Rapid hydrolysis rates of thio- and phosphate esters constrain the origin of metabolism to cool, acidic to neutral environments.

Universal to all life is a reliance on energy carriers such as adenosine triphosphate (ATP) which connect energy-releasing reactions to energy-consuming processes. While ATP is ubiquitously used today, simpler molecules such as thioesters and polyphosphates are hypothesized to be primordial energy carriers. Investigating environmental constraints on the non-enzymatic emergence of metabolism, we find that hydrolysis rates-not hydrolysis energies-differentiate phosphate esters and thioesters. At temperatures consistent with thermophilic microbes, thioesters are favored at acidic pH and phosphate esters at basic pH. Thioacids have a high stability across pH 5-10. The planetary availability of sulfur and phosphate is coincident with these calculations, with phosphate being abundant in alkaline and sulfur in acidic environments. Since both sulfur esters and phosphate esters are uniquely required in metabolism, our results point to a non-thermophilic origin of early metabolism at cool, acidic to neutral environments.

Osmotic energy conversion in serpentinite-hosted deep-sea hydrothermal vents.

Cells harvest energy from ionic gradients by selective ion transport across membranes, and the same principle is recently being used for osmotic power generation from salinity gradients at ocean-river interfaces. Common to these ionic gradient conversions is that they require intricate nanoscale structures. Here, we show that natural submarine serpentinite-hosted hydrothermal vent (HV) precipitates are capable of converting ionic gradients into electrochemical energy by selective transport of Na+, K+, H+, and Cl-. Layered hydroxide nanocrystals are aligned radially outwards from the HV fluid channels, constituting confined nanopores that span millimeters in the HV wall. The nanopores change the surface charge depending on adsorbed ions, allowing the mineral to function as a cation- and anion-selective ion transport membrane. Our findings indicate that chemical disequilibria originating from flow and concentration gradients in geologic environments generate confined nanospaces which enable the spontaneous establishment of osmotic energy conversion.

Primitive purine biosynthesis connects ancient geochemistry to modern metabolism.

An unresolved question in the origin and evolution of life is whether a continuous path from geochemical precursors to the majority of molecules in the biosphere can be reconstructed from modern-day biochemistry. Here we identified a feasible path by simulating the evolution of biosphere-scale metabolism, using only known biochemical reactions and models of primitive coenzymes. We find that purine synthesis constitutes a bottleneck for metabolic expansion, which can be alleviated by non-autocatalytic phosphoryl coupling agents. Early phases of the expansion are enriched with enzymes that are metal dependent and structurally symmetric, supporting models of early biochemical evolution. This expansion trajectory suggests distinct hypotheses regarding the tempo, mode and timing of metabolic pathway evolution, including a late appearance of methane metabolisms and oxygenic photosynthesis consistent with the geochemical record. The concordance between biological and geological analyses suggests that this trajectory provides a plausible evolutionary history for the vast majority of core biochemistry.

Microbially induced precipitation of silica by anaerobic methane-oxidizing consortia and implications for microbial fossil preservation.

Authigenic carbonate minerals can preserve biosignatures of microbial anaerobic oxidation of methane (AOM) in the rock record. It is not currently known whether the microorganisms that mediate sulfate-coupled AOM-often occurring as multicelled consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB)-are preserved as microfossils. Electron microscopy of ANME-SRB consortia in methane seep sediments has shown that these microorganisms can be associated with silicate minerals such as clays [Chen et al., Sci. Rep. 4, 1-9 (2014)], but the biogenicity of these phases, their geochemical composition, and their potential preservation in the rock record is poorly constrained. Long-term laboratory AOM enrichment cultures in sediment-free artificial seawater [Yu et al., Appl. Environ. Microbiol. 88, e02109-21 (2022)] resulted in precipitation of amorphous silicate particles (~200 nm) within clusters of exopolymer-rich AOM consortia from media undersaturated with respect to silica, suggestive of a microbially mediated process. The use of techniques like correlative fluorescence in situ hybridization (FISH), scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDS), and nanoscale secondary ion mass spectrometry (nanoSIMS) on AOM consortia from methane seep authigenic carbonates and sediments further revealed that they are enveloped in a silica-rich phase similar to the mineral phase on ANME-SRB consortia in enrichment cultures. Like in cyanobacteria [Moore et al., Geology 48, 862-866 (2020)], the Si-rich phases on ANME-SRB consortia identified here may enhance their preservation as microfossils. The morphology of these silica-rich precipitates, consistent with amorphous-type clay-like spheroids formed within organic assemblages, provides an additional mineralogical signature that may assist in the search for structural remnants of microbial consortia in rocks which formed in methane-rich environments from Earth and other planetary bodies.

