Plant Adenosine 5′-Phosphosulfate Reductase Is a Novel Iron-Sulfur Protein

Abstract

Adenosine 5-phosphosulfate reductase (APR) catalyzes the two-electron reduction of adenosine 5-phosphosulfate to sulfite and AMP, which represents the key step of sulfate assimilation in higher plants. Recombinant APRs from both Lemna minor and Arabidopsis thaliana were overexpressed in Escherichia coli and isolated as yellow-brown proteins. UV-visible spectra of these recombinant proteins indicated the presence of iron-sulfur centers, whereas flavin was absent. This result was confirmed by quantitative analysis of iron and acid-labile sulfide, suggesting a [4Fe-4S] cluster as the cofactor. EPR spectroscopy of freshly purified enzyme showed, however, only a minor signal at g ‫؍‬ 2.01. Therefore, Mö ssbauer spectra of 57 Fe-enriched APR were obtained at 4.2 K in magnetic fields of up to 7 tesla, which were assigned to a diamagnetic [4Fe-4S] 2؉ cluster. This cluster was unusual because only three of the iron sites exhibited the same Mö ssbauer parameters. The fourth iron site gave, because of the bistability of the fit, a significantly smaller isomer shift or larger quadrupole splitting than the other three sites. Thus, plant assimilatory APR represents a novel type of adenosine 5-phosphosulfate reductase with a [4Fe-4S] center as the sole cofactor, which is clearly different from the dissimilatory adenosine 5-phosphosulfate reductases found in sulfate reducing bacteria.Sulfur is an essential element, which is found in nature mostly in its oxidized inorganic form of sulfate. In living organisms, however, most sulfur is in the reduced form of organic thiols. Plants, yeast, and most prokaryotes are able to reduce sulfate to sulfide and incorporate it into organic compounds. The sulfate assimilation pathway was first resolved in bacteria, such as Escherichia coli and Salmonella typhimurium (1, 2). For reduction sulfate must be activated in two steps; ATP sulfurylase forms adenosine 5Ј-phosphosulfate (APS), 1 which is further phosphorylated by APS kinase to form adenosine 3Ј-phosphate 5Ј-phosphosulfate (PAPS). PAPS is reduced in a thioredoxin-dependent reaction by PAPS reductase (EC 1.8.99) to sulfite. Sulfite is reduced by a NADPH-dependent sulfite reductase (EC 1.8.7.1) to sulfide, which is incorporated into the amino acid skeleton of O-acetyl-L-serine, thus forming cysteine (3). Plants and algae were shown to utilize APS rather than PAPS as sulfonyl donor; the corresponding enzyme was originally called APS sulfotransferase because S-sulfoglutathione was detected among the reaction products (4, 5). APS sulfotransferase was shown to be highly regulated and to play a key role in controlling sulfate reduction in plants (6). Nevertheless, a PAPS-dependent pathway of sulfate reduction could not be excluded, especially when the purification of PAPS reductase from spinach had been reported (7). In attempts to clone plant PAPS reductase by complementation of E. coli CysH mutants, a small family of three cDNA clones was obtained from Arabidopsis thaliana (8, 9). These cDNA clones encoded isoforms of an enzyme with a N-ter...

The Function of the [4Fe-4S] Clusters and FAD in Bacterial and Archaeal Adenylylsulfate Reductases

“…At low salt concentrations (20 mM Tris/Cl, pH 7.6) the maximum activity was observed, which was about 20% higher than under standard conditions (100 mM Tris/Cl, pH 7.6). 6 as electron acceptor (17) at temperatures higher than 75°C proved to be rather difficult (see "Experimental Procedures"). Here we report for the first time activities of APS reductase from A. fulgidus determined as APS reduction in an assay that worked highly reproducibly at temperatures up to 83°C.…”

“…Although both pathways include the same intermediates the APS reductases involved differ with regard to molecular architecture and cofactor composition. Whereas the assimilatory APS reductase is built of two 50-kDa subunits forming a homodimer containing two iron-sulfur centers (5,6), the dissimilatory enzyme is a heterodimer with one ␣-subunit (75 kDa, 1 FAD), and one ␤-subunit (18 kDa, 2 [4Fe-4S]) (7). The dissimilatory APS reductase also converts sulfite plus AMP to APS, providing electrons for anoxygenic photosynthesis and denitrification (8).…”

“…APS oxidation was determined in 50 mM Tris/HCl buffer, pH 7.6, containing 2 mM sodium sulfite, 2 mM AMP, and 0.5 mM EDTA. The reaction was started with K 3 Fe(CN) 6 , final concentration 1 mM. The decrease in absorbance at 420 nm was measured and corrected for the background reaction without enzyme.…”

Spectroscopic Studies on the [4Fe-4S] Cluster in Adenosine 5′-Phosphosulfate Reductase from Mycobacterium tuberculosis

“…A purely structural role also appears unlikely in light of biophysical data obtained on the apo form of APR (6, 13, 16) and the fact that APR and PAPR share a common protein fold (5,11,12). Unfortunately, progress in this area has been hampered by the failure to generate a paramagnetic state of the [4Fe-4S] cluster that can be studied by EPR spectroscopy and related methods (16,17,22).Herein, we report the EPR spectra of MtAPR in the [4Fe-4S] ϩ state. The EPR spectrum of MtAPR displays a rhombic signal but is complex and consists of at least two overlapping S ϭ 1 ⁄ 2 species.…”

“…Other interactions with the iron-sulfur cluster involve Thr-87 and Trp-246. In the active site, the phosphosulfate group of APS is positioned opposite the [4Fe-4S] cluster, and although no atoms intervene, the sulfate moiety is not in direct contact with the [4Fe-4S] cluster.Given the unusual Cys-Cys dyad coordination and its requirement for catalytic activity, defining the function and properties of the iron-sulfur cluster in APR has generated considerable interest (1,5,7,16,17). Most proteins containing [4Fe-4S] clusters are redox-active (18 -21); however, the [4Fe-4S] 2ϩ cluster in APR does not undergo redox changes during the catalytic cycle (1).…”