The role of beta chains in the control of the hemoglobin oxygen binding function: chimeric human/mouse proteins, structure, and function.

By using transgenic methodologies, we have produced a number of mouse/human chimeric hemoglobins containing adult mouse and human embryonic globin chains. A detailed analysis of the oxygen binding properties of these proteins identifies the dominant role played by the specific beta-type globin chains in the control of the oxygen binding characteristics. Further analysis traces the origins of these effects to alterations in the properties of the T states of these proteins. The human zeta/mouse beta chimeric protein has been crystallized, and its structure has been determined by X-ray diffraction to a resolution of 2.1 A with R (R(free)) values of 21.6% (24.9%). Close examination of the structure indicates that the subunit interfaces contain contacts which, although different from those present in either the parent human or the parent mouse proteins, retain the overall stabilizing interactions seen in other R state hemoglobins.     

A Globin Domain in a Neuronal Transmembrane Receptor of Caenorhabditis elegans and Ascaris suum: MOLECULAR MODELING AND FUNCTIONAL PROPERTIES*

We report the structural and biochemical characterization of GLB-33, a putative neuropeptide receptor that is exclusively expressed in the nervous system of the nematode Caenorhabditis elegans. This unique chimeric protein is composed of a 7-transmembrane domain (7TM), GLB-33 7TM, typical of a G-protein-coupled receptor, and of a globin domain (GD), GLB-33 GD. Comprehensive sequence similarity searches in the genome of the parasitic nematode, Ascaris suum, revealed a chimeric protein that is similar to a Phe-Met-Arg-Phe-amide neuropeptide receptor. The three-dimensional structures of the separate domains of both species and of the full-length proteins were modeled. The 7TM domains of both proteins appeared very similar, but the globin domain of the A. suum receptor surprisingly seemed to lack several helices, suggesting a novel truncated globin fold. The globin domain of C. elegans GLB-33, however, was very similar to a genuine myoglobin-type molecule. Spectroscopic analysis of the recombinant GLB-33 GD showed that the heme is pentacoordinate when ferrous and in the hydroxide-ligated form when ferric, even at neutral pH. Flash-photolysis experiments showed overall fast biphasic CO rebinding kinetics. In its ferrous deoxy form, GLB-33 GD is capable of reversibly binding O₂ with a very high affinity and of reducing nitrite to nitric oxide faster than other globins. Collectively, these properties suggest that the globin domain of GLB-33 may serve as a highly sensitive oxygen sensor and/or as a nitrite reductase. Both properties are potentially able to modulate the neuropeptide sensitivity of the neuronal transmembrane receptor.     

Influence of the protein matrix on intramolecular histidine ligation in ferric and ferrous hexacoordinate hemoglobins

Present in most organisms, hexacoordinate hemoglobins (hxHbs) are proteins that have evolved the capacity for reversible bis-histidyl heme coordination. The heme prosthetic group enables diverse protein functionality, such as electron transfer, redox reactions, ligand transport, and enzymatic catalysis. The reactivity of heme is greatly effected by the coordination and noncovalent chemical environment imposed by its connate protein. Of considerable interest is how the hxHb globin fold achieves reversible intramolecular coordination while still enabling high-affinity binding of oxygen, nitric oxide, and other small ligands. Here we explore this question by examining the role of the protein matrix on coordination behavior in a group of hxHbs from animals, plants, and bacteria, including human neuroglobin and cytoglobin, a nonsymbiotic hemoglobin from rice, and a truncated hemoglobin from the cyanobacterium Synechocystis. This is done with a set of experiments measuring the reduction potentials of each wild-type hxHb and its corresponding mutant protein where the reversibly bound histidine (the distal His) has been replaced with a noncoordinating side chain. These reduction potentials, coupled with studies of the mutant proteins saturated with exogenous imidazole, enable us to assess the effects of the protein matrices on histidine coordination. Our results show significant variation among the hxHbs, demonstrating flexibility in the globin moiety's ability to regulate reversible coordination. This regulation is particularly evident in the plant nonsymbiotic hemoglobins, where ferric state histidine coordination affinity is substantially lowered by the protein matrix.     

