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\begin{document}

\title{Structural Evolution of the Ancient Enzyme, Dissimilatory Sulfite
Reductase}



\author[1]{Daniel R. Colman}%
\author[2]{Gilles Labesse}%
\author[3]{Swapna Gurla}%
\author[4]{Johanna Stefanakis}%
\author[5]{Gaetano Montelione}%
\author[1]{Eric S. Boyd}%
\author[6]{Catherine Royer}%
\affil[1]{Montana State University Department of Microbiology and Cell Biology}%
\affil[2]{Centre de Biochimie Structurale}%
\affil[3]{Rutgers Robert Wood Johnson Medical School Department of Biochemistry and Molecular Biology}%
\affil[4]{Bronx High School of Science}%
\affil[5]{Rensselaer Polytechnic Institute Department of Chemistry and Chemical Biology}%
\affil[6]{Department of Biological Sciences Rensselaer Polytechnic Institute Troy NY 12180}%


\vspace{-1em}



  
  \date{June 16, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
Dissimilatory sulfite reductase is an ancient enzyme that has linked the
global sulfur and carbon biogeochemical cycles since at least 3.47 Gya.
While much has been learned about the phylogenetic distribution and
diversity of DsrAB across environmental gradients, far less is known
about the structural changes that occurred to maintain DsrAB function as
the enzyme accompanied diversification of sulfate/sulfite reducing
organisms (SRO) into new environments. Analyses of available crystal
structures of DsrAB from \emph{Archaeoglobus fulgidus} and
\emph{Desulfovibrio vulgaris}, representing early and late evolving
lineages, respectively, show that certain features of DsrAB are
structurally conserved, including active siro-heme binding motifs.
Whether such structural features are conserved among DsrAB recovered
from varied environments, including hot spring environments that host
representatives of the earliest evolving SRO lineage (e.g., MV2-Eury),
is not known. To begin to overcome these gaps in our understanding of
the evolution of DsrAB, structural models from MV2.Eury were generated
and evolutionary sequence co-variance analyses were conducted on a
curated DsrAB database. Phylogenetically diverse DsrAB harbor many
conserved functional residues including those that ligate active
siro-heme(s). However, evolutionary co-variance analysis of monomeric
DsrAB subunits revealed several False Positive Evolutionary Couplings
(FPEC) that correspond to residues that have co-evolved despite being
too spatially distant in the monomeric structure to allow for direct
contact. One set of FPECs corresponds to residues that form a structural
path between the two active siro-heme moieties across the interface
between heterodimers, suggesting the potential for allostery or electron
transfer within the enzyme complex. Other FPECs correspond to structural
loops and gaps that may have been selected to stabilize enzyme function
in different environments. These structural bioinformatics results
suggest that DsrAB has maintained allosteric communication pathways
between subunits as SRO diversified into new environments. The
observations outlined here provide a framework for future biochemical
and structural analyses of DsrAB to examine potential allosteric control
of this enzyme.%
\end{abstract}%



\sloppy


\textbf{Structural Evolution of the Ancient Enzyme, Dissimilatory
Sulfite Reductase}

Daniel R. Colman\textsuperscript{1}, Gilles Labesse\textsuperscript{2},
G.V.T. Swapna\textsuperscript{3}, Johanna Stefanakis\textsuperscript{4},
Gaetano T.

Montelione\textsuperscript{5}, Eric S. Boyd\textsuperscript{1}* and
Catherine A. Royer\textsuperscript{6}*

\textsuperscript{1}Department of Microbiology and Cell Biology, Montana
State University, Bozeman, Montana 59717

\textsuperscript{2}Centre de Biochimie Structurale, CNRS UMR 5048,
Montpellier, France 34090

\textsuperscript{3}Dept of Biochemistry and Molecular Biology, Robert
Wood Johnson Medical School, Rutgers The State University of New Jersey,
Piscataway, NJ, 08854 USA

\textsuperscript{4}The Bronx High School of Science, New York, NY, 10468

\textsuperscript{5}Department of Chemistry and Chemical Biology, and
Center for Biotechnology and Interdisciplinary Studies, Rensselaer
Polytechnic Institute, Troy, NY 12180

\textsuperscript{6}Department of Biological Sciences, Rensselaer
Polytechnic Institute, Troy, NY 12180

JS: stefanakisj@bxscience.edu

DRC: 0000-0002-3253-6833; daniel.colman@montana.edu

GL: 0000-0002-6861-3300;; gilles.labesse@cbs.cnrs.fr

GVTS: 0000-0001-9342-2525; swapna.gurla@gmail.com

GTM: 0000-0002-9440-3059; monteg3@rpi.edu

ESB: 0000-0003-4436-5856; eric.boyd@montana.edu

CAR: 0000-0002-2670-3391; royerc@rpi.edu

Running Title Evolution of Dissimilatory Sulfite Reductase

* - To whom correspondence should be addressed

\textbf{Abstract}

Dissimilatory sulfite reductase is an ancient enzyme that has linked the
global sulfur and carbon biogeochemical cycles since at least 3.47 Gya.
While much has been learned about the phylogenetic distribution and
diversity of DsrAB across environmental gradients, far less is known
about the structural changes that occurred to maintain DsrAB function as
the enzyme accompanied diversification of sulfate/sulfite reducing
organisms (SRO) into new environments. Analyses of available crystal
structures of DsrAB from \emph{Archaeoglobus fulgidus}
and\emph{Desulfovibrio vulgaris} , representing early and late evolving
lineages, respectively, show that certain features of DsrAB are
structurally conserved, including active siro-heme binding motifs.
Whether such structural features are conserved among DsrAB recovered
from varied environments, including hot spring environments that host
representatives of the earliest evolving SRO lineage (e.g., MV2-Eury),
is not known. To begin to overcome these gaps in our understanding of
the evolution of DsrAB, structural models from MV2.Eury were generated
and evolutionary sequence co-variance analyses were conducted on a
curated DsrAB database. Phylogenetically diverse DsrAB harbor many
conserved functional residues including those that ligate active
siro-heme(s). However, evolutionary co-variance analysis of monomeric
DsrAB subunits revealed several False Positive Evolutionary Couplings
(FPEC) that correspond to residues that have co-evolved despite being
too spatially distant in the monomeric structure to allow for direct
contact. One set of FPECs corresponds to residues that form a structural
path between the two active siro-heme moieties across the interface
between heterodimers, suggesting the potential for allostery or electron
transfer within the enzyme complex. Other FPECs correspond to structural
loops and gaps that may have been selected to stabilize enzyme function
in different environments. These structural bioinformatics results
suggest that DsrAB has maintained allosteric communication pathways
between subunits as SRO diversified into new environments. The
observations outlined here provide a framework for future biochemical
and structural analyses of DsrAB to examine potential allosteric control
of this enzyme.

