Introduction
Between 12-29% of the organic carbon that is delivered to the sea floor
is mineralized by biological sulfate
(SO42-)/bisulfite
(HSO3-) reduction (1). As such,
SO42-/HSO3-reducing organisms (SRO) play substantial roles in the global sulfur and
carbon cycles, both today (2, 3) and in the geologic past (4–6).
Dissimilatory reduction of
SO3-/HSO3-to hydrogen sulfide (H2S) in most SRO is catalyzed by
the enzyme dissimilatory sulfite reductase (Dsr) in a reaction that
requires six electrons: SO32- +
6e- + 8H+ H2S + 3
H2O (7). Based on fractionation of sulfur isotopes
preserved in sulfide and sulfate minerals in rocks, dissimilatory
SO42-/HSO3-reduction (presumably via a Dsr-like enzyme) is thought to have evolved
as early as 3.47 billion years ago (6). The earliest evolving SRO may
have reduced
SO3-/HSO3-(8–12), that would have been readily produced through solvation of
sulfur dioxide (SO2) released into the biosphere via
widespread volcanism on early Earth (13). Subsequently, the ability for
SRO to use SO42- as an additional
electron acceptor likely occurred in response to the gradual oxidation
of Earth, culminating in the Great Oxidation Event (GOE) that occurred
~ 2.3 billion years ago (14). Preceding the GOE for
several hundred million years, sustained production of
O2 allowed for oxidative weathering of continental
sulfides that led to the release of
SO42- to oceans (15). At the same
time, sustained production of O2 would have led to a
decrease in the availability of HSO3-,
since it is unstable in the presence of strong oxidants like
O2 and Fe(III) (16), both of which became more abundant
on an oxygenated Earth. The combination of decreased availability of
HSO3- and increased availability of
SO42- may have represented the
selective pressure to recruit ATP sulfurylase (Sat) that catalyzes the
ATP-dependent activation of SO42- to
adenosine 5’-phosphosulfate (APS), and APS reductase (AprAB) that
reduces APS to HSO3-, thereby allowing
for the use of SO42- as an oxidant. In
potential support of this model, the reduction of
SO42- to
HSO3- is an endergonic process
(requires ATP), whereas HSO3-reduction is exergonic and is the major energy conserving step during
SO42- reduction (17). Further evidence
in support of HSO3- reduction
preceding SO42- reduction comes from
physiological studies that reveal higher growth yields in model SRO when
grown with HSO3- relative to those
grown with SO42- (18,
19).. As such, the use of
SO42-, which imparts an additional
energetic burden on SRO, appears to be an adaptation to allow for
respiration of an oxidant, SO42-, that
was much more widely available later in Earth history.
Extant Dsr enzymes are hetero-tetrameric and composed of two highly
homologous A and B subunits thought to have evolved from gene
duplication (20). Electrons for HSO3-reduction derived from small organic molecules (i.e., lactate) or
H2 are thought to be transferred to DsrAB via a third
labile subunit, DsrC, through two C-terminal cysteine residues (7).
Thousands of DsrAB sequences have been generated from cultivars and from
environmental amplicon-based or metagenomic surveys that have been used
to characterize the ecology of SRO and/or to reconstruct their
evolutionary histories (21–24). These studies have revealed that SRO
inhabit a broad range of habitat types, including subsurface,
hydrothermal, soil, and freshwater/marine sediment environments.
Moreover, DsrAB are much more widespread throughout archaeal and
bacterial lineages than suggested from cultivars only a decade ago (23,
24). In particular, metagenomic surveys have significantly expanded the
known taxonomic and genomic backgrounds where DsrAB are found, although
many of the taxa harboring DsrAB are uncultured and thus, the function
of DsrAB in these organisms is inferred from closely related cultivars
or based on phylogenetic clustering among defined DsrAB groups (23, 24).
The recovery of SRO and their corresponding Dsr sequences from
environments that are subject to extremes of pressure, temperature, salt
concentration, and pH indicate that the organisms harboring Dsr have
diversified to function under diverse physiological conditions. For
example, a novel DsrAB-encoding euryarchaeote (within the
Diaforarchaea/Thermoplasmatota group) was recently discovered in
moderately acidic (pH range of ~3.0 to 5.4), high
temperature (~50°C to 75°C) springs in Yellowstone
National Park (YNP) through metagenomic sequencing (11). Several nearly
complete genomes were recovered across multiple springs and years that
were representative of these organisms, and none encoded Sat or AprAB,
consistent with the ability of these organisms to grow with
HSO3-, but not
SO42- (11). Phylogenetic analyses
suggest these euryarchaeote DsrABs to belong to the earliest evolving
lineage that also includes sequences belonging to other thermophilic
Archaea largely found in hydrothermal vents or hot springs, and that
which are generally inferred to respire
HSO3- (but not necessarily
SO42- ). These results add credence to
the notion that Dsr evolved to allow for the reduction of
HSO3- and later diversified to allow
for the reduction of SO42- in habitats
characterized by more modest environmental conditions. However, while it
is clear that DsrAB has substantively diverged at the primary sequence
level, it is unclear if this divergence translates to structural
variation and whether conserved structural features of DsrAB exist that
are invariant to change, irrespective of environmental conditions.
To begin to assess whether structural changes have occurred throughout
the evolutionary history of Dsr, we modeled the structural
characteristics of early-branching archaeal Dsr (i.e., from the newly
characterized euryarchaotes described above) and compared these to those
from an early-diverging group of Dsr (i.e., from the Archaeoglobales)
and from a later-evolving group of Dsr (i.e., from Desulfovibrioand other Deltaproteobacteria). Sequence co-evolution (co-variance)
analysis can provide information about residue-residue contacts and on
functional coupling between distant sites (25–28). Sequence
conservation and co-variance analysis of DsrAB sequences in the context
of representative three-dimensional structures and models across the
family was used to identify functionally-critical direct interactions,
as well as longer-range potential functional couplings consistent with
an intersubunit allosteric network, reminiscent of the negative
cooperativity in the Mo-Fe nitrogenase (29). Observations of structural
evolution are discussed in the context of environmental- and
taxonomic-level adaptations that are likely to have taken place during
the evolution of Dsr and the organisms that encode these enzymes.