Discussion
The diversity of SRO and their DsrAB enzymes has been greatly expanded
through recent cultivation-dependent and cultivation-independent
approaches (11, 23, 24). Together these studies suggest that Dsr likely
emerged to catalyze SO32- reduction
and then diversified (through recruitment of APS and Sat) to catalyze
SO42- reduction and ultimately
HS- oxidation (9–12). The phylogenetic studies
conducted herein suggest that model bacterial SROs implicated as major
players in contemporary biogeochemical S cycling (e.g.,
Deltaproteobacteria and Firmicutes) evolved comparatively recently
whereas early evolving archaeal SROs (and a few taxonomically patchy
bacterial genera) tend to be restricted to hydrothermal or more
nutrient-limited extreme environments (11) where oxidant limitation is
likely pervasive (50). While the ecological drivers of the evolution of
SROs (via Dsr phylogeny) is obscured in the present study by limited
corresponding metadata (e.g., cardinal growth parameters, geochemistry)
associated with these organisms or the environments from where they were
recovered, the broad differences in habitats of early evolving and later
evolving SROs suggests that the ecology of Dsr-harboring SROs has
evolved over time. Yet, it remains unclear if the structure and thus,
functional mechanisms of Dsr have also evolved during its evolutionary
history.
Despite the collective abovementioned observations indicating SRO (and
thus Dsr) diversification across gradients in temperature, pH, salinity,
and pressure, amongst other variables, the phylogenetic studies
conducted herein and elsewhere document a general pattern of vertical
inheritance and a high degree of overall primary sequence conservation
across all DsrAB (11, 23, 24). Consistent with these findings, available
structures and our structural models of selected Dsr enzymes that
represent much of the known sequence diversity of Dsr generated herein
reveal a high degree of structural conservation. Dsr forms a
heterotetrameric structure comprising two heterodimers of DsrA and DsrB,
the latter of which arose from an ancestral gene duplication (22, 51).
The high degree of structural conservation, including at the inferred
quaternary level, suggests that all extant lineages of SROs settled on
this Dsr structural configuration prior to the radiation of SROs.
The evolutionary co-variance in the extensive inferred conservation in
the structure of Dsr revealed a group of three residues in each
heterodimer that form a pathway between the two active site sirohemes.
Based on the homologous monomeric FPECs from both the A. fulgidusand MV2-Eury A subunit queries, and a second set of monomeric FPECs from
the A. fulgidus B subunit query adjacent to two of the three
residues, we conclude that the N393A2/1-T351B1/2 (A. fulgidus )
pair is truly coupled evolutionarily. Given the position of N180B1/2
interacting with the active site heme and the Fe-S center, and its
relatively close proximity to the N393A2/1 residue, we hypothesize that
N180B1/2-N393A2/1 are also evolutionarily coupled. Based on structural
link formed by these residues between the two active sites, we
hypothesize that, beyond simply stabilizing contacts between
heterodimers, the pathway traced by these residues could correspond to
an ancient allosteric pathway (i.e., the “heme road”) that could have
provided the advantage of allosteric control when the heterodimer to
heterotetramer transition occurred. While these heme road residues are
not in direct contact, their interactions are mediated by only one or
two aromatic residues. Consistent with the existence of an allosteric
pathway are the hinge motions predicted by ANM. Inter-domain motions in
multi-chain protein complexes are an increasingly appreciated aspect of
their dynamic structures and thermodynamics, with the potential to
modulate their functions (52–54). Our Gaussian Network normal mode
analysis (40, 41, 49) indicates the possibility of intersubunit
“rocking” in the structure of the DsrAB complex, like that observed
for other dimeric complexes (52). These motions could in principle be
frozen out by crystallization of multiple members of the DsrAB family,
as also observed across crystal structures of dimeric influenza NS1
protein domains (52). In further support of such interfacial dynamics in
DsrAB complexes, which could also relate to the proposed intersubunit
allostery, is the lower quality of the electron density maps noted by
the authors of the A. fulgidus structure between the A2B2, as
opposed to the A1B1, heterodimers (20). This difference in quality could
reflect an asymmetry in the structures of the two heterodimers due to
allosteric interactions. Re-evaluation of the A. fulgidus DsrAB
crystal structure, 3MM5, suggests that the difference between the two
heterodimers in the crystal structure comes from distinct packing. This
allows for greater flexibility that, according to our TLS or ensemble
refinements, appears to be related to rigid-body movements, consistent
with our ANM normal mode analyses.