Keywords: Dissimilatory sulfite reductase, Phylogeny, Evolutionary
covariance, structural modeling,

Introduction

Between 12-29\% of the organic carbon that is delivered to the sea floor
is mineralized by biological sulfate
(SO\textsubscript{4}\textsuperscript{2-})/bisulfite
(HSO\textsubscript{3}\textsuperscript{-}) reduction (1). As such,
SO\textsubscript{4}\textsuperscript{2-}/HSO\textsubscript{3}\textsuperscript{-}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
SO\textsubscript{3}\textsuperscript{-}/HSO\textsubscript{3}\textsuperscript{-}to
hydrogen sulfide (H\textsubscript{2}S) in most SRO is catalyzed by the
enzyme dissimilatory sulfite reductase (Dsr) in a reaction that requires
six electrons: SO\textsubscript{3}\textsuperscript{2-} +
6e\textsuperscript{-} + 8H\textsuperscript{+} H\textsubscript{2}S + 3
H\textsubscript{2}O (7). Based on fractionation of sulfur isotopes
preserved in sulfide and sulfate minerals in rocks, dissimilatory
SO\textsubscript{4}\textsuperscript{2-}/HSO\textsubscript{3}\textsuperscript{-}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
SO\textsubscript{3}\textsuperscript{-}/HSO\textsubscript{3}\textsuperscript{-}(8--12),
that would have been readily produced through solvation of sulfur
dioxide (SO\textsubscript{2}) released into the biosphere via widespread
volcanism on early Earth (13). Subsequently, the ability for SRO to use
SO\textsubscript{4}\textsuperscript{2-} as an additional electron
acceptor likely occurred in response to the gradual oxidation of Earth,
culminating in the Great Oxidation Event (GOE) that occurred
\textasciitilde{} 2.3 billion years ago (14). Preceding the GOE for
several hundred million years, sustained production of
O\textsubscript{2} allowed for oxidative weathering of continental
sulfides that led to the release of
SO\textsubscript{4}\textsuperscript{2-} to oceans (15). At the same
time, sustained production of O\textsubscript{2} would have led to a
decrease in the availability of HSO\textsubscript{3}\textsuperscript{-},
since it is unstable in the presence of strong oxidants like
O\textsubscript{2} and Fe(III) (16), both of which became more abundant
on an oxygenated Earth. The combination of decreased availability of
HSO\textsubscript{3}\textsuperscript{-} and increased availability of
SO\textsubscript{4}\textsuperscript{2-} may have represented the
selective pressure to recruit ATP sulfurylase (Sat) that catalyzes the
ATP-dependent activation of SO\textsubscript{4}\textsuperscript{2-} to
adenosine 5'-phosphosulfate (APS), and APS reductase (AprAB) that
reduces APS to HSO\textsubscript{3}\textsuperscript{-}, thereby allowing
for the use of SO\textsubscript{4}\textsuperscript{2-} as an oxidant. In
potential support of this model, the reduction of
SO\textsubscript{4}\textsuperscript{2-} to
HSO\textsubscript{3}\textsuperscript{-} is an endergonic process
(requires ATP), whereas HSO\textsubscript{3}\textsuperscript{-}reduction
is exergonic and is the major energy conserving step during
SO\textsubscript{4}\textsuperscript{2-} reduction (17). Further evidence
in support of HSO\textsubscript{3}\textsuperscript{-} reduction
preceding SO\textsubscript{4}\textsuperscript{2-} reduction comes from
physiological studies that reveal higher growth yields in model SRO when
grown with HSO\textsubscript{3}\textsuperscript{-} relative to those
grown with SO\textsubscript{4}\textsuperscript{2-} (18,
19)\textsuperscript{.}. As such, the use of
SO\textsubscript{4}\textsuperscript{2-}, which imparts an additional
energetic burden on SRO, appears to be an adaptation to allow for
respiration of an oxidant, SO\textsubscript{4}\textsuperscript{2-}, 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
HSO\textsubscript{3}\textsuperscript{-}reduction derived from small
organic molecules (\emph{i.e.,} lactate) or H\textsubscript{2} 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 \textasciitilde{}3.0 to 5.4), high
temperature (\textasciitilde{}50\selectlanguage{ngerman}°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
HSO\textsubscript{3}\textsuperscript{-}, but not
SO\textsubscript{4}\textsuperscript{2-} (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
HSO\textsubscript{3}\textsuperscript{-} (but not necessarily
SO\textsubscript{4}\textsuperscript{2-} ). These results add credence to
the notion that Dsr evolved to allow for the reduction of
HSO\textsubscript{3}\textsuperscript{-} and later diversified to allow
for the reduction of SO\textsubscript{4}\textsuperscript{2-} 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
\emph{Desulfovibrio}and 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.

Methods

\emph{DsrAB Database Generation and Phylogenetic Analyses}

To evaluate the structural properties that confer Dsr function over
broad geochemical space, an existing DsrAB database was curated from
cultivar and environmental genomes (23). The original database was
constructed to include DsrAB from genomes of cultivars, targeted
PCR-based surveys, and metagenomic data. Thus, many of the sequences
were incomplete, potentially confounding structural modeling
calculations. Therefore, sequence alignments and annotations were used
to curate the database to comprise only full length DsrA and DsrB
sequences. Specifically, sequences demarcated as ``partial'' and those
that were likely obtained via PCR-based methods were removed without
further consideration. Next, individual alignments of DsrA and DsrB were
performed using Clustal Omega (30), guided with primary sequences of
DsrAB from \emph{D. vulgaris} and \emph{A. fulgidus} . Sequences that
were substantially truncated relative to model DsrAB, including those
without start codons, were then removed, resulting in a total of 274
full-length DsrAB sequences. The database will be made available upon
request from the authors. Phylogenetic analysis of DsrAB sequences was
conducted, as previously described (11), and associated metadata was
mapped to the DsrAB phylogeny using environment of sequence origin (from
the original database publication), in addition to taxonomic information
(either from the original database publication or via BLASTp searches of
DsrA subunits against the NCBI nr database). DsrAB sequence homologs
were subjected to structural alignment using the PROMALS3D multiple
sequence and structure alignment server (31). Structures from \emph{D.
vulgaris} (2V4J; (32)) and \emph{A. fulgidus} (3MMC; (20)) served as
threads.

\emph{MV2-Eury Structural Homology Modeling}

Comparative modeling of MV2-Eury Dsr A, B, and C subunits was performed
after careful refinement of a structural alignment using ViTO (33) prior
to model building using MODELLER (34) as a heterohexamer. The model was
based on two templates PDB 3ORI1 (35) and PDB 3MM5 (36). The structure
of the MV2-Eury heterotetramer was also modeled with AlphaFold2 (AF2)
Multimer (37) from Deepmind, Inc. that is enabled for modeling of
complexes, installed on the Rensselaer~ Polytechnic Institute's Center
for Computer Innovation NPL cpu/gpu cluster
(https://secure.cci.rpi.edu/wiki/clusters/NPL\_Cluster/) providing
sufficient random-access memory and accessibility to handle these
relatively-large multi-chain protein complexes.

\emph{Evolutionary Co-variance and Anisotropic Network Analysis}

Evolutionary covariance (EC) analysis (25, 26, 38) was performed using
the EV Couplings server (https://evcouplings.org/ (39)) to identify
instances of False Positive Evolutionary Covariance (FPEC), defined as
significant covariance between residues that are too distant
(\textgreater{}20 Å) to be in direct contact within the monomeric
subunits. Given the strong homology between DsrA and DsrB subunits that
are suspected to have originated from gene duplication, it was not
possible to use the EV Complex Couplings option to identify co-varying
residues at subunit interfaces in the oligomers. Instead, EC analysis
was carried out using the monomeric sequences of the DsrA and DsrB
subunits of \emph{A. fulgidus} , \emph{D. vulgaris,} and MV2-Eury
separately as input. The values of N\textsubscript{eff} (number of
non-redundant sequences in the alignment normalized to the number of
residues) was between 1.16 and 2.84, depending upon the query, with the
number of non-redundant sequences was between 481 and 2786. Queries were
made by separately submitting the A and B subunits from the \emph{A.
fulgidus} and MV2-Eury sequences. Recovered coupling probabilities were
between 80 and 94\%. To model the possible dynamic fluctuations that
might be associated with the putative allosteric pathway that was
identified through EC analysis, we carried out Anisotropic Network
Modeling (ANM) (40) using the webserver from the Bahar group
(http://anm.csb.pitt.edu/cgi-bin/anm2/anm2.cgi; (41)).

