The energy crisis of the last decade has led to new and innovative approaches to combat this pressing issue. One leading candidate as an alternative fuel is hydrogen due to its derivability from water, its high energy density, and its status as a clean fuel. However, the current methods for hydrogen production are not economically feasible to replace fossil fuels because of a reliance on precious metal catalysts. A promising alternative for hydrogen catalysis is through the use of the enzymes hydrogenases, which utilize earth-abundant metals such as nickel and iron. Hydrogenases, however, are limited by their intolerance to oxygen, changes in temperature and pH, and the complexity of their biosynthesis. These factors, among others, leave little potential for industrial application, but hydrogenases can serve as inspiration for structural and functional models. One such model is the metalloprotein rubredoxin. Rubredoxin is a robust, electron-transfer protein with a promiscuous, tetrathiolate active site that binds a variety of metals. When the native iron is replaced with nickel, it mimics the primary coordination of the nickel site in the [NiFe] hydrogenase cofactor. We have demonstrated this construct not only acts as a structural model of hydrogenase, but also a functional mimic as it produces molecular hydrogen through the reduction of protons. The construct’s catalytic capabilities were probed with protein film electrochemistry (PFE), confirming the presence of a proton-coupled electron transfer process and inhibition from carbon monoxide, characteristics shared by the native hydrogenase. Through spectroscopic studies coupled to density functional theory calculations, a theoretical model of the Ni(II)Rd resting state was constructed and validated by resonance Raman spectroscopy. As typical PFE experiments were insufficient to investigate NiRd by standard enzymatic studies, a new quantitative PFE (qPFE) method that utilizes an internal protein film standard was created and validated on Rd. This technique allowed for the observation of mechanistic constraints that suggest a particular mechanism that matches that of the [NiFe] hydrogenase catalysis. This mechanism was validated through electrochemical simulations and DFT calculations. Lastly, this thesis presents preliminary data on a library of secondary sphere mutant variants around the NiRd active site. A wide range of characteristics is observed for the mutants, including an increase in activity by 10-fold and shifts in the catalytic onset potential over an 86 mV range. It has been previously demonstrated that these types of interactions greatly affect catalysis in the native hydrogenase as well, establishing NiRd as a mimic for the secondary sphere interactions of [NiFe] hydrogenase.

The utilization of enzymes as bioelectrocatalysts is of increasing interest due to their advantages in chemical specificity and catalytic rates. Emphasis has been placed on hydrogen producing biocatalysts to overcome drawbacks of current heterogeneous catalysts, including limited availability and poor selectivity. Native enzymes, such as the [NiFe] hydrogenase, have demonstrated extreme proficiency as bi-directional catalysts for proton reduction and hydrogen oxidation, inspiring a variety of small molecule and protein mimics. The utilization of a robust and stable protein scaffold with a similar primary coordination environment as the native enzyme can result in similar activity. Previous reports have demonstrated that nickel-substituted rubredoxin (NiRd) serves as a structural, functional and mechanistic model of the [NiFe] hydrogenase, active towards proton reduction electrochemically and in solution, with an identical primary coordination sphere at the nickel center as the native enzyme. While the mechanism of proton reduction has been experimentally and computationally modeled to be similar to that of the native enzyme, key catalytic intermediates have not yet been isolated and characterized. This project aims to address some of the limitations of this current model, such as significant overpotential and lack of spectroscopic characterization of catalytically competent intermediates. Further, correlation between redox activity and protein structure are investigated by modification of the protein primary sphere coordination site. Primary sphere mutants demonstrate changes in the redox and catalytic behavior dependent on the cysteine site being modified. Drastic changes to the primary coordination sphere are explored using electrochemistry along with optical, multiwavelength resonance Raman, X-ray and electron paramagnetic resonance spectroscopies. This study demonstrates the ability to keep and shut off catalysis, aiding in the understanding of enzyme selectivity and its correlations to active site structure and geometry. Expanding beyond the primary coordination sphere, changes to the secondary sphere have resulted in drastic changes to the turnover frequencies of the enzyme, with modest changes in overpotential. Previous work using electrochemical analysis of a suite of secondary sphere mutants have shown dramatic variation in the catalytic rates and redox activity of NiRd, with some mutants demonstrating a change in the proton reduction mechanism. The observed differences in catalytic and redox activity are hypothesized to arise from changes in the protein structure, dynamics and H-bonding networks. A structural study of a suite of secondary and primary sphere mutants utilizing Nuclear Magnetic Resonance (NMR) spectroscopy directly provides solution-phase information on protein dynamics. The small size of Rd (5.2 kDa) makes it an ideal candidate for NMR interrogations. Additionally, due to the high spin (S=1) active site of the NiIIRd artificial enzyme, and the favorable relaxation properties of pseudo-tetrahedral four coordinate NiII, paramagnetic NMR spectroscopy can be used to gain further insight into the metal electronic structure and the H-bonding network. The information gathered will identify key factors that affect catalysis within a model protein scaffold, allowing for not only rational catalyst design and catalyst optimization, but the development of paramagnetic NMR methodology that allows to probe structural changes by NiRd under catalytically relevant conditions.

