaction centers are poorly understood. One such anaerobic reaction center is found in Heliobacterium modesticaldum. Here, the photosynthetic properties of H. modesticaldum are investigated, especially as they pertain to its unique photochemical reaction center.

The first part of this dissertation describes the optimization of the previously established protocol for the H. modesticaldum reaction center isolation. Subsequently, electron transfer is characterized by ultrafast spectroscopy; the primary electron acceptor, a chlorophyll a derivative, is reduced in ~25 ps, and forward electron transfer occurs directly to a 4Fe-4S cluster in ~650 ps without the requirement for a quinone intermediate. A 2.2-angstrom resolution X-ray crystal structure of the homodimeric heliobacterial reaction center is solved, which is the first ever homodimeric reaction center structure to be solved, and is discussed as it pertains to the structure-function relationship in energy and electron transfer. The structure has a transmembrane helix arrangement similar to that of Photosystem I, but differences in antenna and electron transfer cofactor positions explain variations in biophysical comparisons. The structure is then compared with other reaction centers to infer evolutionary hypotheses suggesting that the ancestor to all modern reaction centers could reduce mobile quinones, and that Photosystem I added lower energy cofactors to its electron transfer chain to avoid the formation of singlet oxygen.

In the second part of this dissertation, hydrogen production rates of H. modesticaldum are quantified in multiple conditions. Hydrogen production only occurs in cells grown without ammonia, and is further increased by removal of N2. These results are used to propose a scheme that summarizes the hydrogen-production metabolism of H. modesticaldum, in which electrons from pyruvate oxidation are shuttled through an electron transport pathway including the reaction center, ultimately reducing nitrogenase. In conjunction, electron microscopy images of H. modesticaldum are shown, which confirm that extended membrane systems are not exhibited by heliobacteria.