\emph{Ensemble refinement}

Crystallographic coordinates and diffraction data for
the\emph{Archaeoglobis fulgidus} complex (PDB3MM5) were downloaded and
subjected to several types of refinement approached using the current
Python-based Hierarchical Environment for Integrated Crystallography
(Phenix) software (42--44). First, the number of TLS groups was
increased using the automatic definition available in Phenix, and some
manual refinement was performed by alternating Coot manual rebuilding
and Phenix minimization. Then, the refined structure was submitted to
ensemble refinement using Phenix\_ensemble with parameters adjusted
according to the default recommendations (pTLS = 1; wxray = 0.5 and Tx
=0.6 ).

Results

\emph{Phylogenetic Reconstruction of DsrAB}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

The curated database of full length DsrAB sequences (n=274) was used to
construct a concatenated DsrA and DsrB phylogeny, as described
previously (11), that was congruent with previous phylogenetic
reconstructions of DsrAB (11, 23, 24). As previously documented,
recently discovered Euryarchaeote DsrAB formed a group with those from
Crenarchaeota, that together formed a basal-branching group among all
DsrAB (Figure 1). All organisms within this first group were recovered
from hydrothermal environments (largely hot springs) and their DsrAB are
inferred to be involved in HSO\textsubscript{3}\textsuperscript{-} or
SO\textsubscript{4}\textsuperscript{2-} reduction (11). An additional
basal-branching second group comprised DsrAB recovered from uncultured
metagenome-assembled-genomes (MAGs) from various organisms and
environments, along with duplicate DsrAB copies within
\emph{Moorella}sp. genomes.

The remainder of DsrAB comprised a large group inclusive of both
reductive- and oxidative-type DsrAB that primarily includes bacterial
DsrAB. Within the ``bacterial'' group, homologs from Archaeoglobales
(Archaea) form a relatively early-evolving group, although the presence
of DsrAB in Archaeoglobales is thought to derive from a horizontal gene
transfer (HGT) event from a bacterial donor (22, 23). Lastly,
the\emph{D. vulgaris} homologs were present within a large cluster
comprising homologs from other putative and characterized
SO\textsubscript{4}\textsuperscript{2-} reducing Deltaproteobacteria.

Mapping of taxonomic information on to the DsrAB phylogenetic tree
revealed general concordance of DsrAB clades with their respective
taxonomic groups, consistent with previous analyses (23, 24). This
indicates that DsrAB are generally vertically inherited, although
several exceptions to this rule are evident including the example of
Archaeoglobales above. The mapping also revealed the broad range of
ecological contexts for SRO and their DsrAB. As documented previously
(11), DsrAB from organisms with subsurface and hydrothermal
environmental origins are particularly prominent near the root of the
tree, suggesting that the earliest DsrAB may derive from oxidant limited
and/or high temperature environments (45). However, general patterns of
ecological distributions beyond these early groups were not readily
apparent. This is likely attributable to the coarseness by which these
original environmental designations were assigned (i.e., by site of
isolation/sequence generation).

\emph{Analysis of Available Dsr Crystal Structures}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

Three dimensional crystal structures have been solved for DsrAB enzymes
from \emph{Archaeoglobus} \emph{fulgidus} (Figure 2A-B) (20,
36),\emph{Desulfovibrio vulgaris} (32) (Figure 2C-D),
\emph{Desulfovibrio gigas} (35), and \emph{Desulfomicrobium norvegicum}
(46) at 2, 2.1, 1.76 and 2.5 \selectlanguage{ngerman}Å resolution, respectively. The A and B
subunits consist of three domains, two of which are structurally
similar. The third is a ferredoxin-like domain, thought to have been
inserted between two beta strands of domain two after the gene
duplication event (20). Each A/B heterodimer in DsrAB harbors two
sirohemes (or one siroheme and a sirohydrochlorin moiety, representing a
siroheme without metal cofactors) and four {[}4Fe-4S{]} clusters that
are presumably involved in electron transfer to
HSO\textsubscript{3}\textsuperscript{-} (present in the \emph{D.
vulgaris} structure Figure 2B, D). A third, cysteine
disulfide-containing labile

subunit, DsrC, was purified and crystalized covalently bound to the
heterotetramer in the \emph{D. vulgaris} structure (32) (grey and purple
in Figure 2B, D) via one of the reduced cysteine residues. In the most
recently proposed model of the
HSO\textsubscript{3}\textsuperscript{-}reduction reaction cycle, DsrC in
reduced form binds to a S\textsuperscript{II} intermediate at the active
site in DsrAB, forming a S\textsuperscript{I} containing
hetero-disulfide. Hydrogen sulfide is then produced via an
S\textsuperscript{0}-containing protein tri-sulfide intermediate,
implicating both cysteine residues in the DsrC C-terminus (7). The final
four electrons required for this latter reaction are proposed to emanate
from the menaquinone pool, likely implicating the membrane DsrMK(JOP)
complex (47), thus coupling HSO\textsubscript{3}\textsuperscript{-}
reduction to proton translocation and energy conservation (7).

Within the DsrAB heterodimer, the A and B subunits are intricately
intertwined in the manner of clasped hands. In addition, the C-terminus
of the A subunit in one heterodimer crosses over the heterodimer
interface to interact extensively with both the A and B subunits of the
other (Figure 2A). While crystallographic domain swapping has been
demonstrated to be artefactual in many cases, the fact that all four
available DsrAB structures (20, 32, 35, 46) exhibit this feature
supports the notion that it is a fundamental feature of the DsrAB
structure. Besides this crossover interaction, the central interface
between heterodimers is quite open, with only limited contacts between
two helices of the B subunits from each heterodimer (Figure 2 D, E).
Interestingly, at this central interface between heterodimers, the
position of the two helices from the B1/B2 subunits responsible for the
contacts has been swapped between the \emph{A. fuglidus} and \emph{D.
vulgaris} structures. All three of the other available structures of
DsrAB (32, 35, 46) show the central interfacial helix positioning of
the\emph{D. vulgaris} structure shown in Figure 2D. Native gel
electrophoresis followed by mass spectrometry measurements made on
preparations of DsrABC from \emph{D. vulgaris} and \emph{D.
norvegicum}revealed that the major oligomeric species were
A\textsubscript{2}B\textsubscript{2}C\textsubscript{2} and
A\textsubscript{2}B\textsubscript{2}C heterohexamer and heteropentamer,
the C subunit being somewhat labile (46). These observations reinforce
the crystallographic results indicating that DsrAB is a heterotetramer.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

It is thought that only two of the siroheme moieties of Dsr (one per
heterodimer) support HSO\textsubscript{3}\textsuperscript{-} reduction
(20). While the \emph{A. fulgidus} heterotetramer (purified and
crystalized anaerobically) comports four siroheme moieties, the \emph{D.
vulgaris} structure (purified aerobically) has only two, the other two
being sirohydrochlorin groups lacking the iron. In this latter
structure, the HSO\textsubscript{3}\textsuperscript{-} ion is found in
interaction only with the siro-hemes (Figure 2B, dark blue). In
the\emph{A. fulgidus} structure, access to the ``bottom'' siroheme is
blocked by tryptophan B119 (Figure 3A), whereas in the \emph{D.
vulgaris} structure, no blocking residue is apparent near the
sirohydrochlorin group (Figure 3B), the corresponding side chain of
threonine B135 facing away from the heme.

