The Ion-Translocating NrfD-Like Subunit of Energy-Transducing Membrane Complexes

Several energy-transducing microbial enzymes have their peripheral subunits connected to the membrane through an integral membrane protein, that interacts with quinones but does not have redox cofactors, the so-called NrfD-like subunit. The periplasmic nitrite reductase (NrfABCD) was the first complex recognized to have a membrane subunit with these characteristics and consequently provided the family's name: NrfD. Sequence analyses indicate that NrfD homologs are present in many diverse enzymes, such as polysulfide reductase (PsrABC), respiratory alternative complex III (ACIII), dimethyl sulfoxide (DMSO) reductase (DmsABC), tetrathionate reductase (TtrABC), sulfur reductase complex (SreABC), sulfite dehydrogenase (SoeABC), quinone reductase complex (QrcABCD), nine-heme cytochrome complex (NhcABCD), group-2 [NiFe] hydrogenase (Hyd-2), dissimilatory sulfite-reductase complex (DsrMKJOP), arsenate reductase (ArrC) and multiheme cytochrome c sulfite reductase (MccACD). The molecular structure of ACIII subunit C (ActC) and Psr subunit C (PsrC), NrfD-like subunits, revealed the existence of ion-conducting pathways. We performed thorough primary structural analyses and built structural models of the NrfD-like subunits. We observed that all these subunits are constituted by two structural repeats composed of four-helix bundles, possibly harboring ion-conducting pathways and containing a quinone/quinol binding site. NrfD-like subunits may be the ion-pumping module of several enzymes. Our data impact on the discussion of functional implications of the NrfD-like subunit-containing complexes, namely in their ability to transduce energy.


INTRODUCTION
All living organisms need energy to fuel life processes. External energy sources, light or chemical compounds, are converted to biologically usable forms of energy, such as adenosine triphosphate (ATP) or electrochemical gradients. According to Peter Mitchell's chemiosmotic hypothesis, the transmembrane difference of the electrochemical potential ( μ) can be established by energytransducing membrane protein complexes that couple the energy released by light or chemical reactions (Gibbs energy change, G) to the translocation of charges (electrons or ions) across the membrane (Mitchell, 1961). The energy stored in the form of the electrochemical potential can drive different energy-requiring reactions of the cells, such as synthesis of cellular components, solute transport or motility.
Energy transducing membrane complexes are usually composed of catalytic subunits and transmembrane proteins that perform translocation of charges, electrons or cations, across the membrane (Calisto et al., 2021). The most common membrane charge-translocating subunits so far observed in energy transduction complexes are the di-heme cytochrome b-like subunits and the so called NrfD-like subunits. The diheme cytochrome b-like subunits are involved in the transport of electrons, whereas the NrfD-like subunits translocate ions. In this way this type of subunits is devoid of redox cofactors but contain ion-conducting pathways.
The NrfD-like subunits are present in many and diverse membrane complexes, widespread in Bacteria and Archaea, that can take part in oxygen, nitrogen, sulfur, arsenate or hydrogen metabolism (Figure 1, Table 1) Refojo et al., 2010Refojo et al., , 2019Marreiros et al., 2016). These subunits thus compose the NrfD family, which was named after the characterization of the periplasmic nitrite reductase (NrfABCD) complex, the first complex recognized to have a NrfD-like subunit (Simon, 2002;Rothery et al., 2008) (Figure 1, NrfABCD). Structures of representatives of the NrfD family were first obtained for the PsrC subunit from the Thermus thermophilus polysulfide reductase (PsrABC) complex (Figures 1, 2, PsrABC) and later for the ActC and ActF subunits from the respiratory alternative complex III (ACIII) from Rhodothermus marinus (Figure 2, ACIII) and Flavobacterium johnsoniae Sun et al., 2018) and for the photosynthetic ACIII from Roseiflexus castenholzii (Shi et al., 2020) (Figure 1, ACIII). The structural data showed these membrane subunits have 8 common transmembrane helixes, organized in two four-helix bundles (TMHs 1-4 and TMHs 5-8) related by a 180 • rotation around an axis perpendicular to the membrane   (Figure 3), and contain one quinone/quinol-binding site close to the periphery of the membrane at the side at which the peripheral subunits are bound to. ActF does not contain any quinone/quinol binding site and it is the only NrfD-like subunit present in a complex in which another subunit of this type (which contains a quinone/quinol-binding site) is present. In addition, for all these subunits, putative ion-conducting pathways were proposed (Calisto et al., 2021). NrfD-like proteins also function as the link of the peripheral subunits to the membrane (Figure 1).
