Reduction of inorganic sulfate to sulfite in prototrophic bacteria occurs with 3-phosphoadenylylsulfate (PAPS) as substrate for PAPS reductase and is the first step leading to reduced sulfur for cellular biosynthetic reactions. The relative efficiency as reductants of homogeneous highly active PAPS reductase of the newly identified second thioredoxin (Trx2) and glutaredoxins (Grx1, Grx2, Grx3, and a mutant Grx1C14S) was compared with the well known thioredoxin (Trx1) from Escherichia coli. Trx1, Trx2, and Grx1 supported virtually identical rates of sulfite formation with a V max ranging from 6.6 units mg ؊1 (Trx1) to 5.1 units mg ؊1 (Grx1), whereas Grx1C14S was only marginally active, and Grx2 and Grx3 had no activity. The structural difference between active reductants had no effect upon K m PAPS (22.5 M). Grx1 effectively replaced Trx1 with essentially identical K m -values: K m trx1 (13.7 M), K m grx1 (14.9 M), whereas the K m trx2 was considerably higher (34.2 M). The results agree with previous in vivo data suggesting that Trx1 or Grx1 is essential for sulfate reduction but not for ribonucleotide reduction in E. coli.Prototrophic bacteria or fungi mainly use inorganic sulfate as the only supply of sulfur for the biosynthesis of amino acids and essential cofactors. Assimilation of sulfate occurs in five enzymatic steps. First, it is activated to adenylylsulfate (APS) 1 and 3Ј-phosphoadenylylsulfate (PAPS) by ATP sulfurylase and APS kinase. PAPS is then reduced to sulfite by PAPS reductase and sulfite is reduced to sulfide by sulfite reductase. Finally, cysteine is formed when sulfide is incorporated into O-acetyl-L-serine (OAS) by OAS-(thiol)lyase. The enzyme 3Ј-phosphoadenylylsulfate (PAPS) reductase (EC 1.8.99.4) catalyzes the first reductive step in this sequence. It uses thioredoxin (Trx) or glutaredoxin (Grx) as in vitro hydrogen donor for the reduction of 3Ј-phosphoadenylylsulfate to free sulfite.PAPS ϩ Trx(SH) 2 3 SO 3 2Ϫ ϩ 2H ϩ ϩ 3Ј-PAP ϩ Trx(S) 2 (Eq. 1) PAPS ϩ Grx(SH) 2 3 SO 3 2Ϫ ϩ 2H ϩ ϩ 3Ј-PAP ϩ Grx(S) 2 (Eq. 2)

Two proteins with similarity to IscA are encoded in the genome of the cyanobacterium Synechocystis PCC 6803. One of them, the product of slr1417 which accounts for 0.025% of the total soluble protein of Synechocystis was over-expressed in E. coli and purified. The purified protein was found to be mainly dimeric and did not contain any cofactor. Incubation with iron ions, cysteine and Synechocystis IscS led to the formation of one [2Fe2S] cluster at an IscA dimer as demonstrated (by the binding of about one iron and one sulfide ion per IscA monomer) by UV/Vis, EPR and Mo¨ssbauer spectroscopy. Mo¨ssbauer spectroscopy furtherindicatedthat the FeSclusterwas bound byfour cysteine residues. Site-directed mutagenesis revealed that of the five cysteine residues only C110 and C112 were involved in cluster binding. It was therefore concluded that the [2Fe2S] cluster is located between the two protomers of the IscA dimer and ligated by C110 and C112 of both protomers. The cluster could be transferred to apo ferredoxin, a [2Fe2S] protein, with a half-time of 10 min. Surprisingly, incubation of cluster-containing IscA with apo adenosine 5¢-phosphosulfate reductase led to a reactivation of the enzyme which requires the presence of a [4Fe4S] cluster. This demonstrates that it is possible to build [4Fe4S] clusters from [2Fe2S] units.