Structure and reactivity of hexacoordinate hemoglobins

The heme prosthetic group in hemoglobins is most often attached to the globin through coordination of either one or two histidine side chains. Those proteins with one histidine coordinating the heme iron are called "pentacoordinate" hemoglobins, a group represented by red blood cell hemoglobin and most other oxygen transporters. Those with two histidines are called "hexacoordinate hemoglobins", which have broad representation among eukaryotes. Coordination of the second histidine in hexacoordinate Hbs is reversible, allowing for binding of exogenous ligands like oxygen, carbon monoxide, and nitric oxide. Research over the past several years has produced a fairly detailed picture of the structure and biochemistry of hexacoordinate hemoglobins from several species including neuroglobin and cytoglobin in animals, and the nonsymbiotic hemoglobins in plants. However, a clear understanding of the physiological functions of these proteins remains an elusive goal.     

Bacterial hemoglobins and flavohemoglobins: versatile proteins and their impact on microbiology and biotechnology

In response to oxygen limitation or oxidative and nitrosative stress, bacteria express three kinds of hemoglobin proteins: truncated hemoglobins (tr Hbs), hemoglobins (Hbs) and flavohemoglobins (flavo Hbs). The two latter groups share a high sequence homology and structural similarity in their globin domain. Flavohemoglobin proteins contain an additional reductase domain at their C-terminus and their expression is induced in the presence of reactive nitrogen and oxygen species. Flavohemoglobins detoxify NO in an aerobic process, termed nitric oxide dioxygenase reaction, which protects the host from various noxious nitrogen compounds. Only a small number of bacteria express hemoglobin proteins and the best studied of these is from Vitreoscilla sp. Vitreoscilla hemoglobin (VHb) has been expressed in various heterologous hosts under oxygen-limited conditions and has been shown to improve growth and productivity, rendering the protein interesting for biotechnology industry. The close interaction of VHb with the terminal oxidases has been shown and this interplay has been proposed to enhance respiratory activity and energy production by delivering oxygen, the ultimate result being an improvement in growth properties.     

Protein fold and structure in the truncated (2/2) globin family

Analysis of amino acids sequences and protein folds has recently unraveled the structural bases and details of several proteins from the recently discovered "truncated hemoglobin" family. The analysis here presented, in agreement with previous surveys, shows that truncated hemoglobins can be classified in three main groups, based on their structural properties. Crystallographic analyses have shown that all three groups adopt a 2-on-2 alpha-helical sandwich fold, resulting from apparent editing of the classical 3-on-3 alpha-helical sandwich of vertebrate and invertebrate conventional globins. Specific structural features distinguish each of the three groups. Among these, a protein matrix tunnel system is typical of group I, a Trp residue at the G8 topological site is conserved in groups II and III, and TyrB10 is almost invariant through the three groups. A strongly intertwined network of hydrogen bonds stabilizes the heme bound ligand, despite variability of the heme distal residues observed in the different proteins considered. Details of ligand recognition in the three groups are discussed at the light of residue conservation and of differing ligand diffusion pathways to the heme. Based on structural analyses of the family-specific fold, we endorse a recent proposal of leaving the "truncated hemoglobins" term, that does not represent properly the observed 2-on-2 alpha-helical sandwich fold, and adopting the simple "2/2Hb" term to concisely address this protein family.     

Lysine as a heme iron ligand: A property common to three truncated hemoglobins from Chlamydomonas reinhardtii

Background

The nuclear genome of Chlamydomonas reinhardtii encodes a dozen hemoglobins of the truncated lineage. Four of these, named THB1–4, contain a single ~130-residue globin unit. THB1, which is cytoplasmic and capable of nitric oxide dioxygenation activity, uses a histidine and a lysine as axial ligands to the heme iron. In the present report, we compared THB2, THB3, and THB4 to THB1 to gain structural and functional insights into algal globins.