In this work we thoroughly analyze the primary structures of the members of the NrfD family and predicted the respective tertiary structures, the data provided allowed a deep and broad discussion on the presence of ion-conducting pathways and quinone/quinol-binding sites, which impacts on the function of the several complexes, namely in their ability to transduce energy. FIGURE 1 | Composition and diversity of NrfD-like subunit-containing complexes. Schematic representation of periplasmic nitrite reductase (NrfABCD), polysulfide reductase (PsrABC), respiratory alternative complex III (ACIII), DMSO reductase (DmsABC), group-2 [Ni-Fe] hydrogenase (Hyd-2), arsenate reductase (ArrABC), tetrathionate reductase (TtrABC), quinone reductase complex (QrcABCD), sulfur reductase complex (SreABC), nine-heme cytochrome complex (NhcABCD), dissimilatory sulfite-reductase complex (DsrMKJOP), multiheme cytochrome c sulfite reductase (MccACD), formate:quinone oxidoreductase (FqoABC), sulfite dehydrogenase (SoeABC). P-side, positive-side of the membrane; N-side, negative-side of the membrane. The NrfD-like subunits (NrfD, PsrC, ActC, ActF, DmsC, HybB, ArrC, ttrC, QrcD, SreC, NhcC, DsrP, MccD, fqoC, and SoeC) are colored, the two-color tones indicate the two structural repeats, each composed of a four-helix bundle (TMHs 1-4 and TMHs 5-8) and related by a 180 • rotation around an axis perpendicular to the membrane. The circle, inside the colored subunits, indicates the presence of a quinone/quinol-binding site. The red arrows represent the pathway for electron transfer. The color code is used in all the following figures and in Tables 1, 2. Cytochrome c-type hemes represented by red sticks; Mo-bisPGD cofactor represented by blue/red sticks with molybdenum shown as a blue sphere. Orange dots stand for the quinone-binding site. P-side, positive-side of the membrane; N-side, negative-side of the membrane.
FIGURE 3 | Fold of homologous membrane subunits ActC and ActF. ActC (pink) and ActF (blue) seen from the membrane (left) and from the top (right). The N-and C-terminal helices (TMHs A-B), which form a helix dimer, are colored in gray. The four-helix bundles are shown in light pink/blue (TMHs 1-4) and dark pink/blue (TMHs 5-8).

Taxonomic Distribution
NrfD-like subunits are the anchor protein of several modular complexes, which take part in a vast array of metabolisms, such as the oxygen, nitrogen, sulfur, arsenate and hydrogen cycles Refojo et al., 2010Refojo et al., , 2019Marreiros et al., 2016) (Figure 1). In this work, we performed sequence alignments and taxonomic profiling to investigate the distribution of the NrfD-like subunit-containing complexes in microbial species. We gathered 4,545 NrfD-like amino acid sequences present in the genomes of 1,822 distinct species (96% Bacteria, 4% Archaea), with an average of 2.5 NrfDlike subunits per organism. In order to reduce the size of the obtained dataset, the 4,545 amino acid sequences were clustered according to their identity (50% identity) and 551 representative sequences were aligned, allowing to generate the correspondent Neighbor-Joining (NJ) dendrogram. From the obtained dendrogram we were able to identify several branches belonging to different groups of NrfD-like subunit-containing complexes (Figure 4). We observed that close to the root of the dendrogram, the sequences of NrfD-like subunits separated into four major groups (Figure 4, dendrogram groups A-D). The amino acid sequences of SreC (21 sequences) (Figure 4, group A) and ArrC (9 sequences) (Figure 4, group B) seem to be less related with the others NrfD-like subunits and constitute two distinct groups. The SreABC complex was biochemically characterized from the sulfur-dependent archaeon A. ambivalens (Laska et al., 2003) and genes coding for SreC subunit were only identified in archaea, in 21 species (27% of Crenarchaeota species) (Figure 5, SreC). ArrAB complex was described as a periplasmic complex, which is associated with the transmembrane ArrC subunit only in few microorganisms (Duval et al., 2008). In agreement, we identified nine genes coding for ArrC subunit, distributed in Gammaproteobacteria (four sequences), Betaproteobacteria (four sequences) and Euryarchaeota (one sequence) species (Figure 5, ArrC).
