Ancient Evolutionary Origin Research Articles

Did gene family expansions during the Eocene-Oligocene boundary climate cooling play a role in Pooideae adaptation to cool climates?

Adaptation to cool environments is a common feature in the core group of the grass subfamily Pooideae (Triticeae and Poeae). This suggest an ancient evolutionary origin of low temperature stress tolerance dating back prior to the initiation of taxonomic divergence of core Pooideae species. Viewing the Pooideae evolution in a palaeo-climatic perspective reveals that taxonomic divergence of the core Pooideae group initiated shortly after a global super-cooling period at the Eocene-Oligocene boundary (approximately 33.5-26 Ma). This global climate cooling altered distributions of plants and animals and must have imposed selection pressure for improved low temperature stress responses. Lineage-specific gene family expansions are known to be involved in adaptation to new environmental stresses. In Pooideae, two gene families involved in low temperature stress response, the C-repeat binding factor (CBF) and fructosyl transferase (FT) gene families, has undergone lineage-specific expansions. We investigated the timing of these gene family expansions by molecular dating and found that Pooideae-specific expansion events in CBF and FT gene families took place during Eocene-Oligocene super-cooling period. We hypothesize that the E-O super-cooling exerted selection pressure for improved low temperature stress response and frost tolerance in a core Pooideae ancestor, and that those individuals with multiple copies of CBF and FT genes were favoured.

Chaperone Requirements for Biosynthesis of the Trypanosome Variant Surface Glycoprotein

Background Trypanosoma brucei does not respond transcriptionally to several endoplasmic reticulum (ER) stress conditions, including tunicamycin or dithiothreitol, indicating the absence of a conventional unfolded protein response. This suggests divergent mechanisms for quality control (QC) of ER protein folding and export may be present in trypanosomes. As the variant surface glycoprotein (VSG) represents ∼90% of trypanosome plasma membrane protein, it is possible that VSG has evolved to fold efficiently to minimize ER folding burden.Methodology/Principal FindingsWe demonstrate the presence of a QC system by pharmacological inhibition of the trypanosome 26S proteasome. This indicates active proteasome-mediated VSG turnover as ∼2.5 fold more VSG is recovered from cell lysates following MG132 inhibition. An in silico scan of the trypanosome genome identified 28 open reading frames likely to encode polypeptides participating in ER nascent chain maturation. By RNA interference we monitored the importance of these gene products to proliferation, VSG abundance and cell morphology. 68% of the cohort were required for normal proliferation, and depletion of most of these factors resulted in increased VSG abundance, suggesting involvement in ERQC and degradation.Conclusions/SignificanceThe retention of genes for, and the involvement of many gene products in, VSG folding indicates a substantial complexity within the pathways required to perform this role. Counterintuitively, for a super-abundant antigen VSG is apparently made in excess. The biosynthetic excess VSG appears to be turned over efficiently by the proteasome, implying that considerable VSG is rejected by the trypanosome ERQC mechanism. Accordingly, the VSG polypeptide is not well optimized for folding, as only ∼30% attains the native state. Finally as much of the core ERQC system is functionally conserved in trypanosomes, the pathway has an ancient evolutionary origin, and was present in the last common eukaryotic ancestor.

Evolution of Protein Binding Modes in Homooligomers

The evolution of protein interactions cannot be deciphered without a detailed analysis of interaction interfaces and binding modes. We performed a large-scale study of protein homooligomers in terms of their symmetry, interface sizes, and conservation of binding modes. We also focused specifically on the evolution of protein binding modes from nine families of homooligomers and mapped 60 different binding modes and oligomerization states onto the phylogenetic trees of these families. We observed a significant tendency for the same binding modes to be clustered together and conserved within clades on phylogenetic trees; this trend is especially pronounced for close homologs with 70% sequence identity or higher. Some binding modes are conserved among very distant homologs, pointing to their ancient evolutionary origin, while others are very specific for a certain phylogenetic group. Moreover, we found that the most ancient binding modes have a tendency to involve symmetrical (isologous) homodimer binding arrangements with larger interfaces, while recently evolved binding modes more often exhibit asymmetrical arrangements and smaller interfaces.

