This thesis analyzes the structure and function of enzymes involved in the biosynthesis of the tetrapyrrole cofactor, siroheme, a critical cofactor used in sulfur and nitrogen metabolism in plants, bacteria, and some archaea. The multifunctional enzyme, siroheme synthase, from Salmonella typhimurium and two newly identified enzymes from Mycobacterium tuberculosis are used to understand how these ancient enzymes function. Additionally, the effects of siroheme deficiency in Escherichia coli are studied in the context of sulfite reductase, an enzyme central to sulfur assimilation. Siroheme synthase (CysG) is a trifunctional enzyme responsible for the three terminal steps of siroheme biosynthesis in Salmonella typhimurium. The enzyme is composed of two functional modules, CysGA that accomplishes the first reaction, and CysGB that accomplishes the final two reactions. Interestingly, the same active site in CysGB is responsible for two very distinct chemistries where in other structural homologs, this is not observed. The work here shows how CysG distinguishes between these reactions to produce siroheme. Point mutagenesis, in vivo complementation assays, spectroscopic activity assays, and X-ray diffraction studies were used to piece together how CysG binds and orients the substrates and intermediates needed to catalyze siroheme. The co-crystal structures of precorrin-2-, sirohydrochlorin-, and cobalt-sirohydrochlorin-bound CysG were solved allowing characterization of the residues involved in binding and how their orientations change throughout catalysis. In Mycobacterium tuberculosis, the enzyme (or enzymes) responsible for siroheme production are unknown even though the siroheme cofactor is present in the bacteria’s sulfur metabolic pathway. This work reports the identification and characterization of two enzymes, MtCysG and MtChe1, that work together to produce siroheme. Molecular cloning teachniques, in vivo complementation assays, spectroscopic activity assays, and X-ray diffraction were used to isolate and identify MtCysG and MtChe1 as the enzymes necessary and sufficient for siroheme production. Interestingly, MtCysG is structurally homologous to Salmonella typhimurium CysG but is not a functional chelatase. Instead, MtChe1 fulfills this function to catalyze siroheme. Assimilatory NADPH-sulfite reductase (SiR) from Escherichia coli catalyzes the six-electron reduction of sulfite to sulfide. Two subunits, one a flavin-binding flavoprotein (SiRFP) and the other an iron-containing hemoprotein (SiRHP), assemble to make a holoenzyme ~800 kDa. How the two subunits assemble is not known. The iron-rich cofactors in SiRHP are unique because they are a covalent arrangement of a Fe4S4 cluster attached through a cysteine ligand to an iron-containing porphyrinoid called siroheme. The link between cofactor biogenesis and SiR stability is also ill-defined. Through hydrogen/deuterium exhchange, biochemical analysis and small-angle X-ray scattering (SAXS) we explore how the holoenzyme assembles and the structure of the N-terminal oligomerization domain of SiRHP. Apo-SiRHP forms a homotetramer, also dependent on its N-terminus, that is unable to assemble with SiRFP. From these results, we propose that homo-tetramerization of apo-SiRHP serves as a quality control mechanism to prevent formation of inactive holoenzyme in the case of limiting cellular siroheme.