Scaling of Protein Function across the Tree of Life.

Scaling laws are a powerful way to compare genomes because they put all organisms onto a single curve and reveal nontrivial generalities as genomes change in size. The abundance of functional categories across genomes has previously been found to show power law scaling with respect to the total number of functional categories, suggesting that universal constraints shape genomic category abundance. Here, we look across the tree of life to understand how genome evolution may be related to functional scaling. We revisit previous observations of functional genome scaling with an expanded taxonomy by analyzing 3,726 bacterial, 220 archaeal, and 79 unicellular eukaryotic genomes. We find that for some functional classes, scaling is best described by multiple exponents, revealing previously unobserved shifts in scaling as genome-encoded protein annotations increase or decrease. Furthermore, we find that scaling varies between phyletic groups at both the domain and phyla levels and is less universal than previously thought. This variability in functional scaling is not related to taxonomic phylogeny resolved at the phyla level, suggesting that differences in cell plan or physiology outweigh broad patterns of taxonomic evolution. Since genomes are maintained and replicated by the functional proteins encoded by them, these results point to functional degeneracy between taxonomic groups and unique evolutionary trajectories toward these. We also find that individual phyla frequently span scaling exponents of functional classes, revealing that individual clades can move across scaling exponents. Together, our results reveal unique shifts in functions across the tree of life and highlight that as genomes grow or shrink, proteins of various functions may be added or lost.

Machine Learning Model Insights into Base-Catalyzed Hydrothermal Lignin Depolymerization.

Lignin, an abundant component of plant matter, can be depolymerized into renewable aromatic chemicals and biofuels but remains underutilized. Homogeneously catalyzed depolymerization in water has gained attention due to its economic feasibility and performance but suffers from inconsistently reported yields of bio-oil and solid residues. In this study, machine learning methods were used to develop predictive models for bio-oil and solid residue yields by using a few reaction variables and were subsequently validated by doing experimental work and comparing the predictions to the results. The models achieved a coefficient of determination (R2) score of 0.83 and 0.76, respectively, for bio-oil yield and solid residue. Variable importance for each model was calculated by two different methodologies and was tied to existing studies to explain the model predictive behavior. Based on the outcome of the study, the creation of concrete guidelines for reporting in lignin depolymerization studies was recommended. Shapley additive explanation value analysis reveals that temperature and reaction time are generally the strongest predictors of experimental outcomes compared to the rest.

One-Pot De Novo Synthesis of [4Fe-4S] Proteins Using a Recombinant SUF System under Aerobic Conditions.

Fe-S clusters are essential cofactors mediating electron transfer in respiratory and metabolic networks. However, obtaining active [4Fe-4S] proteins with heterologous expression is challenging due to (i) the requirements for [4Fe-4S] cluster assembly, (ii) the O2 lability of [4Fe-4S] clusters, and (iii) copurification of undesired proteins (e.g., ferredoxins). Here, we established a facile and efficient protocol to express mature [4Fe-4S] proteins in the PURE system under aerobic conditions. An enzyme aconitase and thermophilic ferredoxin were selected as model [4Fe-4S] proteins for functional verification. We first reconstituted the SUF system in vitro via a stepwise manner using the recombinant SUF subunits (SufABCDSE) individually purified from E. coli. Later, the incorporation of recombinant SUF helper proteins into the PURE system enabled mRNA translation-coupled [4Fe-4S] cluster assembly under the O2-depleted conditions. To overcome the O2 lability of [4Fe-4S] Fe-S clusters, an O2-scavenging enzyme cascade was incorporated, which begins with formate oxidation by formate dehydrogenase for NADH regeneration. Later, NADH is consumed by flavin reductase for FADH2 regeneration. Finally, bifunctional flavin reductase, along with catalase, removes O2 from the reaction while supplying FADH2 to the SufBC2D complex. These amendments enabled a one-pot, two-step synthesis of mature [4Fe-4S] proteins under aerobic conditions, yielding holo-aconitase with a maximum concentration of ∼0.15 mg/mL. This renovated system greatly expands the potential of the PURE system, paving the way for the future reconstruction of redox-active synthetic cells and enhanced cell-free biocatalysis.