Common Avian Infection Plagued the Tyrant Dinosaurs

Background Tyrannosaurus rex and other tyrannosaurid fossils often display multiple, smooth-edged full-thickness erosive lesions on the mandible, either unilaterally or bilaterally. The cause of these lesions in the Tyrannosaurus rex specimen FMNH PR2081 (known informally by the name ‘Sue’) has previously been attributed to actinomycosis, a bacterial bone infection, or bite wounds from other tyrannosaurids.Methodology/Principal FindingsWe conducted an extensive survey of tyrannosaurid specimens and identified ten individuals with full-thickness erosive lesions. These lesions were described, measured and photographed for comparison with one another. We also conducted an extensive survey of related archosaurs for similar lesions. We show here that these lesions are consistent with those caused by an avian parasitic infection called trichomonosis, which causes similar abnormalities on the mandible of modern birds, in particular raptors.Conclusions/SignificanceThis finding represents the first evidence for the ancient evolutionary origin of an avian transmissible disease in non-avian theropod dinosaurs. It also provides a valuable insight into the palaeobiology of these now extinct animals. Based on the frequency with which these lesions occur, we hypothesize that tyrannosaurids were commonly infected by a Trichomonas gallinae-like protozoan. For tyrannosaurid populations, the only non-avian dinosaur group that show trichomonosis-type lesions, it is likely that the disease became endemic and spread as a result of antagonistic intraspecific behavior, consumption of prey infected by a Trichomonas gallinae-like protozoan and possibly even cannibalism. The severity of trichomonosis-related lesions in specimens such as Tyrannosaurus rex FMNH PR2081 and Tyrannosaurus rex MOR 980, strongly suggests that these animals died as a direct result of this disease, mostly likely through starvation.

Organization, Evolution, and Expression Analysis of the Biosynthetic Gene Cluster for Scytonemin, a Cyanobacterial UV-Absorbing Pigment

Cyanobacteria are photosynthetic prokaryotes capable of protecting themselves from UV radiation through the biosynthesis of UV-absorbing secondary metabolites, such as the mycosporines and scytonemin. Scytonemin, a novel indolic-phenolic pigment, is found sequestered in the sheath, where it provides protection to the subtending cells during exposure to UV radiation. The biosynthesis of scytonemin is encoded by a previously identified gene cluster that is present in six cyanobacterial species whose genomes are available. A comparison of these clusters reveals that two major cluster architectures exist which appear to have evolved through rearrangements of large sections, such as those genes responsible for aromatic amino acid biosynthesis and through the insertion of genes that potentially confer additional biosynthetic capabilities. Differential transcriptional expression analysis demonstrated that the entire gene cluster is transcribed in higher abundance after exposure to UV radiation. This analysis helps delineate the cluster boundaries and indicates that regulation of this cluster is controlled by the presence or absence of UV radiation. The findings from an evolutionary phylogenetic analysis combined with the fact that the scytonemin gene cluster is distributed across several cyanobacterial lineages led to our proposal that the distribution of this gene cluster is best explained through an ancient evolutionary origin.

The evolution of pheromonal communication

Small-brained rodents have been the principle focus for pheromonal research and have provided comprehensive insights into the chemosensory mechanisms that underpin pheromonal communication and the hugely important roles that pheromones play in behavioural regulation. However, pheromonal communication does not start or end with the mouse and the rat, and work in amphibians reveals much about the likely evolutionary origins of the chemosensory systems that mediate pheromonal effects. The dual olfactory organs (the main olfactory epithelium and the vomeronasal organ), their receptors and their separate projection pathways appear to have ancient evolutionary origins, appearing in the aquatic ancestors of all tetrapods during the Devonian period and so pre-dating the transition to land. While the vomeronasal organ has long been considered an exclusively pheromonal organ, accumulating evidence indicates that it is not the sole channel for the transduction of pheromonal information and that both olfactory systems have been co-opted for the detection of different pheromone signals over the course of evolution. This has also led to great diversity in the vomeronasal and olfactory receptor families, with enormous levels of gene diversity and inactivation of genes in different species. Finally, the evolution of trichromacy as well as huge increases in social complexity have minimised the role of pheromones in the lives of primates, leading to the total inactivation of the vomeronasal system in catarrhine primates while the brain increased in size and behaviour became emancipated from hormonal regulation.