In group D of the dendrogram (Figure 4, FqoC), we were able to identify 110 amino acid sequences present in an uncharacterized complex, that we tentatively assigned as formate:quinone oxidoreductase complex (Figure 1, FqoABC). Our assignment is based on the observations that the complex is possibly composed of three subunits: two peripheral FqoAB subunits homologous to FdnGH subunits from E. coli formate dehydrogenase complex (Fdn-N) and a FqoC subunit, a NrfDlike subunit with eight predicted TMHs. Genes coding for FqoC were identified in 110 species present in three different dendrogram groups: FqoC1 includes genes coding for FqoC subunit from Actinobacteria (59 species, 7%), Chlorobi (15 species, 88%) and Crenarchaeota (9 species, 12%) phyla; FqoC2 gathers the genes coding for FqoC from Chloroflexi phylum (15 species, 48%); and FqoC3 includes genes coding for FqoC subunit from Deltaproteobacteria phylum (12 species, 13%) (Figure 5, FqoC).

Homology Models
The NrfD-like subunits are transmembrane proteins, with 8 to 10 TMHs, that interact with quinones but do not have redox cofactors. The structures of T. thermophilus PsrC and R. marinus ActC and ActF revealed details of the overall architecture of these NrfD-like subunits: the common 8 TMHs are organized in two four-helix bundles (TMHs 1-4 and TMHs 5-8), which form two structural repeats related by a 180 • rotation around an axis perpendicular to the membrane layer plane Sousa et al., 2018). Two additional TMHs (TMHs A-B), present in ActC and ActF, cross each other at an angle of ∼45 • at the periphery of each subunit   (Figure 3). ActC and ActF subunits have their N-and C-terminal located at N-side of the membrane, while PsrC have both termini located at the P-side of the membrane. T. thermophilus PsrC and R. marinus ActC subunits contain one quinol-binding site at the P-side of the membrane of the first four-helix bundle Sousa et al., 2018) (Figure 2).
We performed structural homology models of the NrfD-like subunits from W. succinogenes PsrABC, E. coli NrfABCD, E. coli DmsABC, E. coli Hyd-2, Salmonella enterica TtrABC, A. ambivalens SreABC, A. aeolicus SoeABC, D. vulgaris QrcABCD, D. vulgaris DsrMKJOP1, A. vinosum DsrMKJOP2, Desulfovibrio desulfuricans NhcABCD, Alkalilimnicola ehrlichii ArrABC, W. succinogenes MccACD and Chlorobaculum tepidum FqoABC complexes. The homology models were calculated in Phyre2 without imposing any template. In all cases the randomly selected template was the structure from R. marinus ActC subunit. The homology models have confidence scores higher than 90%. The final models presented the common 8 TMHs organized in a similar arrangement as those of ActC, ActF and PsrC subunits. As in the case of T. thermophilus PsrC subunit, we predicted only 8 TMHs for W. succinogenes PsrC, E. coli DmsC, E. coli NrfD, A. aeolicus SoeC, W. succinogenes MccD and C. tepidum FqoC subunits. S. enterica TtrC subunit was predicted to have 9 TMHs, with the N-terminal at the P-side and the C-terminal at the N-side of the membrane, the extra TMH is equivalent to TMH B from ActC. The other NrfD-like subunits (E. coli HybB, A. ehrlichii ArrC, D. vulgaris DsrP1, A. vinosum DsrP2, D. vulgaris QrcD, A. ambivalens SreC and D. desulfuricans NhcC) have 10 predicted TMHs, the two extra TMHs are equivalent to TMH A and B of ActC and ActF subunits. The models predict for all NrfDlike subunits the presence of the structural repeats, composed of the two four-helix bundles, harboring putative ion-conducting pathways and the existence of a quinone/quinol-binding site (Figure 1, Table 2).

