Iron-sulfur clusters are present in more than 120 different types of enzymes or proteins and constitute one of the most ancient, ubiquitous and structurally diverse classes of biological prosthetic groups. Although their primary role lies in mediating biological electron transport, iron-sulfur centers are known to constitute the active sites of numerous enzymes and to have important structural and regulatory roles. However, the functional diversity of biological iron-sulfur clusters has yet to be fully defined, and the mechanism of cluster biosynthesis, which is central to cellular iron homeostasis and the regulatory roles of iron-sulfur clusters, is still poorly understood. The long-term goal of this project is a molecular-level understanding of cluster biosynthesis and of the newly emerging roles of biological iron-sulfur clusters in disulfide reduction, initiating radical reactions in S-adenosylmethionine-dependent enzymes, and providing the sulfur for biosynthesis of biotin and lipoic acid. Ultimately this will lead to enhanced understanding of iron homeostasis and human diseases related to iron overload and defects or inhibition of respiratory chain enzymes. The approach involves using molecular biology techniques to effect large scale expression and/or site-specific changes in the target enzymes and proteins, biochemical and enzymatic assays, and the application of biophysical spectroscopic techniques (electron paramagnetic resonance, absorption, magnetic circular dichroism, resonance, absorption, magnetic circular dichroism, resonance Raman, Mossbauer and mass spectrometry) that can probe the nature and detailed properties of iron or iron-sulfur centers during catalytic cycling or cluster biosynthesis. The specific systems to be investigated include the proteins involved with nitrogen-fixation-specific and general iron-sulfur cluster biosynthesis in Azotobacter vinelandii, biotin synthase from Escherichia coli and ferredoxin:thioredoxin reductase from chloroplasts. The objectives are to establish the mechanism of NifU/NifS- and IscU/IscS-mediated iron-sulfur cluster biosynthesis, determine the role of the iron-sulfur cluster in ferredoxin:thioredoxin reductase in mediating reductive cleavage of the active-site disulfide, characterize the cluster transformation that is responsible for providing the sulfur for biotin biosynthesis, determine the mechanism of iron-sulfur cluster-mediated reductive cleavage of S-adenosylmethionine in biotin synthase, and develop an in vitro catalytic system for biotin biosynthesis.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Metallobiochemistry Study Section (BMT)
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Preusch, Peter C
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University of Georgia
Other Domestic Higher Education
United States
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Srivastava, Anurag P; Hardy, Emily P; Allen, James P et al. (2017) Identification of the Ferredoxin-Binding Site of a Ferredoxin-Dependent Cyanobacterial Nitrate Reductase. Biochemistry 56:5582-5592
Dlouhy, Adrienne C; Li, Haoran; Albetel, Angela-Nadia et al. (2016) The Escherichia coli BolA Protein IbaG Forms a Histidine-Ligated [2Fe-2S]-Bridged Complex with Grx4. Biochemistry 55:6869-6879
Subramanian, Sowmya; Duin, Evert C; Fawcett, Sarah E J et al. (2015) Spectroscopic and redox studies of valence-delocalized [Fe2S2](+) centers in thioredoxin-like ferredoxins. J Am Chem Soc 137:4567-80
LaVoie, Stephen P; Mapolelo, Daphne T; Cowart, Darin M et al. (2015) Organic and inorganic mercurials have distinct effects on cellular thiols, metal homeostasis, and Fe-binding proteins in Escherichia coli. J Biol Inorg Chem 20:1239-51
Srivastava, Anurag P; Allen, James P; Vaccaro, Brian J et al. (2015) Identification of Amino Acids at the Catalytic Site of a Ferredoxin-Dependent Cyanobacterial Nitrate Reductase. Biochemistry 54:5557-68
Crack, Jason C; Munnoch, John; Dodd, Erin L et al. (2015) NsrR from Streptomyces coelicolor is a nitric oxide-sensing [4Fe-4S] cluster protein with a specialized regulatory function. J Biol Chem 290:12689-704
Berggren, Gustav; Garcia-Serres, Ricardo; Brazzolotto, Xavier et al. (2014) An EPR/HYSCORE, Mössbauer, and resonance Raman study of the hydrogenase maturation enzyme HydF: a model for N-coordination to [4Fe-4S] clusters. J Biol Inorg Chem 19:75-84
Gao, Huanyao; Subramanian, Sowmya; Couturier, Jérémy et al. (2013) Arabidopsis thaliana Nfu2 accommodates [2Fe-2S] or [4Fe-4S] clusters and is competent for in vitro maturation of chloroplast [2Fe-2S] and [4Fe-4S] cluster-containing proteins. Biochemistry 52:6633-45
Mapolelo, Daphne T; Zhang, Bo; Randeniya, Sajini et al. (2013) Monothiol glutaredoxins and A-type proteins: partners in Fe-S cluster trafficking. Dalton Trans 42:3107-15
Zhang, Bo; Bandyopadhyay, Sibali; Shakamuri, Priyanka et al. (2013) Monothiol glutaredoxins can bind linear [Fe3S4]+ and [Fe4S4]2+ clusters in addition to [Fe2S2]2+ clusters: spectroscopic characterization and functional implications. J Am Chem Soc 135:15153-64

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