Research commentary in brief

BiopolymersVolume 89, Issue 12 p. iv-iv Free Access Research commentary in brief First published: 29 September 2008 https://doi.org/10.1002/bip.21090AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Resolving the Pivotal Role of Argonaut Proteins in RNAi Nils G. Walter The discovery of RNA interference (RNAi)1 in 1998 quickly revolutionized the tool sets of both basic biological research and applied biomedical drug development. Almost weekly new groundbreaking details of the complex set of RNAi pathways involved in the regulation of exogenous and endogenous genes in all higher eukaryotes are being unearthed. No wonder then that biophysicists are struggling to keep up with the rapidly expanding list of key players to elucidate their structure and dynamic interactions, thought critical to tapping RNAi s full biological and biomedical potential. Argonaut (Ago) proteins were previously known to be important for plant development and Drosophila stem-cell division, but stepped into the limelight when their pervasive function in RNAi became apparent.2 Argonauts bind small interfering RNAs (siRNAs) and other non-protein coding RNAs as guide strands to form an RNA-induced silencing complex (RISC), the effector of RNAi. Ago proteins contain four domains, the N-terminal, PAZ, Mid, and PIWI domains. The PAZ domain binds single-stranded RNA (ssRNA), while the RNase H-like PIWI domain carries the cleavage activity of RISC that destroys a target complementary to the bound guide strand. Attesting to their ancient evolutionary origin, Ago proteins are found in all three kingdoms of life, and previous work has characterized the crystal structures of both archaeal and eubacterial Ago proteins in free and siRNA bound form. In a recent article in Nature, Patel, Tuschl, and co-workers describe the structure of a eubacterial Thermus thermophilus argonaute with all four protein domains in complex with either a 21-nucleotide or a 10-nucleotide single-stranded guide DNA analog.3 The 21-mer ligand complex shows the DNA 5′- and 3′-ends anchored by the Mid and PAZ domains, respectively, and maps out the nucleic-acid-binding channel between the PAZ and PIWI domains of Ago. A comparison with the 10-mer ligand complex shows that the 5′-anchor is lost so that both the Mid and PAZ domains significantly pivot (by 22° and 25°, respectively) around the PIWI domain. The authors suggest that a similar conformational change may accompany closing of Ago around the RNA guide strand. In addition, two arginines of Ago pivot nucleotide 11 of the 21-mer guide DNA around nucleotide 10, directly juxtaposed to the expected target cleavage site, highlighting how RISC may predispose a particular segment of the guide-target RNA duplex for subsequent catalysis. In small catalytic RNAs such kinking commonly positions a specific bonds for self-cleavage,4 but it remains to be seen whether the kink or its removal upon target binding plays any role in RNA cleavage by Ago. At any rate, the new crystal structures provide another snapshot on the road to fully deciphering the pivotal role of Ago proteins in RNAi. Nils G. Walter is Associate Professor of Chemistry at the University of Michigan and an Associate Editor of Biopolymers. E-mail: nwalter@umich.edu. References 1 Fire A,Xu S,Montgomery MK,Kostas SA,Driver SE,Mello, CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998 Feb 19; 391(6669): 806– 11. CrossrefCASPubMedWeb of Science®Google Scholar 2 Hutvagner, G and Simard, M.J. 2008. Argonaute proteins: key players in RNA silencing. Nat. Rev. Mol. Cell. Biol. 9: 22– 32. CrossrefCASPubMedWeb of Science®Google Scholar 3 Wang, Y.,Sheng, G.,Juranek, S.,Tuschl, T., and Patel, D.J. 2008. Structure of the guide-strand-containing argonaute silencing complex. Nature in press, doi:10.1038/nature07315. PubMedGoogle Scholar 4 Walter, N.G. 2007. Ribozyme catalysis revisited: is water involved? Mol. Cell 28: 923– 929. CrossrefCASPubMedWeb of Science®Google Scholar Volume89, Issue12December 2008Pages iv-iv ReferencesRelatedInformation

An Ancient Evolutionary Origin of Genes Associated with Human Genetic Diseases

Several thousand genes in the human genome have been linked to a heritable genetic disease. The majority of these appear to be nonessential genes (i.e., are not embryonically lethal when inactivated), and one could therefore speculate that they are late additions in the evolutionary lineage toward humans. Contrary to this expectation, we find that they are in fact significantly overrepresented among the genes that have emerged during the early evolution of the metazoa. Using a phylostratigraphic approach, we have studied the evolutionary emergence of such genes at 19 phylogenetic levels. The majority of disease genes was already present in the eukaryotic ancestor, and the second largest number has arisen around the time of evolution of multicellularity. Conversely, genes specific to the mammalian lineage are highly underrepresented. Hence, genes involved in genetic diseases are not simply a random subset of all genes in the genome but are biased toward ancient genes.