Quinone-Binding Site
The crystal structures of T. thermophilus PsrC co-crystallized with either menaquinone-7 or ubiquinone-1 showed that the quinone-binding site is located in the first four-helix bundle of PsrC (TMHs 1-4) on the P-side of the membrane, in close proximity to the [4Fe-4S] 2+/1+ cluster of PsrB subunit . Highly conserved amino acid charged residues, present in this region, were suggested as essential for quinone-binding and coordination: His21 PsrCT , Asn18 PsrCT and Tyr130 PsrCT in T. thermophilus PsrC and His139 ActC , Asp169 ActC and Asp253 ActC in R. marinus ActC Sousa et al., 2018) (Figure 6, ActC and PsrC T).
To further investigate structurally relevant elements and/or amino acid residues in the quinone-binding site, we analyzed the obtained structural homology models of the NrfD-like subunits and performed sequence alignment to identify the conserved residues involved in the binding and coordination of quinone/quinol molecule.
We identified for all NrfD-like subunits, with the exception of ActF subunit, the only NrfD-like that has been shown not to interact with quinones/quinols and part of a complex in which another NrfD-like subunit is present, the presence of quinone/quinol-binding site in the same spatial position of those observed for T. thermophilus PsrC and R. marinus ActC. These are all located in the first four-helix bundle (TMHs 1-4), close to the P-side of the membrane. In the case of SoeC the quinone/quinol-binding site is also present in the first four-helix bundle (TMHs 1-4) at the same special position of sites present in the other NrfD-like subunits, however it is expecting to be facing the N-site of membrane. This is because the catalytic subunit of SoeABC complex is predicted to be oriented toward the N-side of the membrane (Dahl et al., 2013;Boughanemi et al., 2020) (Figure 1) and thus SoeC would be expected to have an inverted orientation inside the membrane plane comparing to the other NrfD-like proteins.
We observed, within the predicted quinone/quinol-binding site of all NrfD-like subunits analyzed, the presence of 5, on average, amino acid residues (histidine, arginine, aspartate, glutamate, serine, tyrosine, threonine and asparagine) that may be involved in quinone/quinol-binding and stabilization in each protein (Figure 6, Table 2).
Frontiers in Chemistry | www.frontiersin.org FIGURE 6 | Quinone-binding site. Zoomed views of structural models of NrfD-like subunits showing the respective putative quinone/quinol-binding sites, located close to the positive-side (P-side) of the membrane. The SoeC subunit is expected to be in an inverted orientation toward the membrane in relation to the other NrfD-like subunits and thus its quinone/quinol binding site is located close to the negative-side (N-side). The amino acid residues composing the different quinone/quinol-binding sites are indicated and depicted as sticks. Please note that all models are oriented with the P-side of the membrane at the top of each panel, except for SoeC that is oriented with the N-side of the membrane at the top of the respective panel.

Ion Translocation Pathways
NrfD-like subunit-containing complexes were hypothesized to be capable of ion-translocation across the membrane (Calisto et al., 2021) and, in fact, proton-conducting pathways have been identified in the structures of the PsrC, ActC and ActF subunits Sousa et al., 2018;Sun et al., 2018;Shi et al., 2020). Proton-conducting pathways are formed by amino acid residues with side chains that can establish hydrogen bonds, constituting a hydrogen bond network. This allows proton transfer by a Grotthuss-type mechanism, which involves successive breaking and concomitant formation of hydrogen bonds (de Grotthuss, 1806;Cukierman, 2006). In R. marinus ActC and ActF subunits, the putative ionconducting pathway was suggested to be formed by two halfchannels: a N-side half-channel in the first four-helix bundle (TMH 1-4) and a P-side half-channel in the second four-helix bundle (TMH 5-8) . ActC and ActF ionconducting pathways are composed of conserved amino acid residues within all ACIII complexes   (Figure 7, ActC; Table 2).
