The Iron-Sulfur Flavoprotein DsrL as NAD(P)H:Acceptor Oxidoreductase in Oxidative and Reductive Dissimilatory Sulfur Metabolism

Abstract

DsrAB-type dissimilatory sulfite reductase is a key enzyme of microbial sulfur-dependent energy metabolism. Sulfur oxidizers also contain DsrL, which is essential for sulfur oxidation in Allochromatium vinosum. This NAD(P)H oxidoreductase acts as physiological partner of oxidative-type rDsrAB. Recent analyses uncovered that DsrL is not confined to sulfur oxidizers but also occurs in (probable) sulfate/sulfur-reducing bacteria. Here, phylogenetic analysis revealed a separation into two major branches, DsrL-1, with two subgroups, and DsrL-2. When present in organisms with reductive-type DsrAB, DsrL is of type 2. In the majority of cases oxidative-type rDsrAB occurs with DsrL-1 but combination with DsrL-2-type enzymes is also observed. Three model DsrL proteins, DsrL-1A and DsrL-1B from the sulfur oxidizers A. vinosum and Chlorobaculum tepidum, respectively, as well as DsrL-2 from thiosulfate- and sulfur-reducing Desulfurella amilsii were kinetically characterized. DaDsrL-2 is active with NADP(H) but not with NAD(H) which we relate to a conserved YRR-motif in the substrate-binding domains of all DsrL-2 enzymes. In contrast, AvDsrL-1A has a strong preference for NAD(H) and the CtDsrL-1B enzyme is completely inactive with NADP(H). Thus, NAD⁺ as well as NADP⁺ are suitable in vivo electron acceptors for rDsrABL-1-catalyzed sulfur oxidation, while NADPH is required as electron donor for sulfite reduction. This observation can be related to the lower redox potential of the NADPH/NADP⁺ than the NADH/NAD⁺ couple under physiological conditions. Organisms with a rdsrAB and dsrL-1 gene combination can be confidently identified as sulfur oxidizers while predictions for organisms with other combinations require much more caution and additional information sources.

Keywords: DsrAB; DsrL; NAD(P)H; dissimilatory sulfate reduction; dissimilatory sulfur oxidation; sulfite reductase; sulfur metabolism.

FIGURE 1

Common structure of DsrL proteins. All DsrL proteins consist of an amino-terminal ferredoxin domain (orange), a central domain (the NAD(P)-binding domain depicted in red is embedded in the FAD-binding domain highlighted in yellow), a linker domain and a carboxy-terminal ferredoxin domain (orange). Red cubes illustrate [4Fe-4S] clusters.

FIGURE 2

Comparison of DsrL (A) and DsrA (B) trees. Trees were constructed by using the Maximum Likelihood method with 1000 bootstrap resamplings. First, the best amino acid substitution models were calculated in MEGA X (Kumar et al., 2018). For DsrA as well as for DsrL, the Le_Gascuel_2008 model (Le and Gascuel, 2008) had the lowest BIC (Bayesian Information Criterion) score and was considered to describe the substitution pattern the best. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the JTT model, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories [+ G, parameters = 1.1935 and 0.8872 for DsrL and DsrA, respectively)]. The rate variation model allowed for some sites to be evolutionarily invariable ([ + I], 2.11% and 5.17% sites for DsrL and DsrA, respectively). Bootstrap values exceeding 50% are given at branching points. For DsrL and DsrA, the optimal trees with the highest log likelihood –76420.42 and –39346.85, respectively, are shown. The trees are drawn to scale, with branch lengths measured in the number of substitutions per site. Neighbor-joining phylogenies were also calculated and yielded essentially the same results. Complete phylogenetic trees with bootstrap values are available as Supplementary Data Files 1 and 2. It should be noted that some DsrL sequences reported earlier in Actinobacteria bacterium GWC2_53_9, Deltaproteobacteria bacteria RIFOXYA12_FULL_58_15 and RIFOXYB12_FULL_58_9, Candidatus Rokubacteria bacterium RIFCSPLOWO2_02_FULL_68_19, and Nitrospinae bacterium RIFCSPLOWO2_01_FULL_39_10 (Anantharaman et al., 2018) could not be integrated into phylogenetic tree construction because of too many ambiguous residues. A yellow box encloses all oxidative-type DsrA (rDsrA) proteins as well as all DsrL-1 type proteins. A green box features all bacterial reductive-type DsrA and DsrL-2-type proteins. Lineages in blue contain oxidative type DsrA and a DsrL protein of type DsrL-2. All DsrL sequences in group DsrL-2 feature a YRR motif indicative of preference for NADP(H) over NAD(H). Candidatus Acidulodesulfobacterium and Candidatus Acididesulfobacter species are the only exceptions. Here, YRR is replaced by YNK (yellow asterisk). Blue dots indicate the presence of long substrate binding and linker domains. Brown dots highlight DsrL proteins with an arginine instead of cysteine as potential [4Fe-4S] cluster ligand in the N-terminal ferredoxin domain (cf. Figure 3). Organisms highlighted with a black asterisk exhibit leucine or glycine at this position. Green dots indicate organisms containing genes for the sulfurtransferase DsrEFH.