Ancient evolutionary origin of the neural crest gene regulatory network

Ancient evolutionary origin of the neural crest gene regulatory network

Specificity of the innate immune system and diversity of C-type lectin domain (CTLD) proteins in the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans has become an important model for the study of innate immunity. Its immune system is based on several signaling cascades, including a Toll-like receptor, three mitogen-activated protein kinases (MAPK), one transforming growth factor- β (TGF- β), the insulin-like receptor (ILR), and the programmed cell death (PCD) pathway. Furthermore, it also involves C-type lectin domain- (CTLD) containing proteins as well as several classes of antimicrobial effectors such as lysozymes. Almost all components of the nematode immune system have homologs in other organisms, including humans, and are therefore likely of ancient evolutionary origin. At the same time, most of them are part of a general stress response, suggesting that they only provide unspecific defense. In the current article, we re-evaluate this suggestion and explore the level of specificity in C. elegans innate immunity, i.e. the nematode's ability to mount a distinct defense response towards different pathogens. We draw particular attention to the CTLD proteins, which are abundant in the nematode genome (278 genes) and many of which show a pathogen-specific response during infection. Specificity may also be achieved through the differential activation of antimicrobial genes, distinct functions of the immunity signaling cascades as well as signal integration across pathways. Taken together, our evaluation reveals high potential for immune specificity in C. elegans that may enhance the nematode's ability to fight off pathogens.

Ancient Evolutionary Origin of the Neural Crest Gene Regulatory Network

The vertebrate neural crest migrates from its origin, the neural plate border, to form diverse derivatives. We previously hypothesized that a neural crest gene regulatory network (NC-GRN) guides neural crest formation. Here, we investigate when during evolution this hypothetical network emerged by analyzing neural crest formation in lamprey, a basal extant vertebrate. We identify 50 NC-GRN homologs and use morpholinos to demonstrate a critical role for eight transcriptional regulators. The results reveal conservation in deployment of upstream factors, suggesting that proximal portions of the network arose early in vertebrate evolution and have been conserved for >500 million years. We found biphasic expression of neural crest specifiers and differences in deployment of some specifiers and effectors expected to confer species-specific properties. By testing the collective expression and function of neural crest genes in a single, basal vertebrate, we reveal the ground state of the NC-GRN and resolve ambiguities between model organisms.

Hantavirus in Northern Short-tailed Shrew, United States

Phylogenetic analyses, based on partial medium- and large-segment sequences, support an ancient evolutionary origin of a genetically distinct hantavirus detected by reverse transcription–PCR in tissues of northern short-tailed shrews (Blarina brevicauda) captured in Minnesota in August 1998. To our knowledge, this is the first evidence of hantaviruses harbored by shrews in the Americas.

Characterization of two globin genes from the malaria mosquito Anopheles gambiae: Divergent origin of nematoceran haemoglobins

The chironomid midges are the only insects that harbour true haemoglobin in their haemolymph. Here we report the identification of haemoglobin genes in two other nematoceran species. Two paralogous haemoglobin genes (glob1 and glob2) from the malaria mosquito Anopheles gambiae were cloned and sequenced. Furthermore, we identified two orthologous haemoglobin genes in the yellow fever mosquito Aedes aegypti. All four haemoglobins were predicted to be intracellular proteins, with the amino acids required for heme- and oxygen-binding being conserved. In situ-hybridization studies showed that glob1 and glob2 expression in An. gambiae is mainly associated with the tracheal system. This pattern resembles that of other insect intracellular globins. We also observed expression of glob2 in visceral muscles. Phylogenetic analyses showed that the globins of the mosquitoes and the Chironomidae are not orthologous. The chironomid haemoglobins share a recent common origin with the brachyceran glob1 proteins. The mosquito glob1 and glob2 proteins, which separated by gene duplication around 170 million years ago, form a distinct clade of more ancient evolutionary origin within the insects. The glob1 genes have introns in the ancestral globin positions B12.2 and G7.0. An additional intron was observed in Ae. aegypti glob1 helix position E18.0, providing evidence for a recent intron gain event. Both mosquito glob2 genes have lost the B12.2 intron. This pattern must be interpreted in terms of dynamic intron gain and loss events in the globin gene lineage.

