title: Investigation of the iron-sulfur cluster in Mycobacterium tuberculosis APS reductase: implications for substrate binding and catalysis.
abstract: The sulfur assimilation pathway is a key metabolic system in prokaryotes that is required for production of cysteine and cofactors such as coenzyme A. In the first step of the pathway, APS reductase catalyzes the reduction of adenosine 5'-phosphosulfate (APS) to adenosine 5'-phosphate (AMP) and sulfite with reducing equivalents from the protein cofactor, thioredoxin. The primary sequence of APS reductase is distinguished by a conserved iron-sulfur cluster motif, -CC-X( approximately )(80)-CXXC-. Of the sequence motifs that are associated with 4Fe-4S centers, the cysteine dyad is atypical and has generated discussion with respect to coordination as well as the cluster's larger functional significance. Herein, we have used biochemical, spectroscopic, and mass spectrometry analysis to investigate the iron-sulfur cluster and its role in the mechanism of Mycobacterium tuberculosis APS reductase. Site-directed mutagenesis of any cysteine residue within the conserved motif led to a loss of cluster with a concomitant loss in catalytic activity, while secondary structure was preserved. Studies of 4Fe-4S cluster stability and cysteine reactivity in the presence and absence of substrates, and in the free enzyme versus the covalent enzyme-intermediate (E-Cys-S-SO(3)(-)), suggest a structural rearrangement that occurs during the catalytic cycle. Taken together, these results demonstrate that the active site functionally communicates with the iron-sulfur cluster and also suggest a functional significance for the cysteine dyad in promoting site differentiation within the 4Fe-4S cluster.
authors.affiliation: Departments of Chemistry, University of California, Berkeley, California 94702-1460, USA.
authors.forename: Kate S
authors.initials: KS
authors.lastname: Carroll
authors.forename: Hong
authors.initials: H
authors.lastname: Gao
authors.forename: Huiyi
authors.initials: H
authors.lastname: Chen
authors.forename: Julie A
authors.initials: JA
authors.lastname: Leary
authors.forename: Carolyn R
authors.initials: CR
authors.lastname: Bertozzi
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D004795:Enzyme Stability; D005816:Genetic Complementation Test; D007501:Iron / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013879:Thioredoxins / Q000378:metabolism
chemical_list: D007506:Iron-Sulfur Proteins; D013438:Sulfhydryl Compounds; D000249:Adenosine Monophosphate; D013879:Thioredoxins; D007501:Iron; D050862:Oxidoreductases Acting on Sulfur Group Donors; C019618:adenylylsulfate reductase; D003545:Cysteine