Characterization and reconstitution of a 4Fe-4S adenylyl sulfate/phosphoadenylyl sulfate reductase from Bacillus subtilis.
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.
Berndt
Carsten
C
Biochemie der Pflanzen, Fakultät für Biologie, Ruhr Universität, Bochum 44780, Germany.
Lillig
Christopher H
CH
Wollenberg
Markus
M
Bill
Eckhard
E
Mansilla
María C
MC
de Mendoza
Diego
D
Seidler
Andreas
A
Schwenn
Jens D
JD
D001412:Bacillus subtilis / Q000201:enzymology*; D002384:Catalysis; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010100:Oxygen / Q000494:pharmacology; D011994:Recombinant Proteins; D013053:Spectrophotometry; D015204:Spectroscopy, Mossbauer; D013379:Substrate Specificity
D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D010088:Oxidoreductases; C052449:3'-phosphoadenylyl-5'-phosphosulfate reductase; D010100:Oxygen