\emph{Structural Homology Modeling of MV2-Eury DsrAB}

The structural homology model of the MV2-Eury DsrABC sequences (Figure
1E, F), was based on the hetero-hexameric structure from \emph{D.
vulgaris} (32) due the poor quality of the electron density for the
DsrAB structure from \emph{A. fulgidus} . An obvious difference between
the MV2-Eury model and the template is that the \selectlanguage{greek}β-\selectlanguage{english}hairpin of the DsrB
subunit of MV2-Eury that contacts DsrC (Figure 1E) is much shorter than
in \emph{D. vulgaris} , whereas the pseudo-symmetric \selectlanguage{greek}β-\selectlanguage{english}hairpin structure
in the MV2-Eury DsrA subunit near the inactive siroheme (Figure 1E,
bottom, green \& pink) is much longer. Examination of the sequences and
differences between the MV2-Eury Dsr model and the \emph{A}
.\emph{fulgidus} heterotetramer or the \emph{D. vulgaris} hetero-hexamer
structures suggested that much of the sequence variation among these
three homolog types was found at subunit interfaces (Figure S1).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

To complement the homology model in Figure 1, the MV2-Eury DsrAB
heterotetramer was modeled also using AF2 Multimer (37). In this case
the Fe-S centers and siroheme moieties are not present in the modeled
structure. Three models were obtained, with the highest confidence
(highest pLDDT scores) found for model 3 (Figure 4, Figure S2). As can
be seen in the model colored according to the pLDDT score (Figure 4,
Figure S2), the only regions of the complex that were poorly predicted
in the models were in subunit A, residues 50-62 and 397-300. The first
region interacts with the DsrC subunit (Figure 1B), while the second
corresponds to four residues at the end of the first \selectlanguage{greek}β-\selectlanguage{english}strand in a
\selectlanguage{greek}β-\selectlanguage{english}sheet adjacent to the A-subunit ferredoxin domain. Alignment of the
MV2-Eury AF2 model with the homology model shows reasonable agreement
(Figure 4), although the heterodimers in the AF2 model are rotated with
respect to each other around the central axis of the heterodimer
interface. Other notable differences are the orientations of the side
chains at central interface between heterodimers (Figure S3) and a
different orientation of the extended C-terminal A subunit helices that
contact the opposing subunits. Note that the orientation of this helix
is different in the \emph{A. fulgidus} and \emph{D. vulgaris} structures
as well. The region with the low pLDDT scores (residues 50-62 in the A
subunit) in the AF2 model exhibits a similar configuration to that in
the homology model (Figure S3) but is rotated away from the center of
the protein.

\emph{Evolutionary Co-variance Analysis of DsrAB}

Because many of the non-conserved residues in DsrAB are found at subunit
interfaces, Evolutionary co-variance (EC) analysis on the EVCouplings
server (as described in Materials and Methods) (39) was used to reveal
how subunit contacts may have evolved with environment types or
taxonomic groups that are distributed across the DsrAB phylogeny. We
were particularly interested in False Positive Evolutionary Couplings
(FPEC), as these often correspond to inter-subunit contacts within
oligomers. They also can arise from long range allosteric pathways or
dynamic structural heterogeneity (26), although these types of FPECs
represent a small fraction of the total co-varying residues (48).
Numerous FPECs were identified as evolutionarily co-varying residues
that did not correspond to a contact in the 2D residue contact map of
the monomer and were separated in space (within the monomers) by greater
than 20 \selectlanguage{ngerman}Å. Because the DsrA and DsrB subunits are highly homologous,
both in sequence and in structure (Figure S4), both subunits were
present in the multiple sequence alignments (MSAs) used in the
EVCouplings analysis (38, 39). Moreover, because DsrAB complexes are
hetero-tetramers, in principle, putative co-varying pairs of residues
could correspond to eight different possible residue pairs
(Res1A1-Res2A1, Res1B1-Res2B1, Res1A1-Res2B1, Res2A1-Res1B1,
Res1A1-Res2A2, Res1A1-Res2B2, Res2A1-Res1B2, and Res1B1-Res2B2).

\emph{Identification of a Potential Allosteric Pathway in DsrAB}

Two sets of high co-variance probability (\textgreater{}80\%) FPEC pairs
(Table 1, bold) were observed using the \emph{A. fulgidus} DsrA and DsrB
subunits as queries in the EVCoupling calculations. Table 1 lists the
homologous residues in the DsrB (or DsrA) subunits for these FPEC pairs
for the three DsrAB structures in Figure 2, along with the FPEC distance
and the coupling probability. Given the large distances between the
putative co-varying residues in the monomers, and hence the false
positive nature of the pair, the Res1A1-Res2A1 and Res1B1-Res2B1
co-variances are eliminated, leaving six remaining possible pairs of
co-varying residues. Highlighting the four residues from

the two A and two B subunits (eight residues in all) on the DsrAB
structure (Figure 5A-C), it is particularly striking that three out of
four of the possible implicated residues for each of the two sets of
monomer FPEC pairs describe a path from the functional heme in one
heterodimer to that