The Prokaryotes is a comprehensive, multi-authored, peer reviewed reference work on Bacteria and Achaea. This fourth edition of The Prokaryotes is organized to cover all taxonomic diversity, using the family level to delineate chapters. Different from other resources, this new Springer product includes not only taxonomy, but also prokaryotic biology and technology of taxa in a broad context. Technological aspects highlight the usefulness of prokaryotes in processes and products, including biocontrol agents and as genetics tools. The content of the expanded fourth edition is divided into two parts: Part 1 contains review chapters dealing with the most important general concepts in molecular, applied and general prokaryote biology; Part 2 describes the known properties of specific taxonomic groups. Two completely new sections have been added to Part 1: bacterial communities and human bacteriology. The bacterial communities section reflects the growing realization that studies on pure cultures of bacteria have led to an incomplete picture of the microbial world for two fundamental reasons: the vast majority of bacteria in soil, water and associated with biological tissues are currently not culturable, and that an understanding of microbial ecology requires knowledge on how different bacterial species interact with each other in their natural environment. The new section on human microbiology deals with bacteria associated with healthy humans and bacterial pathogenesis. Each of the major human diseases caused by bacteria is reviewed, from identifying the pathogens by classical clinical and non-culturing techniques to the biochemical mechanisms of the disease process. The 4th edition of The Prokaryotes is the most complete resource on the biology of prokaryotes. The following volumes are published consecutively within the 4th Edition: Prokaryotic Biology and Symbiotic Associations Prokaryotic Communities and Ecophysiology Prokaryotic Physiology and Biochemistry Applied Bacteriology and Biotechnology Human Microbiology Actinobacteria Firmicutes Alphaproteobacteria and Betaproteobacteria Gammaproteobacteria Deltaproteobacteria and Epsilonproteobacteria Other Major Lineages of Bacteria and the Archaea

In this timely monograph, the author summarizes the rapidly growing body of knowledge regarding nickel by providing a balanced discussion of its harmful and beneficial effects. Coverage includes a history of nickel; the chemistry of nickel, descriptions of the four known enzymes which contain nickel; and nickel metabolism in microbes, plants, and animals. Taken as a whole, Dr. Hausinger's work will highlight key features of this important element and help define future research.

This volume highlights recent breakthroughs in the interdisciplinary areas of synthetic biology, metabolic engineering and bioprocess engineering for the production of green chemicals. It also presents practical experimental and computational tools for the design, construction and manipulation of cyanobacteria cell factories. The respective contributions cover new technologies in the field, such as novel genetic transformation techniques and bioinformatics analysis methods and address various aspects of cyanobacterial synthetic biology, offering a valuable resource for students and researchers in the fields of industry microbiology and biomedical engineering.

Comprehensive Coordination Chemistry III describes the fundamentals of metal-ligand interactions, provides an overview of the systematic chemistry of this class of compounds, and details their importance in life processes, medicine, industry and materials science. This new edition spans across 9 volumes, 185 entries and 6600 printed pages. Comprehensive Coordination Chemistry III is not just an update of the second edition, it includes a significant amount of new content. In the descriptive sections 3-6, emphasis is placed upon material that has appeared in primary and secondary review literature since the previous edition published. The material in other sections is newly written, with an emphasis on modern aspects of coordination chemistry and the latest developments. The metal-ligand interaction is the link between the award of the 1913 Nobel Prize in Chemistry to Alfred Werner, the father of Coordination Chemistry, the 1987 prize for supramolecular chemistry and the 2016 award for molecular machines. The key role of coordination chemistry in the assembly of hierarchical nano- and micro-dimensioned structures lies at the core of these applications and so this Major Reference Work bridges several sub-disciplines of chemistry, thus targeting a truly interdisciplinary audience. Provides the go-to foundational resource on coordination chemistry research, providing insights into future directions of the field Written and edited by renowned academics and practitioners from various fields and regions this authoritative and interdisciplinary work is of interest to a large audience, including coordination, supramolecular and molecular chemists Presents content that is clearly structured, organized and cross-referenced to allow students, researchers and professionals to find relevant information quickly and easily

Helmut Sigel, Astrid Sigel and Roland K.O. Sigel, in close cooperation with John Wiley & Sons, launch a new Series “Metal Ions in Life Sciences”. The philosophy of the Series is based on the one successfully applied to a previous series published by another publisher, but the move from “biological systems” to “life sciences” will open the aims and scope and allow for the publication of books touching on the interface between chemistry, biology, pharmacology, biochemistry and medicine. Volume 2 focuses on the vibrant research area concerning nickel as well as its complexes and their role in Nature. With more than 2,800 references and over 130 illustrations, it is an essential resource for scientists working in the wide range from inorganic biochemistry all the way through to medicine. In 17 stimulating chapters, written by 47 internationally recognized experts, Nickel and Its Surprising Impact in Nature highlights critically the biogeochemistry of nickel, its role in the environment, in plants and cyanobacteria, as well as for the gastric pathogen Helicobacter pylori, for gene expression and carcinogenensis. In addition, it covers the complex-forming properties of nickel with amino acids, peptides, phosphates, nucleotides, and nucleic acids. The volume also provides sophisticated insights in the recent progress made in understanding the role of nickel in enzymes such as ureases, hydrogenases, superoxide dismutases, acireductone dioxygenases, acetyl-coenzyme A synthases, carbon monoxide dehydrogenases, methyl-coenzyme M reductases...and it reveals the chaperones of nickel metabolism.