The Endo-β-Mannanase gene families in Arabidopsis, rice, and poplar

Mannans are widespread hemicellulosic polysaccharides in plant cell walls. Hydrolysis of the internal beta-1,4-D: -mannopyranosyl linkage in the backbone of mannans is catalyzed by endo-beta-mannanase. Plant endo-beta-mannanase has been well studied for its function in seed germination. Its involvement in other plant biological processes, however, remains poorly characterized or elusive. The completed genome sequences of Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and poplar (Populus trichocarpa) provide an opportunity to conduct comparative genomic analysis of endo-beta-mannanase genes in these three species. In silico sequence analysis led to the identification of eight, nine and 11 endo-beta-mannanase genes in the genomes of Arabidopsis, rice, and poplar, respectively. Sequence comparisons revealed the conserved amino acids and motifs that are critical for the active site of endo-beta-mannanases. Intron/exon structure analysis in conjunction with phylogenetic analysis implied that both intron gain and intron loss has played roles in the evolution of endo-beta-mannanase genes. The phylogenetic analysis that included the endo-beta-mannanases from plants and other organisms implied that plant endo-beta-mannanases have an ancient evolutionary origin. Comprehensive expression analysis of all Arabidopsis and rice endo-beta-mannanase genes showed divergent expression patterns of individual genes, suggesting that the enzymes encoded by these genes, while carrying out the same biochemical reaction, are involved in diverse biological processes.

Ancient evolutionary origin of diversified variable regions demonstrated by crystal structures of an immune-type receptor in amphioxus

Although the origins of genes encoding the rearranging binding receptors remain obscure, it is predicted that their ancestral forms were nonrearranging immunoglobulin-type domains. Variable region-containing chitin-binding proteins (VCBPs) are diversified immune-type molecules found in amphioxus (Branchiostoma floridae), an invertebrate that diverged early in deuterostome phylogeny. To study the potential evolutionary relationships between VCBPs and vertebrate adaptive immune receptors, we solved the structures of both a single V-type domain (to 1.15 A) and a pair of V-type domains (to 1.85 A) from VCBP3. The deduced structures show integral features of the ancestral variable-region fold as well as unique features of variable-region pairing in molecules that may reflect characteristics of ancestral forms of diversified immune receptors found in modern-day vertebrates.

An ancient evolutionary origin of the Rag1/2 gene locus.

The diversity of antigen receptors in the adaptive immune system of jawed vertebrates is generated by a unique process of somatic gene rearrangement known as V(D)J recombination. The Rag1 and Rag2 proteins are the key mediators of this process. They are encoded by a compact gene cluster that has exclusively been identified in animal species displaying V(D)J-mediated immunity, and no homologous gene pair has been identified in other organisms. This distinctly restricted phylogenetic distribution has led to the hypothesis that one or both of the Rag genes were coopted after horizontal gene transfer and assembled into a Rag1/2 gene cluster in a common jawed vertebrate ancestor. Here, we identify and characterize a closely linked pair of genes, SpRag1L and SpRag2L, from an invertebrate, the purple sea urchin (Strongylocentrotus purpuratus) with similarity in both sequence and genomic organization to the vertebrate Rag1 and Rag2 genes. They are coexpressed during development and in adult tissues, and recombinant versions of the proteins form a stable complex with each other as well as with Rag1 and Rag2 proteins from several vertebrate species. We thus conclude that SpRag1L and SpRag2L represent homologs of vertebrate Rag1 and Rag2. In combination with the apparent absence of V(D)J recombination in echinoderms, this finding strongly suggests that linked Rag1- and Rag2-like genes were already present and functioning in a different capacity in the common ancestor of living deuterostomes, and that their specific role in the adaptive immune system was acquired much later in an early jawed vertebrate.