in the other heterodimer. We refer to this pathway as the ``heme road''.
The fourth possible co-varying residue (Table 1) from each heterodimer,
N222A1/2, is in a homologous structural position as N180B in the
structural alignment of the A and B subunits (Figure S4), but in the
heterotetramer N222A1/2 interacts with the ``lower'' inactive,
structural siroheme at a distance of 20.5 Å from the nearest of the
three other possible co-varying residues, whereas the homologous N180 of
the DsrB subunit is proximal to the ``upper'' active siroheme and one of
the nearby Fe-S centers,\selectlanguage{english}
\begin{longtable}[]{@{}lllllllll@{}}
\toprule
\begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Table 1. FPEC pairs for the heme road}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Table 1. FPEC pairs for the heme road}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Table 1. FPEC pairs for the heme road}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Table 1. FPEC pairs for the heme road}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Table 1. FPEC pairs for the heme road}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Table 1. FPEC pairs for the heme road}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Distance} \textbf{in monomer} \selectlanguage{ngerman}\textbf{(Å)}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Distance} \textbf{in monomer} \selectlanguage{ngerman}\textbf{(Å)}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Coupling probability} \textbf{(\%)}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Organisms\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Res1 DsrA\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Res 2 DsrA\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Res1 DsrB\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Res2 DsrB\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{A. fulgidus}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{N393}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\textbf{N222}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{T351}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{N180}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
33.51\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
33.51\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
90\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
90\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{D vulgaris}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
N410\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
N223\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
P368\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
N191\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
MV2-Eury\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\textbf{N369}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\textbf{L204}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
R337\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
S167\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
33.84\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
33.84\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
80\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
80\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{A. fulgidus}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
N392\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
K240\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\textbf{I350}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\textbf{R197}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
23.62\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
23.62\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
94\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
94\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{D vulgaris}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
T409\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
K241\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
D367\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
R208\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
MV2-Eury\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
T368\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
E222\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
L336\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
T184\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
FPEC pairs resulting from EVCouplings analysis are shown in bold. All
corresponding homologous residues in the \emph{A. fulgidus}, \emph{D.
vulgaris} and MV2-Eury A and B chain sequences are shown. Distances are
C\selectlanguage{greek}α-\selectlanguage{english}C\selectlanguage{greek}α \selectlanguage{english}distances. Bolded sequences correspond to FPEC residues from the
queried subunits, italicized and underlined sequences to probable
co-varying pairs (see text).\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
FPEC pairs resulting from EVCouplings analysis are shown in bold. All
corresponding homologous residues in the \emph{A. fulgidus}, \emph{D.
vulgaris} and MV2-Eury A and B chain sequences are shown. Distances are
C\selectlanguage{greek}α-\selectlanguage{english}C\selectlanguage{greek}α \selectlanguage{english}distances. Bolded sequences correspond to FPEC residues from the
queried subunits, italicized and underlined sequences to probable
co-varying pairs (see text).\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
FPEC pairs resulting from EVCouplings analysis are shown in bold. All
corresponding homologous residues in the \emph{A. fulgidus}, \emph{D.
vulgaris} and MV2-Eury A and B chain sequences are shown. Distances are
C\selectlanguage{greek}α-\selectlanguage{english}C\selectlanguage{greek}α \selectlanguage{english}distances. Bolded sequences correspond to FPEC residues from the
queried subunits, italicized and underlined sequences to probable
co-varying pairs (see text).\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
FPEC pairs resulting from EVCouplings analysis are shown in bold. All
corresponding homologous residues in the \emph{A. fulgidus}, \emph{D.
vulgaris} and MV2-Eury A and B chain sequences are shown. Distances are
C\selectlanguage{greek}α-\selectlanguage{english}C\selectlanguage{greek}α \selectlanguage{english}distances. Bolded sequences correspond to FPEC residues from the
queried subunits, italicized and underlined sequences to probable
co-varying pairs (see text).\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
FPEC pairs resulting from EVCouplings analysis are shown in bold. All
corresponding homologous residues in the \emph{A. fulgidus}, \emph{D.
vulgaris} and MV2-Eury A and B chain sequences are shown. Distances are
C\selectlanguage{greek}α-\selectlanguage{english}C\selectlanguage{greek}α \selectlanguage{english}distances. Bolded sequences correspond to FPEC residues from the
queried subunits, italicized and underlined sequences to probable
co-varying pairs (see text).\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
FPEC pairs resulting from EVCouplings analysis are shown in bold. All
corresponding homologous residues in the \emph{A. fulgidus}, \emph{D.
vulgaris} and MV2-Eury A and B chain sequences are shown. Distances are
C\selectlanguage{greek}α-\selectlanguage{english}C\selectlanguage{greek}α \selectlanguage{english}distances. Bolded sequences correspond to FPEC residues from the
queried subunits, italicized and underlined sequences to probable
co-varying pairs (see text).\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
FPEC pairs resulting from EVCouplings analysis are shown in bold. All
corresponding homologous residues in the \emph{A. fulgidus}, \emph{D.
vulgaris} and MV2-Eury A and B chain sequences are shown. Distances are
C\selectlanguage{greek}α-\selectlanguage{english}C\selectlanguage{greek}α \selectlanguage{english}distances. Bolded sequences correspond to FPEC residues from the
queried subunits, italicized and underlined sequences to probable
co-varying pairs (see text).\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
FPEC pairs resulting from EVCouplings analysis are shown in bold. All
corresponding homologous residues in the \emph{A. fulgidus}, \emph{D.
vulgaris} and MV2-Eury A and B chain sequences are shown. Distances are
C\selectlanguage{greek}α-\selectlanguage{english}C\selectlanguage{greek}α \selectlanguage{english}distances. Bolded sequences correspond to FPEC residues from the
queried subunits, italicized and underlined sequences to probable
co-varying pairs (see text).\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
FPEC pairs resulting from EVCouplings analysis are shown in bold. All
corresponding homologous residues in the \emph{A. fulgidus}, \emph{D.
vulgaris} and MV2-Eury A and B chain sequences are shown. Distances are
C\selectlanguage{greek}α-\selectlanguage{english}C\selectlanguage{greek}α \selectlanguage{english}distances. Bolded sequences correspond to FPEC residues from the
queried subunits, italicized and underlined sequences to probable
co-varying pairs (see text).\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

and constitutes the first residue in the heme road.

Residue N393 in the DsrA subunit is aligned to T351 in the DsrB subunit,
but these occupy very different spatial positions (Figure S4). The N393
from the DsrA subunit is part of the C-terminal arm that extends to the
other heterodimer (Figures 1, 5), such that N393A2 inserts itself\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

into the DsrB1 subunit and vice-versa. This is the second residue in the
heme road. The T351 residue in the DsrB subunit is aligned to N393 in
the DsrA subunit in the sequence alignment, but structurally they are
not in homologous positions (Figure S4). T351 is found at the N-terminal
end of a helix in the central interface between heterotetramers in a
T351B1-T351B2 interaction, which is the sole interaction at the central
interface between heterodimers. The distance between the carbonyl oxygen
atoms of the backbone of these two residues is 4.1 \selectlanguage{ngerman}Å. This is the third
residue of the heme road between the two functional siroheme moieties
(Figure 5). Residues N180B1/2 and N393A2/1 are separated by only 10.1 Å,
with two intervening aromatic residues, while a single aromatic residue
separates N393A2/1 from T351B1/2 (Figure 5D). Thus while the heme road
residues are not in direct contact, they are coupled by intervening
aromatic side chains.

In support of evolutionary covariance of heme road residues, EVCoupling
analysis of the MV2-Eury A subunit also indicated coupling between N369A
and L204A, with equally probable residues involved in coupling being
R337 and S167. These residues are the structural and sequence homologues
of the four residues detected as monomeric FPECs by EVCoupling analysis
of the \emph{A. fulgidus} A subunit, three of which constitute the heme
road. The third detected FPEC using the \emph{A. fulgidus} B subunit
sequence as bait, I350B-R197B (Table 1), implicates K240A and N392A as
equally likely to be involved in a residue pair coupling. Residues N392A
and I350B are adjacent to two residues in the heme road, N393A and
T351B, providing support for their evolutionary coupling. Thus,
T351B1-N393A (italicized and underlined in Table 1) are likely to
correspond to actual co-varying residues. Given the relatively close
proximity of N393B1 and N180B1, 10.1 Å, we hypothesize that these two
residues may also be evolutionarily coupled. The aromatic residues
linking the heme road residues, Y348B1, F394A2, in \emph{A. fulgidus}are
replaced in MV2-Eury by hydrophobic residues L334B1 and I360A2, and
F317A1 is replaced by a proline, P166B1, from the B1 rather than A1
subunit.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image10/image10}

\end{center}
\end{figure}

In the \emph{D. vulgaris} structure, the residues making contact between
heterodimers at the central interface are two proline residues
(P368B1/B2) that stack on each other (Table 1, Figure 6A), whereas the
interacting residues in the MV2-Eury homology model based on the
\emph{D. vulgaris} structure are two arginine residues (R337B1/B2)
detected in the EVCoupling analysis as possible monomeric FPECs (Table
1, Figure 6B). This central interfacial residue is found to be an
asparagine in 33.5\% of the sequences and a serine in 7\% of the
sequences. In 42\% of the sequences in the Dsr multiple sequence
alignment (MSA) used by the EV Couplings server, the C-terminus is
truncated prior to this residue at the central interface, which may
correspond to sequencing errors or inclusion of partial sequences in
this database. Note that, in the \emph{D. vulgaris} structure, the B1
and B2 helices at the central interface swap positions with respect to
the \emph{A. fulgidus} structure (Figure 1). The aromatic residues,
F365B1 and Y412A2, in the \emph{D. vulgaris} heme road are switched with
respect to Y348B1 and F394A2 in \emph{A. fulgidus} . Like the MV2-Eury
heme road, the third aromatic residue in \emph{A. fulgidus,} F317A1 is
replaced by a residue from the B1 subunit, I190B1.