Alternating co-synthesis of glycol nucleic acid (GNA) monomers with dicarboxylic acids via drying.

We report the co-polymerization of glycol nucleic acid (GNA) monomers with unsubstituted and substituted dicarboxylic acid linkers under plausible early Earth aqueous dry-down conditions. Both linear and branched co-polymers are produced. Mechanistic aspects of the reaction and potential roles of these polymers in prebiotic chemistry are discussed.

The methanogen core and pangenome: conservation and variability across biology's growth temperature extremes.

Temperature is a key variable in biological processes. However, a complete understanding of biological temperature adaptation is lacking, in part because of the unique constraints among different evolutionary lineages and physiological groups. Here we compared the genomes of cultivated psychrotolerant and thermotolerant methanogens, which are physiologically related and span growth temperatures from -2.5°C to 122°C. Despite being phylogenetically distributed amongst three phyla in the archaea, the genomic core of cultivated methanogens comprises about one-third of a given genome, while the genome fraction shared by any two organisms decreases with increasing phylogenetic distance between them. Increased methanogenic growth temperature is associated with reduced genome size, and thermotolerant organisms-which are distributed across the archaeal tree-have larger core genome fractions, suggesting that genome size is governed by temperature rather than phylogeny. Thermotolerant methanogens are enriched in metal and other transporters, and psychrotolerant methanogens are enriched in proteins related to structure and motility. Observed amino acid compositional differences between temperature groups include proteome charge, polarity and unfolding entropy. Our results suggest that in the methanogens, shared physiology maintains a large, conserved genomic core even across large phylogenetic distances and biology's temperature extremes.

Microbial ecology of a shallow alkaline hydrothermal vent: Strýtan Hydrothermal Field, Eyjafördur, northern Iceland.

Strýtan Hydrothermal Field (SHF) is a submarine system located in Eyjafördur in northern Iceland composed of two main vents: Big Strýtan and Arnarnesstrýtan. The vents are shallow, ranging from 16 to 70 m water depth, and vent high pH (up to 10.2), moderate temperature (T max ∼70°C), anoxic, fresh fluids elevated in dissolved silica, with slightly elevated concentrations of hydrogen and methane. In contrast to other alkaline hydrothermal vents, SHF is unique because it is hosted in basalt and therefore the high pH is not created by serpentinization. While previous studies have assessed the geology and geochemistry of this site, the microbial diversity of SHF has not been explored in detail. Here we present a microbial diversity survey of the actively venting fluids and chimneys from Big Strýtan and Arnarnesstrýtan, using 16S rRNA gene amplicon sequencing. Community members from the vent fluids are mostly aerobic heterotrophic bacteria; however, within the chimneys oxic, low oxygen, and anoxic habitats could be distinguished, where taxa putatively capable of acetogenesis, sulfur-cycling, and hydrogen metabolism were observed. Very few archaea were observed in the samples. The inhabitants of SHF are more similar to terrestrial hot spring samples than other marine sites. It has been hypothesized that life on Earth (and elsewhere in the solar system) could have originated in an alkaline hydrothermal system, however all other studied alkaline submarine hydrothermal systems to date are fueled by serpentinization. SHF adds to our understandings of hydrothermal vents in relationship to microbial diversity, evolution, and possibly the origin of life.

A Cristae-Like Microcompartment in Desulfobacterota.