Structure-function relationships in unusual nonvertebrate globins

Based on the literature and our own results, this review summarizes the most recent state of nonvertebrate myoglobin (Mb) and hemoglobin (Hb) research, not as a general survey of the subject but as a case study. For this purpose, we have selected here four typical globins to discuss their unique structures and properties in detail. These include Aplysia myoglobin, which served as a prototype for the unusual globins lacking the distal histidine residue; midge larval hemoglobin showing a high degree of polymorphism; Tetrahymena hemoglobin evolved with a truncated structure; and yeast flavohemoglobin carrying an enigmatic two-domain structure. These proteins are not grouped by any common features other than the fact they have globin domains and heme groups. As a matter of course, various biochemical functions other than the conventional oxygen transport or storage have been proposed so far to these primitive or ancient hemoglobins or myoglobins, but the precise in vivo activity is still unclear. In this review, special emphasis is placed on the stability properties of the heme-bound O2. Whatever the possible roles of nonvertebrate myoglobins and hemoglobins may be (or might have been), the binding of molecular oxygen to iron(II) must be the primary event to manifest their physiological functions in vivo. However, the reversible and stable binding of O2 to iron(II) is not a simple process, since the oxygenated form of Mb or Hb is oxidized easily to its ferric met-form with the generation of superoxide anion. The metmyoglobin or methemoglobin thus produced cannot bind molecular oxygen and is therefore physiologically inactive. In this respect, protozoan ciliate myoglobin and yeast flavohemoglobin are of particular interest in their very unique structures. Indeed, both proteins have been found to have completely different strategies for overcoming many difficulties in the reversible and stable binding of molecular oxygen, as opposed to the irreversible oxidation of heme iron(II). Such comparative studies of the stability of MbO2 or HbO2 are of primary importance, not only for a full understanding of the globin evolution, but also for planning new molecular designs for synthetic oxygen carriers that may be able to function in aqueous solution and at physiological temperature.     

A phylogenomic profile of globins

Background

Globins occur in all three kingdoms of life: they can be classified into single-domain globins and chimeric globins. The latter comprise the flavohemoglobins with a C-terminal FAD-binding domain and the gene-regulating globin coupled sensors, with variable C-terminal domains. The single-domain globins encompass sequences related to chimeric globins and «truncated» hemoglobins with a 2-over-2 instead of the canonical 3-over-3 α-helical fold.

Results

A census of globins in 26 archaeal, 245 bacterial and 49 eukaryote genomes was carried out. Only ~25% of archaea have globins, including globin coupled sensors, related single domain globins and 2-over-2 globins. From one to seven globins per genome were found in ~65% of the bacterial genomes: the presence and number of globins are positively correlated with genome size. Globins appear to be mostly absent in Bacteroidetes/Chlorobi, Chlamydia, Lactobacillales, Mollicutes, Rickettsiales, Pastorellales and Spirochaetes. Single domain globins occur in metazoans and flavohemoglobins are found in fungi, diplomonads and mycetozoans. Although red algae have single domain globins, including 2-over-2 globins, the green algae and ciliates have only 2-over-2 globins. Plants have symbiotic and nonsymbiotic single domain hemoglobins and 2-over-2 hemoglobins. Over 90% of eukaryotes have globins: the nematode Caenorhabditis has the most putative globins, ~33. No globins occur in the parasitic, unicellular eukaryotes such as Encephalitozoon, Entamoeba, Plasmodium and Trypanosoma.

Conclusion

Although Bacteria have all three types of globins, Archaeado not have flavohemoglobins and Eukaryotes lack globin coupled sensors. Since the hemoglobins in organisms other than animals are enzymes or sensors, it is likely that the evolution of an oxygen transport function accompanied the emergence of multicellular animals.     