Examination of the heme road residues in the structural alignment of the
MV2-Eury homology and AF2 models reveals that they are slightly shifted
between the two models, with S167 of the B1/2 subunits in the AF2 model
exhibiting a steric clash between models with two of the Fe-S centers in
the homology model. The two residues constituting the central interface
between heterodimers in MV2-Eury B1/2 subunits, R337B1-R337B2, are
superimposed at the level of the backbone between the homology and AF2
models, but the side chains point away from each other in the AF2 model,
while they interact closely with each other in the homology model
(Figure S3). The positioning of the A-subunit C-terminal heme road
residue, N369A1/2, in MV2-Eury is quite similar between the homology and
AF2 models, despite the very different positioning of the C-terminal
helix (Figure 4). The three residues in each heterodimer comprising the
heme road on either side of the central interface are not in direct
contact, supporting the notion that the co-variance is not uniquely
structural in nature. Results of the evolutionary coupling using
different subunits from different homologues as bait supports the notion
that at least one pair of these residues, T351B1-N393A2 and vice versa
in \emph{A. fulgidus} , is truly co-varying. The positioning of N180B1/2
in interaction with the siroheme and relatively close to N393A2/1 is
suggestive that this constitutes a second co-varying pair. Based on
these observations, we hypothesize that the heme road represents a
pathway of cooperative communication between active sites within the
DsrAB heterotetramer. The aromatic residues packed between them (Figure
5D) could help modulate information transfer in this putative allosteric
route. Alternatively, given the relatively short distances between the
heme road residues and the intervening aromatic residues, we cannot rule
out that this represents a pathway for electron transfer between
heterodimer active sites.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image12/image12}

\end{center}
\end{figure}

To model the possible dynamic fluctuations that might be associated with
this putative allosteric pathway, we carried out Anisotropic Network
Modeling (ANM) (40, 41). ANM is based on a Gaussian network that
considers protein structures as elastic networks in which the nodes
correspond to the C\selectlanguage{greek}α \selectlanguage{english}atoms, connected by identical spring constants and
in which a Kirchhoff matrix is used to represent the topology of
internal contacts. ANM has been used to successfully model the known
dynamical modes of the allosteric transition in hemoglobin (49). The
three most prominent normal modes obtained from the ANM calculation
on\emph{A. fulgidus} DsrAB reveal significant motion of one heterodimer
with respect to the other, in addition to internal modes within monomers
and heterodimers (Figure 7, Movies S1-S4). The red squares along the
diagonal in the correlation matrix (Figure S5) indicate motions within
each domain of each subunit. Within subunits, individual domains exhibit
anti-correlated motions (blue). Off diagonal correlations (red) are
observed between subunits within a heterodimer and between heterodimers.
For example, motions of domain 1 of the DsrA1 subunit are correlated
with the ferredoxin domain of the DsrB1 subunit (Figure S5, yellow
circles) and with the domain 1 of the DsrB2 subunit (Figure S5, green
circle). The largest motions as evaluated by the calculated B-factors
(Figure S6), were observed in the slowest mode for the ferredoxin
domains of the DsrA1/2 and DsrB1/2 subunits, and in a region of the
DsrB1/2 subunits that is near the structural heme and adjacent to one of
the FPEC heme road residues, N180B. The point of contact between the two
heterodimers is the FPEC heme road residue, T351. It corresponds to a
pivot point (low B-factor) for the most significant modes.
Interestingly, the shifts in the backbone observed between the two
models of the MV2-Eury sequence, particularly apparent in the ferredoxin
domains (Figure 6), mimic the rocking like conformational changes
associated with the first two normal modes (Figure 7).

In parallel, we resumed the crystallographic refinement using current
Phenix software to evaluate the impact of simulating more numerous but
smaller segments for TLS refinement of the \emph{Archeoglobus
fulgidus}DsrAB crystal structure, PDB3MM5 (20). Changing from two to
twenty-one TLS groups did not affect significantly Rwork/Rfree factors
(shifting from 15.8 to 16.1 and from 18.8 to 18.5, respectively) and did
not yield a much nicer electron density for one highly flexible
heterodimer while the other one is very well resolved and mainly rigid.
This result suggested that the overall flexibility relates mainly to
rigid body movement of the whole heterodimers. Our ensemble refinement
based on 34 conformational states slightly improves the agreement with
diffraction data (Rwork: 15.3 from 16.1 and Rfree: 18.5 from 18.5) and
suggested some flexibility does occur in the crystal state. Consistent
with the notion of dynamic displacements of one heterodimer relative to
the other, ensemble refinement of the 3mm5 crystal structure yielded one
highly resolved heterodimer (but for only small loops actually
flexible), while the second appeared more flexible as a whole with some
external segments appearing poorly resolved (see Fig S7). This
asymmetric behavior relates to the odd crystal packing that is almost
absent for the flexible heterodimer while crystal organization relies on
contacts involving the other apparently more rigid heterodimer. The
B-factors recapitulate the same picture with one `cold' heterodimer and
the second heterodimer getting `hotter' from their common interface to
its outward surface..

\emph{Inserts and gaps near the structural heme}

\textbf{Table 2. FPEC pairs corresponding to inserts/gaps in the DsrB
and DsrA subunits}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
Organisms & Res1 DsrB & Res 2 DsrB & Res1 DsrA & Res2
DsrA\tabularnewline
Eury & K302 & F106 & R334 & M143\tabularnewline
AF & \textbf{K316} & \textbf{D120} & F358 & F161\tabularnewline
DV & K333 & F130 & N375 & T162\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image14/image14}

\end{center}
\end{figure}

EVCoupling analysis also revealed another FPEC pair involving residues
(D120/K316) near the structural siroheme moiety (in \emph{A. fulgidus} )
(bold in Table 2). This pair was identified as an FPEC pair when
compared against a contact map of the \emph{D. vulgaris} DsrAB X-ray
crystal structure. However, when highlighted on the structure of
\emph{A. fulgidus} DsrAB, the residues are in contact. Hence it is a
true contact for some members of the protein family, and a false
predicted contact for others. Aligning the three structures of DsrAB
from \emph{A. fulgidus} , \emph{D. vulgaris,} and the model of MV2-Eury
(Figure 8) reveals an antiparallel \selectlanguage{greek}β-\selectlanguage{english}hairpin/loop insert in the \emph{A.
fulgidus} DsrB subunit (Figure 8, light pink) which brings aspartate
120B within 7.4 \selectlanguage{ngerman}Å of lysine 316B and also makes contact with the end of
a helix in the DsrA subunit. This insert includes a tryptophan residue
(in CPK color) that blocks access to the structural heme in the \emph{A.
fulgidus} structure (see also Figure 3). The MV2-Eury and \emph{D.
vulgaris} DsrB subunits (Figure 8, cyan, deep teal) lack this extension.
The phenylalanine residue in these sequences, that corresponds to the
aspartate in the FPEC pair residue D120B/K316B in \emph{A. fulgidus,} is
far (\textasciitilde{}27 Å) from its co-evolving partner residue (Figure
8, red, raspberry sticks on the right side), thus providing a false
positive signal. Interestingly, this insert in the DsrB subunit of
\emph{A. fulgidus} (Figure 3, pink) is absent from all other DsrB
sequences (Fig. 9), posing the question of why the co-evolutionary
coupling probability was high (99\%) for this pair.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image16/image16}

\end{center}
\end{figure}

The\emph{D. vulgaris} sequence exhibits another insert in the DsrB
subunit (Figure 8, magenta), just prior to that observed in the \emph{A.
fulgidus} sequence that makes contact with a different helix in the DsrA
subunit. This insert is missing in all early-branching DsrAB (Figure 9,
outer circle, purple) and is present in nearly all Deltaproteobacteria,
in addition to a few other lineages with evidence for HGT of DsrAB from
Deltaproteobacteria to other lineages (i.e., Thermodesulfobacteria and
some Firmicutes (23); Figure 9). Thus, this adaptation appears to have
evolved in the Deltaproteobacteria and has been retained throughout this
lineage, in addition to maintenance in other lineages following HGT.
These results suggest that these adaptations were a consequence of the
ecophysiological lifestyles of late-evolving Deltaproteobacteria.