We were able to identify, in all NrfD-like subunits, amino acid residues that may constitute a N-side half-channel in the first four-helix bundle (TMH 1-4) and a P-side half-channel in the second four-helix bundle (TMH 5-8). The amino acid residues that compose these putative ion-conducting pathways are conserved within each subunit: ActC, ActF, HybB, ArrC, DsrP, QrcD, SreC, NrfD and NhcC subunits (Figure 7, Table 2). However, in PsrC, DmsC, TtrC, SoeC, MccD and FqoC subunits, the two half-channels were not so easy to define, as we observed less conserved residues.
In T. thermophilus PsrC structure, the putative ion-conducting pathway was suggested to be formed by a N-side half-channel in the second four-helix bundle (TMH 5-8) and a P-side halfchannel in the first four-helix bundle (TMH 1-4) . Of the proposed residues in the N-side halfchannel at TMH 5-8 (Glu224 PsrC_T , Thr220 PsrC_T , Arg177 PsrC_T ) , only Arg177 PsrC_T residue is conserved among other PsrC T subunits. Mutation of Asp218 PsrC_W , the equivalent to Arg177 PsrC_T in W. succinogenes PsrC, and Ser192 PsrC_W residues, both located at TMH 5-8 in the N-side half-channel, resulted in inhibition or reduction of polysulfide respiration, respectively . Nevertheless, we identified conserved amino acid residues (Figure 7, PsrC T; Table 2) that could form two half-channels, resembling those identified in ActC and ActF subunits. Our identification is supported by substitution studies of amino acid residues located at TMH 1-4 on our proposed N-side half-channel (Tyr106 PsrC_W and Glu146 PsrC_W ) and located at TMH 5-8 on the P-side half-channel (Glu225 PsrC_W , Ser185 PsrC_W , Ser188 PsrC_W and Tyr310 PsrC_W ) of W. succinogenes PsrC model, resulted in strains with a compromised polysulfide respiration (Dietrich and Klimmek, 2002). These data suggest those residues may be important for proton translocation, since PsrABC catalyzes an endergonic reaction dependent of electrochemical potential (Dietrich and Klimmek, 2002;Calisto et al., 2021).
The presence of an ion-conducting pathway in HybB is also supported by the observations that replacement of Arg89 HybB , Tyr99 HybB , Glu148 HybB and His184 HybB in E. coli HybB (Nside half-channel TMH 5-8) significantly decreased hydrogen oxidation . Addition of a protonophore increases hydrogen oxidation in these mutated strains , showing that ion translocation and catalytic activity are coupled.
Previously for QrcD, only the N-side half-channel, present in the first four-helix bundle (TMH 1-4) as observed for ActC and ActF subunits, was proposed to be present (Duarte et al., 2018). This N-side half-channel was hypothesized to translocate protons from the N-side of the membrane to the quinone-binding site, for quinone reduction (Duarte et al., 2018). However, we identified conserved amino acid residues that could be part of a P-side halfchannel in the second four-helix bundle (TMH 5-8) (Figure 7, QrcD; Table 2).
FIGURE 7 | Ion-translocation pathway. Structural models of NrfD-like subunits with amino acid residues putatively involved in ion-translocation. The top left side panel contains a general schematic representation of NrfD-like subunits. Each protein is composed by eight common TMH that form two four-helix bundle (TMHs 1-4, light FIGURE 7 | gray [lighter colors in the following panels) and TMHs 5-8, dark gray (darker colors in the following panels)] related by a 180 • rotation around an axis perpendicular to the membrane. The light blue line schematizes the ion-conducting pathway and the red dot indicates the presence of the arginine residue (Arg395 ActC ), possibly acting as the gate of the pathway. The orange circle points to the quinone/quinol-binding site, which is in close proximity to the [4Fe-4S] +2/+1 cluster ([3Fe-4S] 1+/0 in ActC) (cube) present at the peripheral subunit. The other panels contain the structural models obtained for the 8 common TMHs, the amino acid residues, putatively involved ion-pathways are indicated and depicted as sticks along the light blue line and are highlighted in black (conserved residues in respective sequence alignments), gray (amino acid residues present in the model able to conduct protons, but not conserved in respective sequence alignments) and red (equivalent to Arg395 ActC ). Please note that all models are oriented with the P-side of the membrane at the top of each panel, except for SoeC that is oriented with the N-side of the membrane at the top of the respective panel (see more in text and in legend of Figure 6).