Novel flavohemoglobins of mycobacteria

Flavohemoglobins (flavoHbs) constitute a distinct class of chimeric hemoglobins in which a globin domain is coupled with a ferredoxin reductase such as FAD- and NADH-binding modules. Structural features and active site of heme and reductase domains are highly conserved in various flavoHbs. A new class of flavoHbs, displaying crucial differences in functionally conserved regions of heme and reductase domains, have been identified in mycobacteria. Mining of microbial genome data indicated that the occurrence of such flavoHbs might be restricted to a small group of microbes unlike conventional flavoHbs that are widespread among prokaryotes and lower eukaryotes. One of the representative flavoHbs of this class, encoded by Rv0385 gene (MtbFHb) of Mycobacterium tuberculosis, has been cloned, expressed, and characterized. The ferric and deoxy spectra of MtbFHb displayed a hexacoordinate state indicating that its distal site may be occupied by an intrinsic amino acid or an external ligand and it may not be involved in nitric oxide detoxification. Phylogenetic analysis revealed that mycobacterial flavoHbs constitute a separate cluster distinct from conventional flavoHbs and may have novel function(s).     

Androglobin: a chimeric globin in metazoans that is preferentially expressed in Mammalian testes

Comparative genomic studies have led to the recent identification of several novel globin types in the Metazoa. They have revealed a surprising evolutionary diversity of functions beyond the familiar O(2) supply roles of hemoglobin and myoglobin. Here we report the discovery of a hitherto unrecognized family of proteins with a unique modular architecture, possessing an N-terminal calpain-like domain, an internal, circular permuted globin domain, and an IQ calmodulin-binding motif. Putative orthologs are present in the genomes of many metazoan taxa, including vertebrates. The calpain-like region is homologous to the catalytic domain II of the large subunit of human calpain-7. The globin domain satisfies the criteria of a myoglobin-like fold but is rearranged and split into two parts. The recombinantly expressed human globin domain exhibits an absorption spectrum characteristic of hexacoordination of the heme iron atom. Molecular evolutionary analyses indicate that this chimeric globin family is phylogenetically ancient and originated in the common ancestor to animals and choanoflagellates. In humans and mice, the gene is predominantly expressed in testis tissue, and we propose the name "androglobin" (Adgb). Expression is associated with postmeiotic stages of spermatogenesis and is insensitive to experimental hypoxia. Evidence exists for increased gene expression in fertile compared with infertile males.     

The crystal structure of Synechocystis hemoglobin with a covalent heme linkage

The x-ray crystal structure of Synechocystis hemoglobin has been solved to a resolution of 1.8 A. The conformation of this structure is surprisingly different from that of the previously reported solution structure, probably due in part to a covalent linkage between the heme 2-vinyl and His117 that is present in the crystal structure but not in the structure solved by NMR. Synechocystis hemoglobin is a hexacoordinate hemoglobin in which the heme iron is coordinated by both the proximal and distal histidines. It is also a member of the "truncated hemoglobin" family that is much shorter in primary structure than vertebrate and plant hemoglobins. In contrast to other truncated hemoglobins, the crystal structure of Synechocystis hemoglobin displays no "ligand tunnel" and shows that several important amino acid side chains extrude into the solvent instead of residing inside the heme pocket. The stereochemistry of hexacoordination is compared with other hexacoordinate hemoglobins and cytochromes in an effort to illuminate factors contributing to ligand affinity in hexacoordinate hemoglobins.     

Structure of the Hemoglobin-IsdH Complex Reveals the Molecular Basis of Iron Capture by Staphylococcus aureus