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 SO\textsubscript{3}\textsuperscript{2-} reduction
and then diversified (through recruitment of APS and Sat) to catalyze
SO\textsubscript{4}\textsuperscript{2-} reduction and ultimately
HS\textsuperscript{-} 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 \emph{A.
fulgidus}and MV2-Eury A subunit queries, and a second set of monomeric
FPECs from the \emph{A. fulgidus} B subunit query adjacent to two of the
three residues, we conclude that the N393A2/1-T351B1/2 (\emph{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 \emph{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 \emph{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\textsuperscript{-} 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\textsuperscript{-}shuttling) stress imposed by extreme conditions
(e.g., temperature, pH and pressure) (50) where the kinetics of
HSO\textsubscript{3}\textsuperscript{-} 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 \emph{A. fulgidus} Dsr
and the loss of the iron in the sirohydrochlorin moieties in \emph{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
SO\textsubscript{4}\textsuperscript{2-} reduction but that rather are
limited to SO\textsubscript{3}\textsuperscript{2-} reduction. Such
investigations would enable a better understanding of the evolution of
Dsr as it transitioned from early
SO\textsubscript{3}\textsuperscript{2-} respiring organisms to the
SO\textsubscript{4}\textsuperscript{2-} reducers that are widespread in
anoxic environments on Earth today.

Acknowledgments

We thank Drs. Kelly Brock and Chris Sander for assistance with sequence
co-variance analysis using EvFold. DRC and ESB acknowledge support from
the National Science Foundation (EAR-1820658) and the NASA EPSCoR
program (MT-19-EPSCoR-0020). GTM acknowledges support by NIH Grant
R35-GM141818.

Competing Interest Statement

GTM is a founder of Nexomics Biosciences. This affiliation does not
present a competing interest with respect to this study. The other
authors declare no competing interests.

Modeling and evolutionary covariance data will be made available upon
request.



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\begin{longtable}[]{@{}lllllllll@{}}
\toprule
\begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Table 1. FPEC pairs for the heme road}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Table 1. FPEC pairs for the heme road}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Table 1. FPEC pairs for the heme road}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Table 1. FPEC pairs for the heme road}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Table 1. FPEC pairs for the heme road}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Table 1. FPEC pairs for the heme road}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Distance} \textbf{in monomer} \selectlanguage{ngerman}\textbf{(Å)}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Distance} \textbf{in monomer} \selectlanguage{ngerman}\textbf{(Å)}\strut
\end{minipage} & \begin{minipage}[b]{0.11\columnwidth}\raggedright\strut
\textbf{Coupling probability} \textbf{(\%)}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Organisms\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Res1 DsrA\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Res 2 DsrA\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Res1 DsrB\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
Res2 DsrB\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.11\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{A. fulgidus}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{N393}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\textbf{N222}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{T351}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{N180}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
33.51\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
33.51\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
90\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
90\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{D vulgaris}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
N410\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
N223\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
P368\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
N191\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
MV2-Eury\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\textbf{N369}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\textbf{L204}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
R337\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
S167\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
33.84\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
33.84\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
80\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
80\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{A. fulgidus}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
N392\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
K240\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\textbf{I350}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\textbf{R197}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
23.62\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
23.62\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
94\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
94\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\emph{D vulgaris}\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
T409\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
K241\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
D367\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
R208\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
MV2-Eury\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
T368\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
E222\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
L336\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
T184\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
FPEC pairs resulting from EVCouplings analysis are shown in bold. All
corresponding homologous residues in the \emph{A. fulgidus}, \emph{D.
vulgaris} and MV2-Eury A and B chain sequences are shown. Distances are
C\selectlanguage{greek}α-\selectlanguage{english}C\selectlanguage{greek}α \selectlanguage{english}distances. Bolded sequences correspond to FPEC residues from the
queried subunits, italicized and underlined sequences to probable
co-varying pairs (see text).\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
FPEC pairs resulting from EVCouplings analysis are shown in bold. All
corresponding homologous residues in the \emph{A. fulgidus}, \emph{D.
vulgaris} and MV2-Eury A and B chain sequences are shown. Distances are
C\selectlanguage{greek}α-\selectlanguage{english}C\selectlanguage{greek}α \selectlanguage{english}distances. Bolded sequences correspond to FPEC residues from the
queried subunits, italicized and underlined sequences to probable
co-varying pairs (see text).\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
FPEC pairs resulting from EVCouplings analysis are shown in bold. All
corresponding homologous residues in the \emph{A. fulgidus}, \emph{D.
vulgaris} and MV2-Eury A and B chain sequences are shown. Distances are
C\selectlanguage{greek}α-\selectlanguage{english}C\selectlanguage{greek}α \selectlanguage{english}distances. Bolded sequences correspond to FPEC residues from the
queried subunits, italicized and underlined sequences to probable
co-varying pairs (see text).\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
FPEC pairs resulting from EVCouplings analysis are shown in bold. All
corresponding homologous residues in the \emph{A. fulgidus}, \emph{D.
vulgaris} and MV2-Eury A and B chain sequences are shown. Distances are
C\selectlanguage{greek}α-\selectlanguage{english}C\selectlanguage{greek}α \selectlanguage{english}distances. Bolded sequences correspond to FPEC residues from the
queried subunits, italicized and underlined sequences to probable
co-varying pairs (see text).\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
FPEC pairs resulting from EVCouplings analysis are shown in bold. All
corresponding homologous residues in the \emph{A. fulgidus}, \emph{D.
vulgaris} and MV2-Eury A and B chain sequences are shown. Distances are
C\selectlanguage{greek}α-\selectlanguage{english}C\selectlanguage{greek}α \selectlanguage{english}distances. Bolded sequences correspond to FPEC residues from the
queried subunits, italicized and underlined sequences to probable
co-varying pairs (see text).\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
FPEC pairs resulting from EVCouplings analysis are shown in bold. All
corresponding homologous residues in the \emph{A. fulgidus}, \emph{D.
vulgaris} and MV2-Eury A and B chain sequences are shown. Distances are
C\selectlanguage{greek}α-\selectlanguage{english}C\selectlanguage{greek}α \selectlanguage{english}distances. Bolded sequences correspond to FPEC residues from the
queried subunits, italicized and underlined sequences to probable
co-varying pairs (see text).\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
FPEC pairs resulting from EVCouplings analysis are shown in bold. All
corresponding homologous residues in the \emph{A. fulgidus}, \emph{D.
vulgaris} and MV2-Eury A and B chain sequences are shown. Distances are
C\selectlanguage{greek}α-\selectlanguage{english}C\selectlanguage{greek}α \selectlanguage{english}distances. Bolded sequences correspond to FPEC residues from the
queried subunits, italicized and underlined sequences to probable
co-varying pairs (see text).\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
FPEC pairs resulting from EVCouplings analysis are shown in bold. All
corresponding homologous residues in the \emph{A. fulgidus}, \emph{D.
vulgaris} and MV2-Eury A and B chain sequences are shown. Distances are
C\selectlanguage{greek}α-\selectlanguage{english}C\selectlanguage{greek}α \selectlanguage{english}distances. Bolded sequences correspond to FPEC residues from the
queried subunits, italicized and underlined sequences to probable
co-varying pairs (see text).\strut
\end{minipage} & \begin{minipage}[t]{0.08\columnwidth}\raggedright\strut
FPEC pairs resulting from EVCouplings analysis are shown in bold. All
corresponding homologous residues in the \emph{A. fulgidus}, \emph{D.
vulgaris} and MV2-Eury A and B chain sequences are shown. Distances are
C\selectlanguage{greek}α-\selectlanguage{english}C\selectlanguage{greek}α \selectlanguage{english}distances. Bolded sequences correspond to FPEC residues from the
queried subunits, italicized and underlined sequences to probable
co-varying pairs (see text).\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 2. FPEC pairs corresponding to inserts/gaps in the DsrB
and DsrA subunits}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
Organisms & Res1 DsrB & Res 2 DsrB & Res1 DsrA & Res2
DsrA\tabularnewline
Eury & K302 & F106 & R334 & M143\tabularnewline
AF & \textbf{K316} & \textbf{D120} & F358 & F161\tabularnewline
DV & K333 & F130 & N375 & T162\tabularnewline
\bottomrule
\end{longtable}



