Evolutionary history of redox metal-binding domains across the tree of life Arye Harela, Yana Brombergb, Paul G. Falkowskia,c,1, and Debashish Bhattacharyad aEnvironmental Biophysics and Molecular Ecology Program, Institute of Marine and Coastal Science, bDepartment of Biochemistry and Microbiology, and dDepartment of Ecology, Evolution, and Natural Resources, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901; and cDepartment of Earth and Planetary Sciences, Rutgers University, The State University of New Jersey, Piscataway, NJ 08854 Contributed by Paul G. Falkowski, March 4, 2014 (sent for review October 23, 2013; reviewed by Edward F. DeLong and Michael Lynch) Oxidoreductases mediate electron transfer (i.e., redox) reactions diverged family members (2, 14, 15). The evolutionary relation- across the tree of life and ultimately facilitate the biologically driven ships of protein families identified using profile HMMs may be fluxes of hydrogen, carbon, nitrogen, oxygen, and sulfur on Earth. reconstructed with similarity networks. These networks are com- The core enzymes responsible for these reactions are ancient, often posed of vertices (nodes), which represent protein sequences, small in size, and highly diverse in amino acid sequence, and many connected by edges, representing similarity above a specified cut- require specific transition metals in their active sites. Here we re- off. Protein similarity networks offer an appealing alternative to construct the evolution of metal-binding domains in extant oxidor- phylogenetic approaches that rely on simultaneous multiple se- eductases using a flexible network approach and permissive profile quence alignments to reconstruct strictly bifurcating trees. By in- alignments based on available microbial genome data. Our results corporating different metrics (e.g., pairwise alignment of profiles or suggest there were at least 10 independent origins of redox domain sequence-to-profile alignments), network analysis provides a flexi- families. However, we also identified multiple ancient connections ble approach to access the composition of domains in ancient c between Fe2S2- (adrenodoxin-like) and heme- (cytochrome )binding protein families. In this study we applied a flexible network ap- domains. Our results suggest that these two iron-containing redox proach (11, 12) and permissive profile alignments (2, 14, 15) on families had a single common ancestor that underwent duplication microbial genome data to reconstruct the evolutionary history of and divergence. The iron-containing protein family constitutes ∼50% oxidoreductase metal-binding domains. Our results suggest that of all metal-containing oxidoreductases and potentially catalyzed re- whereas there were at least 10 independent origins of redox do- dox reactions in the Archean oceans. Heme-binding domains seem to be derived via modular evolutionary processes that ultimately form the main families one core family of iron-containing oxidoreductases backbone of redox reactions in both anaerobic and aerobic respiration came to dominate the electron fluxes across the planet before the and photosynthesis. The empirically discovered network allows us to evolution of oxygen. This family continues to represent the core of peer into the ancient history of microbial metabolism on our planet. biologically catalyzed electron transfer reactions on Earth. Results and Discussion iron–sulfur | Great Oxidation/Oxygenation Event | biogeochemical cycles | core pathways Profile Alignments Reveal at Least 10 Origins of Transition-Metal Redox Domains. By aligning sequence profiles of metal-binding redox domains (16) (102 HMM domain profiles with five or xidoreductases are anciently derived enzymes that mediate more sequences; Methods) we constructed a network of vertices, electron transfer (i.e., redox) reactions across the tree of life O with edges between them indicating domain similarity. This ap- and ultimately came to facilitate biologically driven fluxes of proach revealed 10 distinct (disconnected; Methods) subnetworks hydrogen, carbon, nitrogen, oxygen, and sulfur on Earth (1). It has (grouping 71 domains) and 31 isolated domains (Fig. 1, Fig. S1, been suggested that an ancestral pool of peptide modules may have and Table S1). Domains binding different ligands (e.g., Fe S given rise to the first protein folds that were dispersed into dif- 4 4 with Fe S ) were connected to each other only in the largest ferent superfamilies (2–4). Some of these peptide modules are part 2 2 of the limited set of building blocks (i.e., the “redox enzyme con- struction kit”) that gave rise to many oxidoreductases (5). Given Significance this Darwinian model of “descent with modification” for amino acid sequences in the