The proposed heme road allosteric pathway could serve to allow for
communication between the two active sites, one in each heterodimer,
during the delivery of 2 e- from DsrC to one of them.
Such an interaction might inhibit DsrC binding and electron injection
into one heterodimer, while the other is active. . Interestingly, it has
recently been proposed that DsrD, a small protein that is found in
late-evolving SROs, acts as an allosteric regulator of DsrAB (55). This
is the case for negative cooperativity in the function of the Mo-Fe
nitrogenase heterotetramer (29), which exhibits similar rocking normal
modes as DsrAB. Examples of statistically detected evolutionarily
conserved pathways of energetic coupling within proteins have been
reported for several proteins, including PDZ domains, GPCRs,
chymotrypsin, lectin and hemoglobin (27, 56–59). We emphasize the
importance of experimental validation of putative allosteric networks
revealed by statistical analysis of sequence co-evolution. Such
validation can be accomplished by combinations of approaches including
H/D exchange mass spectrometry (60) and/or NMR (61), and spectroscopic
approaches coupled with mutagenesis (62). We note that given the
distances, we cannot rule out that the heme road corresponds to a
pathway for electron transfer between active sites, although it is
difficult to rationalize its utility.
While this proposed pathway warrants experimental scrutiny, the
co-evolution at the positions putatively involved also indicates that
this pathway was likely established prior to the radiation of all DsrAB,
although the precise residues involved in these interactions vary across
Dsr enzyme types. Consequently, the presence of a slightly modified
allosteric pathway may have allowed fine-tuning of Dsr activity in the
context of different physiological backgrounds, including those that
operate under chronic energy (dissimilatory e-shuttling) stress imposed by extreme conditions (e.g., temperature, pH
and pressure) (50) where the kinetics of
HSO3- reduction and thus growth of SRO
are likely to be much slower. The combination of the evolutionary
coupling analysis and ANM dynamic “normal mode” calculations provide
an intriguing set of residues to probe for their involvement in
structure-function relationships in future experiments.
In addition to the aforementioned putative allosteric pathway, it is
possible that the presence of the W119 residue in A. fulgidus Dsr
and the loss of the iron in the sirohydrochlorin moieties in D.
vulgaris Dsr represent two separate mechanisms to limit function to the
“top” siroheme in each heterodimer, adjacent to the binding site of
the DsrC putative electron carrying subunit. Evolution of the gaps and
inserts in the region of the structural hemes may have contributed to
their loss of function. Clearly, detailed comparative enzymatic assays
will be required to demonstrate the structural, as opposed to
functional, role of the “bottom” siroheme”.
Collectively, the combination of phylogenetic and structural
bioinformatics studies of Dsr conducted herein point to the need for
comparative experimental studies to test the present hypotheses in order
to fully understand the functional differences encompassed within the
diversity of these enzymes. This is particularly true for the two
most-basal branching lineages of Dsr from SRO that are most reminiscent
of the ancestral Dsr enzymes that likely shaped sulfur and carbon
biogeochemical cycles on early Earth and that continue to shape these
cycles in thermal environments. The vast majority of these taxa
(inclusive of MV2-Eury) are either known only from
cultivation-independent environmental genomics studies or have
very-limited cultivation information. Thus, future efforts should first
be made to domesticate these SROs and optimize cultivation conditions to
enable more thorough investigations of their physiology, ecology, and
enzymology. In particular, these efforts should focus on SROs that are
physiologically unlike canonical SROs that conduct
SO42- reduction but that rather are
limited to SO32- reduction. Such
investigations would enable a better understanding of the evolution of
Dsr as it transitioned from early
SO32- respiring organisms to the
SO42- reducers that are widespread in
anoxic environments on Earth today.