Although the ion-conducting pathways present in the different NrfD-like subunits are not composed by the same amino acid residues, they are localized at the same spatial position. Noticeably, we identified a conserved arginine residue (Arg395 ActC , Arg239 PsrC_T , Arg305 PsrC_W , Arg329 HybB , Arg271 DmsC , Arg324 ArrC , Arg358 QrcD , Arg306 NrfD , Arg280 TtrC , Arg345 NhcC , Arg297 SoeC , Arg306 MccD , Arg293 FqoC ) located in middle of the membrane (in TMH 8) in a position that coincides with that at which the two proton half-channels converge (Figure 7). In Desulfovibrio vulgaris DsrP this is occupied by a lysine (Lys329 DsrP1 ). We hypothesized that the residue at this position may perform a gate keeping role for proton translocation across the membrane. In fact, water molecules around Arg239 PsrC_T were observed in the structure of PsrC from T. thermophilus  and mutation of Arg305 PsrC_W resulted in inhibition of polysulfide respiration (Dietrich and Klimmek, 2002). Although, we hypothesized a relevant function for this arginine (or Lys329 DsrP1 ) residue, it is not conserved in ActF, DsrP2 and SreC. In these subunits the gating role may be performed by a conserved serine residue (Ser238 ActF , Ser204 DsrP2 and Ser235 SreC ), which is located in middle of the membrane in TMH 5, also coinciding with the convergence of the two half-channels (Figure 7).
The members of the NrfD family are transmembrane proteins (8 to 10 TMHs) characterized by the presence of structural repeats, composed of two four-helix bundles, harboring iontranslocation pathways and a quinone/quinol-binding site.
The quinone/quinol-binding site of NrfD-like subunits is located at the P-side of the membrane in the first four-helix bundle (TMHs 1-4), always in vicinity of the peripheral ironsulfur subunit. The peripheral subunits of SoeABC complex are hypothesized to be located at the N-side (Dahl et al., 2013;Boughanemi et al., 2020) and thus SoeC would be expected to be in an inverted orientation in the membrane when comparing to the other NrfD-like proteins. In this way, its quinone-binding site would be present on the N-side of the membrane. We identified, in TMH 2 close to the entry of the quinone/quinol pocket, a serine residue (Ser164 ActC ) that appears to be important for interaction with quinone/quinol molecule, since this serine residue is strictly conserved in all NrfD-like subunits that interact with quinone/quinol. Our structural models reinforce the possible existence of ion-translocation pathways in all NrfD-like subunits and the contribution of the NrfD-like subunit-containing complexes to energy transduction. NrfD-like subunit-containing complexes may perform energy transduction by an indirect-coupling mechanism and may generate or consume electrochemical potential (Calisto et al., 2021). W. succinogenes PsrABC activity was shown to be dependent on electrochemical potential (Dietrich and Klimmek, 2002), while QrcABCD activity was shown to be coupled to the formation of electrochemical potential (Duarte et al., 2018). The ion-translocation pathways, observed in NrfD-like subunits, are formed by amino acid residues that may establish hydrogen bonds, allowing proton translocation by a Grotthuss-type mechanism (de Grotthuss, 1806;Cukierman, 2006). A semiconserved arginine amino acid residue, located in the middle of the membrane at the intersection of the two ion half-channels, was here suggested to play an important role as gate keeper in ion-translocation. In ActF, DsrP2 and SreC the gating role may be performed by a conserved serine.
The data here presented indicate that NrfD-like subunits are possibly the ion translocating modules of different enzymes, involved in a vast array of metabolisms, such as the oxygen, nitrogen, sulfur, arsenate and hydrogen cycles. All NrfD-like subunits containing complexes may be thus energy transducing membrane machines that contribute to energy conservation in vast range of organisms and under multiple growth conditions.
The dendrogram was constructed using RAxML toll and the Neighbor-Joining (NJ) method at the CIPRES gateway portal (Miller et al., 2010). The obtained dendrogram was visualized and manipulated in Dendroscope (Huson et al., 2007).

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AUTHOR CONTRIBUTIONS
FC and MP conceived the study, analyzed the results, and wrote the manuscript. All authors contributed to the article and approved the submitted version.