The mammalian α D-globin gene lineage and a new model for the molecular evolution of α-globin gene clusters at the stem of the mammalian radiation

We have explored the evolution of the α-globin gene family by comparative sequence and phylogenetic analyses of mammalian α-globin genes. Our analyses reveal the existence of a new α-globin gene lineage in mammals that is related to the α D-globin genes of birds, squamates and turtles. The gene is located in the middle of the α-globin gene cluster of a marsupial, Sminthopsis macroura and of humans. It exists in a wide variety of additional mammals, including pigs, cows, cats, and dogs, but is a pseudogene in American marsupials. Evolutionary analyses suggest that the gene has generally evolved under purifying selection, indicative of a functional gene. The presence of mRNA products in humans, pigs, and cows also suggest that the gene is expressed and likely to be functional. The analyses support the hypothesis that the α D-globin gene lineage has an ancient evolutionary origin that predates the divergence of amniotes. The structural similarity of α-globin gene clusters of marsupials and humans suggest that an eight gene cluster (5′- ζ2- ζ1-α D-α3-α2-α1-θ-ω-3′), including seven α-like genes and one β-like globin gene ( ω- globin) existed in the common ancestor of all marsupial and eutherian mammals. This basic structure has remained relatively stable in marsupials and in the lineage leading to humans, although ω- globin has been lost from the α-globin gene cluster of humans.

Arousal Mechanisms: Speedy Flies Don’t Sleep at Night

Alertness and behavioral performance depend on an animal's level of arousal. In vertebrates, reinforcement and maintenance of arousal in the cortex are ensured by diffuse inputs from neurons releasing biogenic amine neuromodulators. Fruit flies similarly use dopamine for arousal control, indicating an ancient evolutionary origin of this essential feature of the functioning brain.

Permutation Pattern Discovery in Biosequences

Functionally related genes often appear in each other's neighborhood on the genome; however, the order of the genes may not be the same. These groups or clusters of genes may have an ancient evolutionary origin or may signify some other critical phenomenon and may also aid in function prediction of genes. Such gene clusters also aid toward solving the problem of local alignment of genes. Similarly, clusters of protein domains, albeit appearing in different orders in the protein sequence, suggest common functionality in spite of being nonhomologous. In the paper, we address the problem of automatically discovering clusters of entities, be they genes or domains: we formalize the abstract problem as a discovery problem called the (pi)pattern problem and give an algorithm that automatically discovers the clusters of patterns in multiple data sequences. We take a model-less approach and introduce a notation for maximal patterns that drastically reduces the number of valid cluster patterns, without any loss of information, We demonstrate the automatic pattern discovery tool on motifs on E. Coli protein sequences.

Reversible hexa‐ to penta‐coordination of the heme Fe atom modulates ligand binding properties of neuroglobin and cytoglobin

Neuroglobin (Ngb) and cytoglobin (Cygb) are two recently discovered intracellular members of the vertebrate hemoglobin (Hb) family. Ngb, predominantly expressed in nerve cells, is of ancient evolutionary origin and is homologous to nerve-globins of invertebrates. Cygb, present in many different tissues, shares common ancestry with myoglobin (Mb) and can be traced to early vertebrate evolution. Ngb is held to facilitate O2 diffusion to the mitochondria and to protect neuronal cells from hypoxic-ischemic insults, may be an oxidative stress-responsive sensor protein for signal transduction, and may carry out enzymatic activities, such as NO/O2 scavenging. Cygb is linked to collagen synthesis, may provide O2 for enzymatic reactions, and may be involved in a ROS(NO)-signaling pathway(s). Ngb and Cgb display the classical three-over-three alpha-helical fold of Hb and Mb, and are endowed with a hexa-coordinate heme-Fe atom, in their ferrous and ferric forms, having the heme distal HisE7 residue as the endogenous ligand. Reversible hexa- to penta-coordination of the heme Fe atom modulates ligand binding properties of Ngb and Cygb. Moreover, Ngb and Cygb display a tunnel/cavity system within the protein matrix held to facilitate ligand channeling to/from the heme, multiple ligand copies storage, multi-ligand reactions, and conformational transitions supporting ligand binding.