FIGURE 3

Partial sequence alignments of DsrL proteins. DsrL-1 group proteins are highlighted in yellow and proteins belonging to group DsrL-2 are highlighted in green. DsrL from Allochromatium vinosum, Magnetospirillum gryphiswaldense and Thiobacillus denitrificans falls within the DsrL-1A branch, while the proteins from Chlorobaculum tepidum, Deltaproteobacteria bacterium UBA12577 and Nitrospirae bacterium CG2_20_70_394 are representatives of the DsrL-1B group. The DsrL-2 proteins from Chlorobium phaeobacteroides BS1, Desulforhopalus sp. IMCC35007 and Nitrospirae bacterium RBG_19FT_COMBO_42_15 stem from organisms with oxidative-type rDsrA, while the DsrL-2 from Candidatus Sulfopaludibacter sp. SbA3, the Chocolate Pot Hot Spring Metagenome and Desulfurella amilsii are encoded together with reductive bacterial-type DsrA. (A) The amino-terminal region binding two [4Fe-4S] clusters is shown. Predicted iron ligands are highlighted in orange and yellow for the distal and the proximal cluster, respectively. (B) Iron-liganding cysteines in the two [4Fe-4S] cluster-binding carboxy-terminal ferredoxin domain are marked in yellow and orange for each of the clusters. (C) The NAD(P)-binding domain of the different DsrL proteins is compared revealing part of an extension for the DsrL-1A proteins and the presence of the YRR motif indicative of interaction with NADP in DsrL-2-type enzymes. (D) Part of the linker domain connecting the major protein body with the carboxy-terminal ferredoxin domain showing an extension for DsrL-1A-type enzymes. The respective regions in the structurally characterized and DsrL-related protein NfnB from Thermotoga maritima (Demmer et al., 2015) are shown for comparison in panels A,C.

FIGURE 4

Two different views on the overlayed modeled structures of AvDsrL-1A (beige), CtDsrL-1B (light blue) and DaDsrL-2 (violet). For clarity, FAD, NAD and [4Fe-4S] clusters in the amino-terminal ferredoxin-domain are only shown for the protein from A. vinosum. All these prosthetic groups/substrates were modeled at the equivalent positions in CtDsrL-1B and DaDsrL-2. (A) The view shown here illustrates the different possible positions for the carboxy-terminal ferredoxin domains shown in the left part of the figure. (B) The view provided here highlights in red and green, respectively, the extensions present in the substrate-binding domain (amino acids 302–333) and the linker domain (amino acids 485-532) of AvDsrL-1A.

FIGURE 5

(A) SDS-PAGE of DsrL proteins after streptactin-based affinity purification. Lane 1, 30 μg AvDsrL1-I, lane 2, 25 μg CbDsrL1-II, lane 3, 25 μg DaDsrL2. (B–D) UV vis spectra of recombinant DsrL proteins. (B) AvDsrL-1A, (C) CtDsrL-1B, (D) DaDsrL-2. Spectra were normalized to 40 μM. Solid lines, spectra of proteins as isolated. Dashed lines, proteins reduced by addition of 1–4 times molar excess of titanium(III) citrate.

FIGURE 6

EPR spectra of the as-isolated and reduced AvDsrL-1A (A) and DaDsrL-2 (B), EPR spectra taken during the redox titration of AvDsrL-1A (C) and redox titration curve following the g value of 1.94 (D). The best fit to the experimental data was achieved by assuming reduction of four Fe-S centers in a 3:1 ratio with a E_m value of –330 mV and –390 mV, respectively.

FIGURE 7

NAD(P)H:acceptor oxidoreductase activity of recombinant (A) AvDsrL-1A, (B) CtDsrL-1B, and (C) DaDsrL-2. Activities were determined under anoxic conditions applying optimal pH and temperature for each enzyme. MTT was used as artificial electron acceptor in the NADH/NADPH oxidizing direction and reduced methylviologen served as electron donor for assays with NAD⁺/NADP⁺ as substrate. Filled circles: NADH, open circles: NAD⁺, open boxes: NADPH, filled boxes: NADP⁺.