The active centers of adenylylsulfate reductase from Desulfovibrio gigas

Abstract

In order to utilize sulfate as the terminal electron acceptor, sulfate-reducing bacteria are equipped with a complex enzymatic system in which adenylylsulfate (AdoPSO,) reductase plays one of the major roles, reducing AdoPS04 (the activated form of sulfate) to sulfite, with release of AMP. The enzyme has been purified to homogeneity from the anaerobic sulfate reducer Desulfovihrio gigas. The protein is composed of two non-identical subunits (70 kDa and 23 kDa) and is isolated in a multimeric form ( z 400 kDa). It is an iron-sulfur, flavincontaining protein, with one FAD moiety, eight iron atoms and a minimum molecular mass of 93 kDa.Low-temperature EPR studies were performed to characterize its redox centers. In the native state, the enzyme showed an almost isotropic signal centered at g = 2.02 and only detectable below 20 K. This signal represented a minor species (0.10 -0.25 spins/mol) and showed line broadening in the enzyme isolated from 57Fe-grown cells. Addition of sulfite had a minor effect on the EPR spectrum, but caused a major decrease in the visible region of the optical spectrum (around 392 nm). Further addition of AMP induced only a minor change in the visible spectrum whereas major changes were seen in the EPR spectrum; the appearance of a rhombic signal at g values 2.096, 1.940 and 1.890 (reduced Fe-S center I) observable below 30 K and a concomitant decrease in intensity of the g = 2.02 signal were detected. Effects of chemical reductants (ascorbate, H,/hydrogenase-reduced methyl viologen and dithionite) were also studied. A short time reduction with dithionite (15 s) or reduction with methyl viologen gave rise to the full reduction of center I (with slightly modified g values at 2.079, 1.939 and 1.897), and the complete disappearance of the g = 2.02 signal. Further reduction with dithionite produces a very complex EPR spectrum of a spin-spin-coupled nature (observable below 20 K), indicating the presence of at least two iron-sulfur centers, (centers I and 11).Mossbauer studies on "Fe-enriched D. gigas Ado P S 0 4 reductase demonstrated unambiguously the presence of two 4Fe clusters. Center I1 has a redox potential < 400 mV and exhibits spectroscopic properties that are characteristic of a ferredoxin-type [4Fe-4S] cluster. Center I exhibits spectra with atypical Mossbauer parameters in its reduced state and has a midpoint potential around 0 mV, which is distinct from that of a ferredoxin-type [4Fe-4S] cluster, suggesting a different structure and/or a distinct cluster-ligand environment.Inorganic sulfur usually enters biosynthetic pathways at the oxidation level of sulfate or sulfide. Plants, animals and microorganisms are able to incorporate sulfate into several organic compounds. Microorganisms and plants can reduce sulfate to the level of sulfide which is subsequently incorporated into sulfur-containing amino acids and coenzymes [I]. This process is known as assimilatory sulfate reduction. However, an important group of anaerobic bacteria, the sulfatereducing bacteria, can utilize sulfate as a...

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

“…The molecular mass of oxidized APSR was measured at 20°C in Tris-HCl buffer (pH 7.6, 25°C) by ultracentrifugation, which showed a major peak at about 500 kDa, together with other impurities. The ratio of the UV absorption (A 278 /A 392 ) of the protein obtained from the final step was about 4.89, which differed slightly from the previous report of that of 4.97 (21). SDS-PAGE indicated the presence of two subunits with molecular masses of 70 kDa and 20 kDa, respectively.…”

“…The phase was solved by the molecular replacement method with the program MOLREP (36) in the CCP4 suite (4) using the monomer structure of APSR from A. fulgidus (Protein Data Bank [PDB] accession number, 1JNR) as a search model, with sequence similarities of 64% for the ␣-subunit and 82% for the ␤-subunit (7). The molecular replacement solution was found and confirmed that the space group is P3 1 21 and that there are six molecules in an asymmetric unit. After rigid-body refinement using the CNS version 1.2 program (2) in a resolution range of 30 to 3.5 Å, the R and R free factors were 50.6% and 51.8%, respectively.…”

“…Cluster II, coordinated by residues Cys10, Cys13, Cys21, and Cys57, is near the surface of the ␤-subunit and is thought to accept electrons from an unknown electron donor to transfer electrons further to cluster I. The reduction potentials of clusters I and II are reported to be 0 mV and less than Ϫ400 mV, respectively (21). Electrons are transferred from the negative potential to the positive potential, and this driving force determines the efficiency of the electron transfer.…”

“…Multiple forms of APSR in Desulfovibrio vulgaris were ob-served in buffers under varied conditions (1) and were found in the cytoplasm of cells (18). APSR from D. gigas was first purified by Lampreia et al (21) and showed a molecular mass of 400 kDa comprised of ␣-and ␤-subunits, corresponding to the molecular masses of 70 kDa and 23 kDa, respectively. One flavin adenine dinucleotide (FAD) and two [4Fe-4S] clusters per APSR have been observed and characterized by electron paramagnetic resonance and Mössbauer spectroscopy.…”

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

“…2, A and C), respectively, by an EPR-monitored redox titration. These values are close to those reported for the Fe-S center I in APS reductase of D. vulgaris (Ϫ19 mV) (12) and of Desulfovibrio gigas (-50 mV) (22). The midpoint potentials of center I are unusually high for [4Fe-4S] clusters, which normally range between Ϫ200 and Ϫ500 mV (23).…”