active sites of enzymes, we analyzed a set of Oxidoreductases mediate the biological production of chemical core of oxidoreductase catalytic domains to elucidate origin(s) and energy and regulate the flow of essential elements in all or- evolutionary patterns of biological electron transfer reactions. ganisms and ecosystems, yet their evolutionary history is poorly Previous analysis suggests that the catalytic domains evolved in understood. Here we present a network analysis of all known microbes long before the Great Oxidation Event (GOE) ca. 2.4 metal-containing oxidoreductases across the tree of life. Mem- billion y ago (6). However, owing to their ancient provenance, bers of this network seem to have driven microbial metabolism often small size, and high divergence, the evolutionary history in the Archean oceans. Our analysis reveals that oxidoreductases of these domains is challenging to reconstruct. For example, are polyphyletic and derived from a minimum of 10 different a recent attempt to reconstruct the phylogeny of oxidoreductase ancient protein families with distantly related domains. How- domains based on structural data (7) was limited to pairwise dis- ever, we find substantial evidence that two apparently distinct tance analysis (without an underlying model of structure evolution) and ubiquitous iron-containing families of oxidoreductases con- taining Fe S and hemes arose from a single common ancestor. and implied a monophyletic origin of all metal-binding domains. 2 2 To address the evolutionary history of oxidoreductases, we used Author contributions: A.H. and D.B. designed research; A.H. performed research; D.B. hidden Markov model (HMM) (8–10) profile-to-profile alignments provided conceptual insights; A.H., Y.B., P.G.F., and D.B. analyzed data; and A.H. wrote and protein similarity networks (11, 12) to study the metal-binding the paper. domains. HMMs (10) are a class of probabilistic models generally Reviewers: E.F.D., Massachusetts Institute of Technology; and M.L., Indiana University. applicable to linear sequences (13). Because profile HMMs cap- The authors declare no conflict of interest. ture family-specific information, including functionally and struc- 1To whom correspondence should be addressed. E-mail: [email protected]. turally important residues, they are more sensitive and accurate This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. than sequence alignments alone when searching for deeply 1073/pnas.1403676111/-/DCSupplemental. 7042–7047 | PNAS | May 13, 2014 | vol. 111 | no. 19 www.pnas.org/cgi/doi/10.1073/pnas.1403676111 Downloaded by guest on September 25, 2021 subnetwork (Fig. 1A); however, this subnetwork did not contain Ancient Domains Arose in Thermophilic Anaerobic Prokaryotes and domains binding molybdenum, tungsten, manganese, or copper. Subsequently Diverged Following the Rise of Oxygen. To test the These results strongly imply that oxidoreductases (assigned to hypothesis that oxidoreductase transition metal-binding domains EC class 1) are polyphyletic (Fig. 1). that share a common ancestor may have diversified via envi- ronmental selection, we studied their distribution with respect to One Diverged Subnetwork Implies a Common Ancestor for Iron-Binding tolerance of oxygen and elevated temperatures. Domain families Domains. The largest subnetwork contains edges that connect dif- dominated by sequences from anaerobes (>50%) and thermo- ferent ligand-binding protein domains. Both profile-vs.-profile and philes (>70%) are found in the largest subnetwork (Fig. 1B and position-specific iterative (PSI)-BLAST alignments connect the SI Methods) and are presumably relics of the oldest core motifs. following binding domains: Fe S to Fe S ,iron–sulfur (Fe S , 4 4 2 2 4 4 In contrast, one-half of the domain families (14 of 28) in other Fe S ) to four cysteine iron domains (FeS ), and iron–sulfur 2 2 4 subnetworks are dominated by sequences from aerobic prokar- (Fe4S4,Fe2S2) to hemes (Fig. S2). In contrast, alignments of yotes. Ten of these domains are present in enzymes whose func- Fe4S4-binding domains with nitrogenase (FeFe, MoFe, VFe, or 8Fe-7S) are supported only by the more sensitive profile align- tions require molecular oxygen [e.g., oxygenases (monooxygenase c ments (Fig. 1A), implying a more distant relationship. This analysis and dioxygenase) and cytochrome oxidase]. Our results suggest suggests a monophyletic origin of iron-binding domains that we that some domains found in the largest subnetwork arose early in postulate to have evolved from FexSx to hemes (discussed below). a thermophilic anaerobic prokaryotic ancestor (17, 18) and sub- This core set of domains is distantly related to domains of nitro- sequently diverged as the oxidation state