\textbf{Figure legends}

\textbf{Figure 1} . DsrAB phylogenetic reconstruction showing the
taxonomic affiliation of Dsr-hosting organisms and their environmen-tal
origin. The Maximum Likelihood reconstruction was conducted on a
concatenated alignment of DsrA and DsrB subunits from a curated database
comprising the previously recognized primary homolog groups. The scale
bar shows the expected substitutions per site. The environmental origin
of Dsr homologs is shown based on available metadata associated with the
previously published data-base (Muller et al. 2015) or from metadata
associated with MAGs from metagenomes. Taxonomic classification is given
based on information in the aforementioned database or by
\textgreater{}80\% amino acid homology to Dsr from cultivars/genomes
with taxonomic annotation.

\textbf{Figure 2.} Available Crystal Structures of Dsr and a Homology
Model of an Early Evolving Dsr From a Sulfite Reducing Euryarchaeote
(MV2). A-C) side view; D-F) top view; A and D) \emph{Archaeoglobus.
fulgidus} heterotetramer -- pdb id: 3mmc, B and E) Desulfovibrio.
vulgaris hetero-hexamer - pdb id: 2v4j, C and F) homology model of
MV2-Eury hetero-hexamer. Chain colors are: A1 (green), B1 (cyan), A2
(light pink), B2 (light blue), C1 and C2 where present (purple and
white, respectively). Sirohemes (red), siro-hydrochlorine (salmon), and
Fe-S centers (yellow) are sticks or spheres. The sulfite substrate is
shown in the D. vulgaris structure (B) in dark blue spheres.

\textbf{Figure 3} . Close-up view of the ``lower'' prosthetic group in
the structure of Dsr. A) A. fulgidus and B) D. vulgaris Chain colors are
: A1 (green), B1 (cyan), Sirohemes (red), siro-hydrochlorine (salmon)
and Fe-S centers (yellow) are sticks or spheres. Trp 119 and Thr 135 are
shown in spheres and colored by element.

\textbf{Figure 4} . AlphaFold2 model of MV2.Eury DsrAB. The sequence is
colored for pLDDT score (descried in the text), with dark blue
corresponding to high confidence prediction (96\%) and red to low
confidence prediction (43\%).The view is a top view of the structure as
in Figure 1F. The light blue to red region on the center left and right
correspond to residues 50-62 if the A subunit which interact with the
DsrC electron donating subunit (Figure 1E, F). The upper left, bottom
right and center red regions are the chain termini.

\textbf{Figure 5} . The heme road FPEC residues in the structure of
DsrAB from \emph{A. fulgidus} . A) Side view B) top view, C) slab of a
zoom from the top view Distances between FPEC residues are shown in
yellow. Chain colors are: A1 (green), B1 (cyan), A2 (light pink), B2
(light blue). Sirohemes (red sticks) and Fe-S centers (yellow) are also
shown. Heme road FPEC residues (blue spheres) tracing a connection
between functional hemes are from left to right N180B1, N393A2, T351B1,
T351B2, N393A1 and N180B2. N222A1/2 (also blue spheres) interacts with
the propionate group of the structural (non-functional) heme visible in
(A) in a lower plane. D) Network of aromatic residues connect-ing the
residues of the heme road (dark blue) for the left half of the heme
road. Three aromatic residues, F317A1, F394A2 and Y348B1, directly
connect the heme road residues. These interactions are stabilized from
above by N246B1. N246B1 also contacts C244B1 that makes contact with the
second Fe-S center distal to the siroheme. Two heme road residues T351B2
and N393A1 of the second heterodimer also appear on the right of the
figure.

\textbf{Figure 6} . Comparison of the heme road FPEC residues in DsrAB.
A). \emph{D. vulgaris} B), the homology model of MV2-Eury. Slab of a
zoom from the top views. Chain colors are: A1 (green), B1 (cyan), A2
(light pink), B2 (light blue). Sirohemes (red sticks) and Fe-S centers
(yellow) are also shown. FPEC residues (blue spheres) tracing a
connection between functional hemes in the D. vulgaris structure (A) are
from left to right N191B1 (blue spheres), N410A2 (blue spheres), P368B1
(CPK spheres), P368B2 (CPK spheres), N410A1 (blue spheres) and N191B2
(blue spheres) make up the heme road. The corresponding residues in the
MV2.Eury model (B) are from left to right S167B1 (blue spheres), N369A2
(blue spheres), R337B1 (CPK spheres), R337B2 (CPK spheres), N369A1 (blue
spheres) and S167B2 (blue spheres). Note that the sulfite ion is shown
in darer blue bound to the heme in the \emph{D. vulgaris} structure (A).

\textbf{Figure 7} . Internal water molecules in the A1B1 heterodimer of
the \emph{A. fulgidus} structure of DsrAB (A) Full heterodimer and (B)
Zoom in the siroheme. Chain colors are: A1 (green), B1 (cyan). Sirohemes
(red sticks) and Fe-S centers (yellow) are also shown. Internal water
molecules are shown as red spheres. The tryptophan residue blocking
access to the structural (lower) heme is shown in CPK spheres.

\textbf{Figure 8} . Zoom view of the overlaid structures of DsrAB
from\emph{A. fulgidus} (cyan), \emph{D. vulgaris} (dark cyan, and MV2
Eury (pale cyan. The DsrB subunit insert in the A. fulgidus structure is
in light pink, while the bacterial B loop is magenta. The DsrA subunit
insert in the D. vulgaris structure is wheat. The rest of the DsrA1
subunits are green. Some of the FPEC residues (K and F/D/F) are
indicated in red stick and distances between them (7.4 and 26.7 \selectlanguage{ngerman}Å are
indicated). The lysine residues of the FPEC form a salt bridge with a
glutamate from the DsrA subunit (2.7 Å).

\textbf{Figure 9} . DsrAB phylogenetic reconstruction showing the
taxonomic affiliation of Dsr-hosting organisms and the presence of DsrA
and DsrB structural inserts. The Maximum Likelihood reconstruction was
conducted on a concatenated alignment of DsrA and DsrB subunits from a
curated database comprising the previously recognized primary homolog
groups. Scale bar shows the expected substitutions per site. Taxonomic
classification is given based on information in the database (Muller et
al. 2015) or by \textgreater{}80\% amino acid homology to Dsr from
cultivars/genomes with taxonomic annotation. DsrA or DsrB inserts were
identified based on structural characterizations and subsequent
identification within DsrA or DsrB alignments.

\textbf{Hosted file}

\verb`Mode1_movie1.mpg`  available at \url{https://authorea.com/users/457922/articles/554572-structural-evolution-of-the-ancient-enzyme-dissimilatory-sulfite-reductase}

\textbf{Hosted file}

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