Some Alphaproteobacteria contain intracytoplasmic membranes (ICMs) and proteins homologous to those responsible for the mitochondrial cristae, an observation which has given rise to the hypothesis that the Alphaproteobacteria endosymbiont had already evolved cristae-like structures and functions. However, our knowledge of microbial fine structure is still limited, leaving open the possibility of structurally homologous ICMs outside the Alphaproteobacteria. Here, we report on the detailed characterization of lamellar cristae-like ICMs in environmental sulfate-reducing Desulfobacterota that form syntrophic partnerships with anaerobic methane-oxidizing (ANME) archaea. These structures are junction-bound to the cytoplasmic membrane and resemble the form seen in the lamellar cristae of opisthokont mitochondria. Extending these observations, we also characterized similar structures in Desulfovibrio carbinolicus, a close relative of the magnetotactic D. magneticus, which does not contain magnetosomes. Despite a remarkable structural similarity, the key proteins involved in cristae formation have not yet been identified in Desulfobacterota, suggesting that an analogous, but not a homologous, protein organization system developed during the evolution of some members of Desulfobacterota. IMPORTANCE Working with anaerobic consortia of methane oxidizing ANME archaea and their sulfate-reducing bacterial partners recovered from deep sea sediments and with the related sulfate-reducing bacterial isolate D. carbinolicus, we discovered that their intracytoplasmic membranes (ICMs) appear remarkably similar to lamellar cristae. Three-dimensional electron microscopy allowed for the novel analysis of the nanoscale attachment of ICMs to the cytoplasmic membrane, and these ICMs are structurally nearly identical to the crista junction architecture seen in metazoan mitochondria. However, the core junction-forming proteins must be different. The outer membrane vesicles were observed to bud from syntrophic Desulfobacterota, and darkly stained granules were prominent in both Desulfobacterota and D. carbinolicus. These findings expand the taxonomic breadth of ICM-producing microorganisms and add to our understanding of three-dimensional microbial fine structure in environmental microorganisms.

An evolutionary history of the CoA-binding protein Nat/Ivy.

Nat/Ivy is a diverse and ubiquitous CoA-binding evolutionary lineage that catalyzes acyltransferase reactions, primarily converting thioesters into amides. At the heart of the Nat/Ivy fold is a phosphate-binding loop that bears a striking resemblance to that of P-loop NTPases-both are extended, glycine-rich loops situated between a ß-strand and an α-helix. Nat/Ivy, therefore, represents an intriguing intersection between thioester chemistry, a putative primitive energy currency, and an ancient mode of phospho-ligand binding. Current evidence suggests that Nat/Ivy emerged independently of other cofactor-utilizing enzymes, and that the observed structural similarity-particularly of the cofactor binding site-is the product of shared constraints instead of shared ancestry. The reliance of Nat/Ivy on a ß-α-ß motif for CoA-binding highlights the extent to which this simple structural motif may have been a fundamental evolutionary "nucleus" around which modern cofactor-binding domains condensed, as has been suggested for HUP domains, Rossmanns, and P-loop NTPases. Finally, by dissecting the patterns of conserved interactions between Nat/Ivy families and CoA, the coevolution of the enzyme and the cofactor was analyzed. As with the Rossmann, it appears that the pyrophosphate moiety at the center of the cofactor predates the enzyme, suggesting that Nat/Ivy emerged sometime after the metabolite dephospho-CoA.

Evidence for the emergence of ß-trefoils by 'Peptide Budding' from an IgG-like ß-sandwich.

As sequence and structure comparison algorithms gain sensitivity, the intrinsic interconnectedness of the protein universe has become increasingly apparent. Despite this general trend, ß-trefoils have emerged as an uncommon counterexample: They are an isolated protein lineage for which few, if any, sequence or structure associations to other lineages have been identified. If ß-trefoils are, in fact, remote islands in sequence-structure space, it implies that the oligomerizing peptide that founded the ß-trefoil lineage itself arose de novo. To better understand ß-trefoil evolution, and to probe the limits of fragment sharing across the protein universe, we identified both 'ß-trefoil bridging themes' (evolutionarily-related sequence segments) and 'ß-trefoil-like motifs' (structure motifs with a hallmark feature of the ß-trefoil architecture) in multiple, ostensibly unrelated, protein lineages. The success of the present approach stems, in part, from considering ß-trefoil sequence segments or structure motifs rather than the ß-trefoil architecture as a whole, as has been done previously. The newly uncovered inter-lineage connections presented here suggest a novel hypothesis about the origins of the ß-trefoil fold itself-namely, that it is a derived fold formed by 'budding' from an Immunoglobulin-like ß-sandwich protein. These results demonstrate how the evolution of a folded domain from a peptide need not be a signature of antiquity and underpin an emerging truth: few protein lineages escape nature's sewing table.

Complex History of Aerobic Respiration and Phototrophy in the Chloroflexota Class Anaerolineae Revealed by High-Quality Draft Genome of Ca. Roseilinea mizusawaensis AA3_104.

