The objectives of this work were to identify and characterize iron-sulfur (Fe-S) cluster-bound forms of monothiol glutaredoxin (Grx)-BolA complexes from Azotobacter vinelandii and to investigate the novel system for Fe-S cluster assembly in the gastric pathogenic bacterium Helicobacter pylori. The approach involved in vitro characterization of the type and properties of protein-bound Fe-S clusters using UV-visible absorption/CD, resonance Raman and EPR spectroscopy, coupled with protein identification and oligomeric state determinations using mass spectrometry and analytical gel filtration, respectively. Grxs have emerged as a major class of [2Fe-2S] cluster trafficking proteins in prokaryotic and eukaryotic Fe-S cluster assembly systems. In eukaryotic model organisms, the cluster donor/acceptor specificity of homodimeric [2Fe-2S]-(Grx)2 complexes can be modulated by replacement of one Grx by a BolA protein to form heterodimeric [2Fe-2S]-Grx-BolA complexes. BolA binding also enables assessment of cytosolic Fe levels by sensing [2Fe-2S] cluster loading of Grxs and facilitates control of the Fe regulon. The regulation of iron within all living organisms is tightly regulated and is essential for maintaining organismal health. However, it is currently unclear if Fe-S cluster-bound Grx-BolA complexes are important in bacteria, since current information about the role of Grx-BolA complexes comes from eukaryotic model systems, and bacterial Grx-BolA complexes are largely unstudied. Of the four possible Fe-S cluster containing Grx-BolA complexes in A. vinelandii, three have now been identified and characterized: GrxNif-YrbA, Grx5-YrbA, and Grx5-BolA. Both [2Fe-2S]-GrxNif-YrbA and [2Fe-2S]-Grx5-YrbA exhibit rapid rates of formation, indicating these Grx-BolA complexes are physiologically relevant. The results also show that the GrxNif-YrbA and Grx5-YrbA complexes can also bind linear [3Fe-4S] clusters. While linear [3Fe-4S] cluster-bound forms are unlikely to be physiologically relevant, it is important to be able to recognize in vitro formation of linear [3Fe-4S] cluster-bound forms of Grx-BolA heterodimers. Over 50% of the world population is infected with the gastrointestinal bacteria H. pylori, and traditional antimicrobial treatments are only 80% effective. Moreover, the rapidly evolving antibiotic resistant strains of H. pylori are difficult to treat due to the diverse array of resistance that can vary by region. Hence the discovery that H. pylori is unique among gastrointestinal bacteria in having a partial NIF (Nitrogen Fixing) Fe-S cluster assembly system offers a new antimicrobial target. The core assembly machinery comprises a cysteine desulfurase NifS and a NifU scaffold protein that was shown to interact with 26 of the 36 target Fe-S proteins in H. pylori, based on bacterial two hybrid analysis. This work also identified a Nfu cluster trafficking protein that binds both [2Fe-2S] and [4Fe-4S] clusters and interacts with 14 of the 36 target Fe-S proteins in H. pylori, based on bacterial two hybrid analysis. Since no other potential cluster trafficking type proteins, such as A-type carriers or glutaredoxins, are found in the H. pylori proteome these results implicate Nfu as a major cluster trafficking protein in H. pylori. Characterization and cluster transfer studies were also carried out on a novel 2[4Fe-4S] ferredoxin protein, HP0277, which has a selective interaction towards Nfu.