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Abstract
Iron-sulfur clusters are one of the most ubiquitous and diverse types of prosthetic groups in biology. Iron-sulfur cluster biosynthesis is a universal and highly regulated biological process due to the toxic nature of free iron and sulfide and much of the current understanding arises from the study of prokaryotic organisms in which iron-sulfur cluster assembly genes are organized in operons. The objectives of the research described in this dissertation were to understand the biosynthesis of iron-sulfur clusters by investigating gene products from the nitrogen fixation (nif) and the iron sulfur cluster assembly (isc) operons from the aerobic nitrogen-fixing bacterium Azotobacter vinelandii. Analytical and spectroscopic (UV-visible absorption, Mssbauer, and resonance Raman) studies of Nifcysteine desulfurase (NifS)-mediated cluster assembly on NifU and IscA show both to be scaffold proteins capable of sequential assembly of [2Fe-2S] and [4Fe-4S] clusters. Subsequent genetic, biochemical and spectroscopic studies involving wild-type and site-specific variants of NifU and fragments containing the N-terminal and central/C-terminal domains, revealed that NifU contains two distinct scaffold domains. The N-terminal domain assembles a labile [4Fe-4S] cluster at the subunit interface via reductive coupling of two [2Fe-2S] clusters and the C-terminal domain assembles [4Fe-4S] clusters directly. Moreover, [4Fe-4S] clusters assembled in the N-terminal and C-terminal domains of NifU can be rapidly transferred to activate the nitrogenase Fe protein, thereby providing the first well documented examples of intact [4Fe-4S] cluster transfer from a scaffold protein to an acceptor protein. Support from the proposal that [4Fe-4S] clusters can be formed via reductive coupling of two [2Fe-2S] clusters at a subunit interface was obtained via studies of nitrogenase Fe protein. Spectroscopic and crystallographic studies of wild-type nitrogenase Fe protein and a variant that mimics the ATP-bound form revealed evidence for glycerol-induced, redox-activated cycling between two [2Fe-2S] rhombs and a [4Fe-4S] cluster. Mass spectrometry was used to investigate the mechanism of cluster biosynthesis using IscS and wild-type and Cys-to-Ala variants of IscU (NifS and NifU homologs, respectively). IscS was shown to be capable of transferring multiple sulfane sulfur atoms to IscU, but the transfer does not involve a specific conserved cysteine residue or formation of an IscS-IscU disulfide complex. Index Words: Iron-sulfur proteins; Iron-sulfur cluster-assembly; Cysteine desulfurase; Scaffold protein; Iron homeostasis; Nitrogen fixation; Mssbauer; Resonance Raman; Mass Spectrometry.