Noncovalent complexes of APS reductase from M. tuberculosis: delineating a mechanistic model using ESI-FTICR MS.
ESI-FTICR MS was utilized to characterize a 4Fe-4S containing protein Mycobacterium tuberculosis APS reductase. This enzyme catalyzes the reduction of APS to sulfite and AMP with reducing equivalents from the protein cofactor, thioredoxin. Under nondenaturing conditions, a distribution of the apoprotein, a 2Fe-2S intermediate, and the 4Fe-4S holoprotein were observed. Accurate mass measurements indicated an oxidation state of +2 for the 4Fe-4S cluster, with no disulfide bond in the holoenzyme. Gas-phase stability of the 4Fe-4S cluster was investigated using both in-source and collision induced dissociation, which provided information regarding the relative gas-phase binding strength of iron towards protein ligands and inorganic sulfides. Noncovalent complexes of the holoprotein with several ligands, including APS, thioredoxin, and AMP, were also investigated. Calculated values of dissociation constants for the complexes indicate that AMP binds with a higher affinity to the enzyme intermediate than to the free enzyme. The implications of the binary and ternary complexes observed by gas-phase noncovalent interactions in the mechanism of APS reduction are discussed.
Gao
Hong
H
Section of Molecular Cell Biology, University of California, Davis, California 95616, USA.
Leary
Julie
J
Carroll
Kate S
KS
Bertozzi
Carolyn R
CR
Chen
Huiyi
H
D000249:Adenosine Monophosphate / Q000737:chemistry / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000201:enzymology*; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000032:analysis / Q000378:metabolism*; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism
D007506:Iron-Sulfur Proteins; D000249:Adenosine Monophosphate; D013879:Thioredoxins; D050862:Oxidoreductases Acting on Sulfur Group Donors; C019618:adenylylsulfate reductase