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The effect of added iron on the growth of Clostridium botulinum type A in a chemically defined medium was studied. Growth of C. botulinum was supported by an iron level of 0.05 ug/ml with maximum growth observed at a level of 3 ug iron/ml. Electron paramagnetic resonance (EPR) studies were conducted to detect the presence of iron-sulfur centers and iron-nitric oxide complexes in untreated and nitrite treated cell-free extracts of C. botulinum type A. Untreated extracts of C. botulinum exhibited EPR signals in the oxidized and reduced states characteristic of a "HiPiP-type" iron-sulfur center (g=2.02) in the oxidized state and a reduced signal at g=l.94, characteristic of a reduced iron-sulfur center. Extracts of C. botulinum treated with nitrite exhibited an EPR signal at g=2.035, characteristic of iron-nitrosyl complexes, with the simultaneous disappearance of the the signal at g=l.94. This indicates that nitrite reacts with the iron-sulfur centers in botulinal cells to form iron-nitrosyl complexes. Addition of ascorbate with nitrite intensified the EPR signal at g=2.035, probably by enhancing the reduction of nitrite to nitric oxide. A cytochrome c reduction method was used for the determination of ferredoxin activity in untreated and nitrite treated cells of C. botulinum type A from which ferredoxin had been partially purified. Untreated extracts of C. botulinum reduced cytochrome c which demonstrates ferredoxin activity within the cells. Treatment of the cells with nitrite at a level of 1000 ppm for 45 min was found to inhibit ferredoxin activity by 90%. Boiling the partially purified ferredoxin from the untreated cells for 5 min inactivated the protein. Pyruvate-ferredoxin oxidoreductase activity in partially purified extracts of nitrite treated and untreated cells of C. botulinum was determined by assaying for FAD reduction and acylhydroxamate formation. Nitrite treated cells exhibited an inhibition of 70% of FAD reducing activity and 80% inhibition of acylhydroxamate formation when compared to the untreated cells. Boiling inhibited the activity of partially purified oxidoreductase activity by more than 90% in both the assays.

Thesis (Ph. D. in Biochemistry)--Vanderbilt University, Dec. 2005. / Title from title screen. Includes bibliographical references.

Mitochondrial dysfunction has been implicated for a wide range of human diseases. One of the major biosynthetic processes in human mitochondria is the biogenesis of Iron-Sulfur (Fe-S) clusters which primarily involves in electron transfer reactions during oxidative phosphorylation (OXPHOS). Defects in Fe-S cluster biogenesis process leads to mitochondrial dysfunction and that eventually results in various human mitochondrial disorders. One of the major mitochondrial disorders associated with Fe-S cluster biogenesis impairment is exercise intolerance disorder ISCU myopathy, which is a result of loss of function of Fe-S cluster scaffold protein ISCU. Our biochemical results using yeast model system and HeLa cells lines suggests that ISCU Myopathy results in defective Fe-S cluster biogenesis in mitochondrial compartment. As a result, electron transport chain (ETC) complexes demonstrate significant reduction in their redox properties, leading to loss of cellular respiration. Furthermore, in ISCU Myopathy, mitochondria display enhancement in iron levels and reactive oxygen species, thereby causing oxidative stress leading to impairment in the mitochondrial functions. On the other hand, in mammalian mitochondria, the initial step of Fe-S cluster assembly process is assisted by NFS1-ISD11 complex, which delivers sulfur to the scaffold protein ISCU during Fe-S cluster synthesis. In humans, loss of ISD11 function leads to development of respiratory distress disorder, Combined Oxidative Phosphorylation Deficiency 19 (COXPD19). Our study maps the important ISD11 amino acid residues critical for in vivo Fe-S cluster biogenesis. Importantly, mutation of these critical ISD11 residues to alanine leads to its compromised interaction with NFS1, which results in reduced stability and enhanced aggregation of NFS1 in the mitochondria. Moreover, our findings highlight that, COXPD19 associated R68L ISD11 mutant displays reduced affinity to form a stable sub-complex with NFS1, thereby fails to prevent NFS1 aggregation, resulting impairment of Fe-S cluster biogenesis. The prime affected machinery is the ETC complex which demonstrates compromised redox properties, causing diminished mitochondrial respiration in COXPD19 patients. In summary, our findings provide compelling evidence that respiration defect due to impaired biogenesis of Fe-S clusters in ISCU myopathy patients, leads to manifestation of complex clinical symptoms. Additionally, our study highlights the role of ISD11 protein in Fe-S cluster biogenesis and maps the surface residues of ISD11 protein that are involved in interaction with sulfur donor protein NFS1. Moreover, we have demonstrated the molecular basis of disease progression of COXPD19 as a result of R68L ISD11 mutation.