Staphylococcus aureus causes life-threatening disease in humans. The S. aureus surface protein iron-regulated surface determinant H (IsdH) binds to mammalian hemoglobin (Hb) and extracts heme as a source of iron, which is an essential nutrient for the bacteria. However, the process of heme transfer from Hb is poorly understood. We have determined the structure of IsdH bound to human Hb by x-ray crystallography at 4.2 Å resolution, revealing the structural basis for heme transfer. One IsdH molecule is bound to each α and β Hb subunit, suggesting that the receptor acquires iron from both chains by a similar mechanism. Remarkably, two near iron transporter (NEAT) domains in IsdH perform very different functions. An N-terminal NEAT domain binds α/β globin through a site distant from the globin heme pocket and, via an intervening structural domain, positions the C-terminal heme-binding NEAT domain perfectly for heme transfer. These data, together with a 2.3 Å resolution crystal structure of the isolated N-terminal domain bound to Hb and small-angle x-ray scattering of free IsdH, reveal how multiple domains of IsdH cooperate to strip heme from Hb. Many bacterial pathogens obtain iron from human hemoglobin using proteins that contain multiple NEAT domains and other domains whose functions are poorly understood. Our results suggest that, rather than acting as isolated units, NEAT domains may be integrated into higher order architectures that employ multiple interaction interfaces to efficiently extract heme from host proteins.     

Organization of non-vertebrate globin genes.

The organization of non-vertebrate globin genes exhibits substantially more variability than the three-exon, two-intron structure of the vertebrate globin genes. (1) The structures of genes of the single-domain globin chains of the annelid Lumbricus and the mollusc Anadara, and the globin gene coding for the two-domain chains of the clam Barbatia, are similar to the vertebrate plan. (2) Genes for single-domain chains exist in bacteria and protozoa. Although the globin gene is highly expressed in the bacterium Vitreoscilla, the putative globin gene hmp in E. coli, which codes for a chimeric protein whose N-terminal moiety of 139 residues contains 67 residues identical to the Vitreoscilla globin, may be either unexpressed or expressed at very low levels, despite the presence of normal regulatory sequences. The DNA sequence of the globin gene of the protozoan Paramecium, determined recently by Yamauchi and collaborators, appears to consist of two exons separated by a short intron. (3) Among the lower eukaryotes, the yeasts Saccharomyces and Candida have chimeric proteins consisting of N-terminal globin and C-terminal flavoprotein moieties of about the same size. The structure of the gene for the chimeric protein of Saccharomyces exhibits no introns. According to Riggs, the presence of chimeric proteins in E. coli and other prokaryotes, such as Alcaligenes and Rhizobium, as well as in yeasts, suggests a previously unrecognized evolutionary pathway for hemoglobin, namely that of a multipurpose heme-binding domain attached to a variety of unrelated proteins with diverse functions. (4) The published globin gene sequences of the insect larva Chironomus have an intron-less structure and are present as clusters of multiple copies; the expression of the globin genes is tissue and developmental stage-specific. Furthermore, the expression of many of these genes has not yet been demonstrated despite the presence of apparently normal regulatory sequences in the two flanking regions. Unexpectedly, Bergtrom and collaborators have recently shown that at least three Ctt globin II beta genes contain putative introns. (5) Pohajdak and collaborators have found a seven-exon and six-intron structure for the globin gene of the nematode Pseudoterranova which codes for a two-domain globin chain. Although the second and fourth introns of the N-terminal domain correspond to the two introns found in vertebrate globin genes, the position of the third intron is close to that of the central intron in plant hemoglobins.(ABSTRACT TRUNCATED AT 400 WORDS)     

The Interplay between Molten Globules and Heme Disassociation Defines Human Hemoglobin Disassembly