Roseilinea is a novel lineage of Chloroflexota known only from incomplete metagenome-assembled genomes (MAGs) and preliminary enrichments. Roseilinea is notable for appearing capable of anoxygenic photoheterotrophy despite being only distantly related to well-known phototrophs in the Chloroflexia class such as Chloroflexus and Roseiflexus. Here, we present a high-quality MAG of a member of Roseilinea, improving our understanding of the metabolic capacity and phylogeny of this genus, and resolving the multiple instances of horizontal gene transfer that have led to its metabolic potential. These data allow us to propose a candidate family for these organisms, Roseilineaceae, within the Anaerolineae class.

Electrochemically induced metal- vs. ligand-based redox changes in mackinawite: identification of a Fe3+- and polysulfide-containing intermediate.

Under anaerobic conditions, ferrous iron reacts with sulfide producing FeS, which can then undergo a temperature, redox potential, and pH dependent maturation process resulting in the formation of oxidized mineral phases, such as greigite or pyrite. A greater understanding of this maturation process holds promise for the development of iron-sulfide catalysts, which are known to promote diverse chemical reactions, such as H+, CO2 and NO3- reduction processes. Hampering the full realization of the catalytic potential of FeS, however, is an incomplete knowledge of the molecular and redox processess ocurring between mineral and nanoparticulate phases. Here, we investigated the chemical properties of iron-sulfide by cyclic voltammetry, Raman and X-ray absorption spectroscopic techniques. Tracing oxidative maturation pathways by varying electrode potential, nanoparticulate n(Fe2+S2-)(s) was found to oxidize to a Fe3+ containing FeS phase at -0.5 V vs. Ag/AgCl (pH = 7). In a subsequent oxidation, polysulfides are proposed to give a material that is composed of Fe2+, Fe3+, S2- and polysulfide (Sn2-) species, with its composition described as Fe2+1-3xFe3+2xS2-1-y(Sn2-)y. Thermodynamic properties of model compounds calculated by density functional theory indicate that ligand oxidation occurs in conjunction with structural rearrangements, whereas metal oxidation may occur prior to structural rearrangement. These findings together point to the existence of a metastable FeS phase located at the junction of a metal-based oxidation path between FeS and greigite (Fe2+Fe3+2S2-4) and a ligand-based oxidation path between FeS and pyrite (Fe2+(S2)2-).

HydG, the "dangler" iron, and catalytic production of free CO and CN-: implications for [FeFe]-hydrogenase maturation.

The organometallic H-cluster of the [FeFe]-hydrogenase consists of a [4Fe-4S] cubane bridged via a cysteinyl thiolate to a 2Fe subcluster ([2Fe]H) containing CO, CN-, and dithiomethylamine (DTMA) ligands. The H-cluster is synthesized by three dedicated maturation proteins: the radical SAM enzymes HydE and HydG synthesize the non-protein ligands, while the GTPase HydF serves as a scaffold for assembly of [2Fe]H prior to its delivery to the [FeFe]-hydrogenase containing the [4Fe-4S] cubane. HydG uses l-tyrosine as a substrate, cleaving it to produce p-cresol as well as the CO and CN- ligands to the H-cluster, although there is some question as to whether these are formed as free diatomics or as part of a [Fe(CO)2(CN)] synthon. Here we show that Clostridium acetobutylicum (C.a.) HydG catalyzes formation of multiple equivalents of free CO at rates comparable to those for CN- formation. Free CN- is also formed in excess molar equivalents over protein. A g = 8.9 EPR signal is observed for C.a. HydG reconstituted to load the 5th "dangler" iron of the auxiliary [4Fe-4S][FeCys] cluster and is assigned to this "dangler-loaded" cluster state. Free CO and CN- formation and the degree of activation of [FeFe]-hydrogenase all occur regardless of dangler loading, but are increased 10-35% in the dangler-loaded HydG; this indicates the dangler iron is not essential to this process but may affect relevant catalysis. During HydG turnover in the presence of myoglobin, the g = 8.9 signal remains unchanged, indicating that a [Fe(CO)2(CN)(Cys)] synthon is not formed at the dangler iron. Mutation of the only protein ligand to the dangler iron, H272, to alanine nearly completely abolishes both free CO formation and hydrogenase activation, however results show this is not due solely to the loss of the dangler iron. In experiments with wild type and H272A HydG, and with different degrees of dangler loading, we observe a consistent correlation between free CO/CN- formation and hydrogenase activation. Taken in full, our results point to free CO/CN-, but not an [Fe(CO)2(CN)(Cys)] synthon, as essential species in hydrogenase maturation.

Changes in ATP Sulfurylase Activity in Response to Altered Cyanobacteria Growth Conditions.