Properties of purified recombinant adenosine 3'-phosphate 5'-phosphosulfate (PAdoPS) reductase from Escherichia coli were investigated. The Michaelis constants for reduced thioredoxin and PAdoPS are 23 pM and 10 pM, respectively; the enzyme has a V,,,,,, of 94-99 pmol min.. mg and a molecular activity/catalytically active dimer of 95 s-'. Adenosine 3',5'-bisphosphate (PAdoP) inhibits competetively ( K , 4 pM) with respect to PAdoPS; adenosine 2',5'-bisphosphate and sulfite are not inhibitory. Alkylation by SH-group inhibitors irreversibly inactivates the enzyme.The structural gene (cysH) encodes for a small polypeptide with a single Cys residue located in a conserved cluster (KXECGVLH) of amino acids. Involvement of the only Cys and of Tyr209 in the reduction of PAdoPS to sulfite was investigated by site-specific mutagenesis: cysH was mutated by single-strand-overlay extension PCR; the mutated genes were cloned in pBTacl and expressed in E. coli RL 22 (AcysHIJ). Homogenous Cys239Ser and Tyr209Phe mutant PAdoPS reductases were investigated for altered catalytic properties. Mutation of the single Cys reduced V,,,,, by a factor of 4.5X 10' (V,,.,, = 0.02-0.013 pmol min-' mg ') with marginal effects on K,, for PAdoPS (19 pM) and reduced thioredoxin (14 pM). Mutation of Tyr209 drastically affected saturation with thioredoxin (K,,, 1 .S pM) and decreased V,,,.,, (0.22-0.25 pmol min-' mg I ) in addition to a small increase in K,, for PAdoPS (31 pM).Chromophores as prosthetic groups were absent from recombinant PAdoPS reductase. Difference absorption spectra between reduced and oxidized forms of wild-type and mutated proteins indicated that, in addition to Cys239 and Tyr209, an unidentified Trp (Ain,:,x 292 nm) appears to be involved in the reduction.The data suggest a special ping-pong mechanism with PAdoPS reacting with the reduced enzyme isomer in a Theorell-Chance type mechanism.

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 M Trx-saturating, 10.7 M Grx-saturating) than for APS (K m APS 28.7 M Trx-saturating, 105 M 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 ؋ 10 4 -3.6 ؋ 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. Mö ssbauer 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 oxygensensitive switch that suppresses SO 3 formation under aerobiosis.Plants, fungi, and many bacteria reduce inorganic sulfate to sulfide to cover their need for the element sulfur. This assimilatory type of sulfate reduction has two reductive steps in which sulfate is reduced to sulfite and sulfite is reduced further to sulfide. The reduction of sulfate to sulfite requires two electrons at a redox potential of Ϫ517 mV, which is considered too high for physiological electron carriers. Chemical activation of the sulfate by forming a mixed anhydride between phosphoric and sulfuric acid as in adenylyl sulfate (APS) 1 or 3Ј-phosphoadenylyl sulfate (PAPS) lowers the potential to mE 0 ϭ Ϫ60 mV, which is covered by thiols or pyrimidine nucleotides. The reduction is catalyzed by APS-and PAPS reductases. These proteins have a similar core protein of ϳ30 kDa in which the reactive center is located in a conserved ECG(L/I)H motif. Small redox proteins like thioredoxin (Trx) and glutaredoxin (Grx) are the physiological reductants for this group of reductases, whereas the APS reductases from higher plants also use monothiols like glutathione. Plant APS reductases that can use glutathione are larger than the Trx/Grx-dependent reductases because of an extension at their C terminus of ϳ16 kDa. This extension with its YAPWCXXC motif shows structural and functional similarities to thioredoxins or glutaredoxins (1-3). The core protein of the plant APS reductase and many of the PAPS reductase homologues found in different bacteria of the phytosphere contain a second CXXC and a Cys-Cys double cysteine motif. APS reductases of the plant type differ from PAPS reductases of the Trx-Grx type in that th...

The sequence conservation between ATP sulphurylases, a subfamily of ATP PPases, and PAPS reductase and the similarities in both their mechanisms and folds, suggest an evolutionary link between the ATP PPases and PAPS reductases. Together with the N type ATP PPases, PAPS reductases and ATP sulphurylases are proposed to form a new family of homologous enzymes with adenine nucleotide alpha-hydrolase activity. The open, reduced form of PAPS reductase is able to bind PAPS, whereas the closed oxidized form cannot. A movement between the two monomers of the dimer may allow this switch in conformation to occur.