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

Abstract

The iron-sulfur flavoenzyme adenylylsulfate (adenosine 5-phosphosulfate, APS) reductase catalyzes reversibly the 2-electron reduction of APS to sulfite and AMP, a key step in the biological sulfur cycle. APS reductase from one archaea and three different bacteria has been purified, and the molecular and catalytic properties have been characterized. The EPR parameters and redox potentials (؊60 and ؊520 mV versus NHE) have been assigned to the two [4Fe-4S] clusters I and II observed in the three-dimensional structure of the enzyme from Archaeoglobus fulgidus (Fritz, G., Roth, A., Schiffer, A., Bü chert, T., Bourenkov, G. Adenosine 5Ј-phosphosulfate (APS)1 reductase is a key enzyme involved in the pathways of sulfate reduction and sulfide oxidation. In higher plants the so-called assimilatory pathway of sulfate reduction provides sulfide for the biosynthesis of sulfur containing amino acids and cofactors, whereas in the dissimilatory pathway sulfate serves as a terminal electron acceptor of an anaerobic respiratory electron transfer chain in bacteria and archaea (2). Prior to reduction the sulfate molecule has to be activated via ATP sulfurylase (E.C. 2.7.7.4) to APS as shown in Scheme 1.Hydrolytic cleavage of the S-O-P moiety in APS yields Ϸ 80 kJ mol Ϫ1 (3), which is among the highest values reported so far for a X-O-P bond in a biological molecule. This energy is utilized by APS reductase (E.C. 1.8.99.2) in the reductive transformation of APS to sulfite and AMP. The calculated standard reduction potential E 0 Ј(APS/AMPϩHSO 3 Ϫ ) is Ϸ Ϫ60 mV versus Ϫ516 mV for E 0 Ј(SO 4 2Ϫ /HSO 3 Ϫ ), i.e. the potential is shifted by 450 mV (4). Sulfite is subsequently reduced by sulfite reductase (E.C. 1.8.7.1) to hydrogen sulfide. 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). First reports on APS reductase appeared already several decades ago (9), nevertheless the role of its cofactors in the reaction is still not fully understood. The observation of a covalent flavin-sulfite adduct suggested that FAD might be involved in catalysis. However, many flavoproteins with different functions form such an adduct. Especially the flavin-dependent oxidases bind sulfite with high affinity (10). Therefore, the involvement of a FAD-sulfite adduct in catalysis remained questionable. The number and relative arrangement of the [4Fe-4S] clusters in APS reductase has also been debated. Whereas Lampreia et al. described APS reductase as an enzyme with two [4Fe-4S] clusters in close proximity (11), Verhagen et al. proposed just one iron-sulfur cen...

Crystal Structure of Adenylylsulfate Reductase fromDesulfovibrio gigasSuggests a Potential Self-Regulation Mechanism Involving the C Terminus of the β-Subunit

“…The absorption features between 340 nm and 500 nm are due to the [4Fe-4S] clusters and FAD. These features in the UV-visible spectrum are similar to those previously reported for APSR from various Desulfovibrio species and A. fulgidus (1,8).…”

“…Electron input to the FAD catalyzes the cleavage of APS, releasing AMP and sulfite. Although there have been a number of mechanisms proposed to explain the catalytic cleavage of APS to AMP and sulfite (7,8,13,20,34), many features of the postulated mechanism remain unsettled, including the proteinogenic hydrogen acceptor in the reaction, the conformational change in the enzyme induced by reduction/oxidation of the FAD cofactor, and the reasons for the observed multiple forms of APSR. The divergence between A. fulgidus and Desulfovibrio species also suggests an obvious distinction in the phylogeny of the ␣-and ␤-subunits of APSR.…”

A Post-Genomic View of the Ecophysiology, Catabolism and Biotechnological Relevance of Sulphate-Reducing Prokaryotes

“…Two crystal structures have been determined for AprBA from SRP, one from A. fulgidus (Fritz, Roth, et al, 2002) and one from Desulfovibrio gigas (Chiang et al, 2009). The AprA subunit is a flavoprotein that binds an FAD cofactor and has a fold similar to the fumarate reductase/aspartate oxidase family, while the AprB subunit binds two [4Fe-4S] 2+/1+ clusters in a ferredoxin-like domain (Fritz, Büchert, & Kroneck, 2002;Fritz, Roth, et al, 2002). One of these clusters is located close to the protein surface and receives electrons from the physiological electron donor, which are then transferred to the second buried cluster that delivers them to the FAD active site.…”

“…One of these clusters is located close to the protein surface and receives electrons from the physiological electron donor, which are then transferred to the second buried cluster that delivers them to the FAD active site. A detailed mechanism for this reaction, based on the initial proposal by Michaels, Davidson, and Peck (1970), has been derived from the structural and spectroscopic characterization of different enzymatic states (Fritz, Büchert, et al, 2002;Parey, Fritz, et al, 2013;Schiffer, Fritz, Kroneck, & Ermler, 2006). The key step involves a nucleophilic attack of the N5 atom of reduced FAD on the sulphur atom of APS to form a FAD-APS intermediate, which decomposes to AMP and the FAD-sulphite adduct.…”

Key Bacterial Multi-Centered Metal Enzymes Involved in Nitrate and Sulfate Respiration

“…The two electrons required for APS reduction are transferred via two clusters from the surface of the protein to FAD. The exceptionally large difference in reduction potential of these clusters, -60 mV, -520 mV [Fritz et al, 2002a] is explained by interactions of the clusters with the protein matrix ( fi g. 6 ) [Fritz et al, 2002b].…”