We investigated variations in cell growth and ATP Sulfurylase (ATPS) activity when two cyanobacterial strains-Synechocystis sp. PCC6803 and Synechococcus sp. WH7803-were grown in conventional media, and media with low ammonium, low sulfate and a high CO2/low O2 atmosphere. In both organisms, a transition and adaptation to the reconstructed environmental media resulted in a decrease in ATPS activity. This variation appears to be decoupled from growth rate, suggesting the enzyme is not rate-limiting in S assimilation and raising questions about the role of ATPS redox regulation in cell physiology and throughout Earth history.

The Thermosynechococcus Genus: Wide Environmental Distribution, but a Highly Conserved Genomic Core.

Cyanobacteria thrive in diverse environments. However, questions remain about possible growth limitations in ancient environmental conditions. As a single genus, the Thermosynechococcus are cosmopolitan and live in chemically diverse habitats. To understand the genetic basis for this, we compared the protein coding component of Thermosynechococcus genomes. Supplementing the known genetic diversity of Thermosynechococcus, we report draft metagenome-assembled genomes of two Thermosynechococcus recovered from ferrous carbonate hot springs in Japan. We find that as a genus, Thermosynechococcus is genomically conserved, having a small pan-genome with few accessory genes per individual strain as well as few genes that are unique to the genus. Furthermore, by comparing orthologous protein groups, including an analysis of genes encoding proteins with an iron related function (uptake, storage or utilization), no clear differences in genetic content, or adaptive mechanisms could be detected between genus members, despite the range of environments they inhabit. Overall, our results highlight a seemingly innate ability for Thermosynechococcus to inhabit diverse habitats without having undergone substantial genomic adaptation to accommodate this. The finding of Thermosynechococcus in both hot and high iron environments without adaptation recognizable from the perspective of the proteome has implications for understanding the basis of thermophily within this clade, and also for understanding the possible genetic basis for high iron tolerance in cyanobacteria on early Earth. The conserved core genome may be indicative of an allopatric lifestyle-or reduced genetic complexity of hot spring habitats relative to other environments.

Ten Years of Collaborative Progress in the Quest for Orthologs.

Accurate determination of the evolutionary relationships between genes is a foundational challenge in biology. Homology-evolutionary relatedness-is in many cases readily determined based on sequence similarity analysis. By contrast, whether or not two genes directly descended from a common ancestor by a speciation event (orthologs) or duplication event (paralogs) is more challenging, yet provides critical information on the history of a gene. Since 2009, this task has been the focus of the Quest for Orthologs (QFO) Consortium. The sixth QFO meeting took place in Okazaki, Japan in conjunction with the 67th National Institute for Basic Biology conference. Here, we report recent advances, applications, and oncoming challenges that were discussed during the conference. Steady progress has been made toward standardization and scalability of new and existing tools. A feature of the conference was the presentation of a panel of accessible tools for phylogenetic profiling and several developments to bring orthology beyond the gene unit-from domains to networks. This meeting brought into light several challenges to come: leveraging orthology computations to get the most of the incoming avalanche of genomic data, integrating orthology from domain to biological network levels, building better gene models, and adapting orthology approaches to the broad evolutionary and genomic diversity recognized in different forms of life and viruses.

Repressor Activity of SqrR, a Master Regulator of Persulfide-Responsive Genes, Is Regulated by Heme Coordination.

Reactive sulfur species (RSS) are involved in bioactive regulation via persulfidation of proteins. However, how cells regulate RSS-based signaling and RSS metabolism is poorly understood, despite the importance of universal regulation systems in biology. We previously showed that the persulfide-responsive transcriptional factor SqrR acts as a master regulator of sulfide-dependent photosynthesis in proteobacteria. Here, we demonstrated that SqrR also binds heme at a near one-to-one ratio with a binding constant similar to other heme-binding proteins. Heme does not change the DNA-binding pattern of SqrR to the target gene promoter region; however, DNA-binding affinity of SqrR is reduced by the binding of heme, altering its regulatory activity. Circular dichroism spectroscopy clearly showed secondary structural changes in SqrR by the heme binding. Incremental change in the intracellular heme concentration is associated with small, but significant reduction in the transcriptional repression by SqrR. Overall, these results indicate that SqrR has an ability to bind heme to modulate its DNA-binding activity, which may be important for the precise regulation of RSS metabolism in vivo.