Characterization and reconstitution of a 4Fe-4S adenylyl sulfate/phosphoadenylyl sulfate reductase from Bacillus subtilis

J Biol Chem. 2004 Feb 27;279(9):7850-5. doi: 10.1074/jbc.M309332200. Epub 2003 Nov 19.


CysH1 from Bacillus subtilis encodes a 3'-phospho/adenosine-phosphosulfate-sulfonucleotide reductase (SNR) of 27 kDa. Recombinant B. subtilis SNR is a homodimer, which is bispecific and reduces adenylylsulfate (APS) and 3'-phosphoadenylylsulfate (PAPS) alike with thioredoxin 1 or with glutaredoxin 1 as reductants. The enzyme has a higher affinity for PAPS (K(m)PAPS 6.4 microm Trx-saturating, 10.7 microm Grx-saturating) than for APS (K(m) APS 28.7 microm Trx-saturating, 105 microm Grx-saturating) at a V(max) ranging from 280 to 780 nmol sulfite mg(-1) min(-1). The catalytic efficiency with PAPS as substrate is higher by a factor of 10 (K(cat)/K(m) 2.7 x 10(4)-3.6 x 10(4) liter mol(-1) s(-1). B. subtilis SNR contains one 4Fe-4S cluster per polypeptide chain. SNR activity and color were lost rapidly upon exposure to air or upon dilution. Mössbauer and absorption spectroscopy revealed that the enzyme contained a 4Fe-4S cluster when isolated, but degradation of the 4Fe-4S cluster produced an inactive intermediate with spectral properties of a 2Fe-2S cluster. Activity and spectral properties of the 4Fe-4S cluster were restored by preincubation of SNR with the iron-sulfur cluster-assembling proteins IscA1 and IscS. Reconstitution of the 4Fe-4S cluster of SNR did not affect the reductive capacity for PAPS or APS. The interconversion of the clusters is thought to serve as oxygen-sensitive switch that suppresses SO(3) formation under aerobiosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacillus subtilis / enzymology*
  • Catalysis
  • Electron Spin Resonance Spectroscopy
  • Escherichia coli
  • Iron-Sulfur Proteins / chemistry
  • Oxidation-Reduction
  • Oxidoreductases / chemistry*
  • Oxidoreductases / genetics
  • Oxidoreductases / metabolism*
  • Oxygen / pharmacology
  • Recombinant Proteins
  • Spectrophotometry
  • Spectroscopy, Mossbauer
  • Substrate Specificity


  • Iron-Sulfur Proteins
  • Recombinant Proteins
  • Oxidoreductases
  • 3'-phosphoadenylyl-5'-phosphosulfate reductase
  • Oxygen