Novel structure and redox chemistry of the prosthetic groups of the iron-sulfur flavoprotein sulfide dehydrogenase from Pyrococcus furiosus; evidence for a [2Fe-2S] cluster with Asp(Cys)3 ligands

Abstract

The consecutive structural genes for the iron-sulfur flavoenzyme sulfide dehydrogenase, sudB and sudA, have been identified in the genome of Pyrococcus furiosus. The translated sequences encode a heterodimeric protein with an alpha-subunit, SudA, of 52598 Da and a beta-subunit, SudB, of 30686 Da. The alpha-subunit carries a FAD, a putative nucleotide binding site for NADPH, and a [2Fe-2S]2+,+ prosthetic group. The latter exhibit EPR g-values, 2.035, 1.908, 1.786, and reduction potential, Em,8 = +80 mV, reminiscent of Rieske-type clusters; however, comparative sequence analysis indicates that this cluster is coordinated by a novel motif of one Asp and three Cys ligands. The motif is not only found in the genome of hyperthermophilic archaea and hyperthermophilic bacteria, but also in that of mesophilic Treponema pallidum. The beta-subunit of sulfide dehydrogenase contains another FAD, another putative binding site for NADPH, a [3Fe-4S]+,0 cluster, and a [4Fe-4S]2+,+ cluster. The 3Fe cluster has an unusually high reduction potential, Em,8 = +230 mV. The reduced 4Fe cluster exhibits a complex EPR signal, presumably resulting from magnetic interaction of its S = 1/2 spin with the S=2 spin of the reduced 3Fe cluster. The 4Fe cluster can be reduced with deazaflavin/EDTA/light but not with sodium dithionite; however, it is readily reduced with NADPH. SudA is highly homologous to KOD1-GO-GAT (or KOD1-GltA), a single-gene encoded protein in Pyrococcus kodakaraensis, which has been putatively identified as hyperthermophilic glutamate synthase. However, P. furiosus sulfide dehydrogenase does not have glutamate synthase activity. SudB is highly homologous to HydG, the gamma-subunit of P. furiosus NiFe hydrogenase. The latter enzyme also has sulfide dehydrogenase activity. The P. furiosus genome contains a second set of consecutive genes, sudY and sudX, with very high homology to the sudB and sudA genes, and possibly encoding a sulfide dehydrogenase isoenzyme. Each subunit of sulfide dehydrogenase is a primary structural paradigm for a different class of iron-sulfur flavoproteins.

Novel Genes of the dsr Gene Cluster and Evidence for Close Interaction of Dsr Proteins during Sulfur Oxidation in the Phototrophic Sulfur Bacterium Allochromatium vinosum

“…Only the first cysteine residue of the typical CXXC thioredoxin reductase active-site motif (76) is conserved at the respective position in DsrL. At their amino-terminus the small subunits of glutamate synthases carry two putative [Fe-S] clusters, the first with four and the second with three or four [Fe-S] binding cysteine residues (25,54). The second cysteine of the first cluster is missing in DsrL.…”

“…The second cysteine of the first cluster is missing in DsrL. Among the proteins related to DsrL is SudA, the large ␣-subunit of sulfide dehydrogenase (SudAB) from Pyrococcus furiosus (25). It catalyzes the reduction of polysulfide to H 2 S and also functions as a ferredoxin:NADP oxidoreductase.…”

“…On the basis of its amino acid sequence, it is more likely that DsrL is able to transfer two reducing equivalents between NADPH ϩ H ϩ /NADP ϩ and an as yet unidentified acceptor/donor via the protein-bound FAD cofactor. Several related proteins function as NADPH:feredoxin oxidoreductases (6,25). With its additional C-terminal ferredoxin domain, DsrL would carry this electron-transporting unit within the same polypeptide chain.…”

The Subnanometer Resolution Structure of the Glutamate Synthase 1.2-MDa Hexamer by Cryoelectron Microscopy and Its Oligomerization Behavior in Solution

“…Furthermore, the location and the role of the [4Fe-4S] 1ϩ/2ϩ clusters of ␤GltS only rely on indirect observations. Interestingly, the sequence of ␤GltS and, in particular, the spacing of its N-terminal conserved cysteine residues are similar to those of several other proteins or protein domains (1), among which only the Pyrococcus furiosus sulfide dehydrogenase ␣ subunit (11) and the bovine dihydropyrimidine dehydrogenase (DPD) (12)(13)(14) have been characterized to different extents. Site-directed mutagenesis experiments allowed us to confirm the location of the DPD-like [4Fe-4S] clusters of GltS within ␤GltS.…”

Genetic Examination of Initial Amino Acid Oxidation and Glutamate Catabolism in the Hyperthermophilic Archaeon Thermococcus kodakarensis

“…Glutamate synthase from T. kodakarensis has also been characterized (57). The function of this protein, however, will need further examination, as closely related homologs from Pyrococcus species have been demonstrated to exhibit different roles, such as the electron transfer component of sulfide dehydrogenase (58,59). One more gene that should be studied is a putative glutamate decarboxylase gene.…”