Hemoglobin functions as a tetrameric oxygen transport protein, with each subunit containing a heme cofactor. Its denaturation, either in vivo or in vitro, involves autoxidation to methemoglobin, followed by cofactor loss and globin unfolding. We have proposed a global disassembly scheme for human methemoglobin, linking hemin (ferric protoporphyrin IX) disassociation and apoprotein unfolding pathways. The model is based on the evaluation of circular dichroism and visible absorbance measurements of guanidine-hydrochloride-induced disassembly of methemoglobin and previous measurements of apohemoglobin unfolding. The populations of holointermediates and equilibrium disassembly parameters were estimated quantitatively for adult and fetal hemoglobins. The key stages are characterized by hexacoordinated hemichrome intermediates, which are important for preventing hemin disassociation from partially unfolded, molten globular species during early disassembly and late-stage assembly events. Both unfolding experiments and independent small angle x-ray scattering measurements demonstrate that heme disassociation leads to the loss of tetrameric structural integrity. Our model predicts that after autoxidation, dimeric and monomeric hemichrome intermediates occur along the disassembly pathway inside red cells, where the hemoglobin concentration is very high. This prediction suggests why misassembled hemoglobins often get trapped as hemichromes that accumulate into insoluble Heinz bodies in the red cells of patients with unstable hemoglobinopathies. These Heinz bodies become deposited on the cell membranes and can lead to hemolysis. Alternatively, when acellular hemoglobin is diluted into blood plasma after red cell lysis, the disassembly pathway appears to be dominated by early hemin disassociation events, which leads to the generation of higher fractions of unfolded apo subunits and free hemin, which are known to damage the integrity of blood vessel walls. Thus, our model provides explanations of the pathophysiology of hemoglobinopathies and other disease states associated with unstable globins and red cell lysis and also insights into the factors governing hemoglobin assembly during erythropoiesis.     

Structure-Function Relationships in Unusual Nonvertebrate Globins

Based on the literature and our own results, this review summarizes the most recent state of nonvertebrate myoglobin (Mb) and hemoglobin (Hb) research, not as a general survey of the subject but as a case study. For this purpose, we have selected here four typical globins to discuss their unique structures and properties in detail. These include Aplysia myoglobin, which served as a prototype for the unusual globins lacking the distal histidine residue; midge larval hemoglobin showing a high degree of polymorphism; Tetrahymena hemoglobin evolved with a truncated structure; and yeast flavohemoglobin carrying an enigmatic two-domain structure. These proteins are not grouped by any common features other than the fact they have globin domains and heme groups. As a matter of course, various biochemical functions other than the conventional oxygen transport or storage have been proposed so far to these primitive or ancient hemoglobins or myoglobins, but the precise in vivo activity is still unclear.

In this review, special emphasis is placed on the stability properties of the heme-bound O₂. Whatever the possible roles of nonvertebrate myoglobins and hemoglobins may be (or might have been), the binding of molecular oxygen to iron(II) must be the primary event to manifest their physiological functions in vivo. However, the reversible and stable binding of O₂ to iron(II) is not a simple process, since the oxygenated form of Mb or Hb is oxidized easily to its ferric met-form with the generation of superoxide anion. The metmyoglobin or methemoglobin thus produced cannot bind molecular oxygen and is therefore physiologically inactive. In this respect, protozoan ciliate myoglobin and yeast flavohemoglobin are of particular interest in their very unique structures. Indeed, both proteins have been found to have completely different strategies for overcoming many difficulties in the reversible and stable binding of molecular oxygen, as opposed to the irreversible oxidation of heme iron(II). Such comparative studies of the stability of MbO₂ or HbO₂ are of primary importance, not only for a full understanding of the globin evolution, but also for planning new molecular designs for synthetic oxygen carriers that may be able to function in aqueous solution and at physiological temperature.     

Structure-guided recombination creates an artificial family of cytochromes P450

Creating artificial protein families affords new opportunities to explore the determinants of structure and biological function free from many of the constraints of natural selection. We have created an artificial family comprising ˜3,000 P450 heme proteins that correctly fold and incorporate a heme cofactor by recombining three cytochromes P450 at seven crossover locations chosen to minimize structural disruption. Members of this protein family differ from any known sequence at an average of 72 and by as many as 109 amino acids. Most (>73%) of the properly folded chimeric P450 heme proteins are catalytically active peroxygenases; some are more thermostable than the parent proteins. A multiple sequence alignment of 955 chimeras, including both folded and not, is a valuable resource for sequence-structure-function studies. Logistic regression analysis of the multiple sequence alignment identifies key structural contributions to cytochrome P450 heme incorporation and peroxygenase activity and suggests possible structural differences between parents CYP102A1 and CYP102A2.     

Structural and thermodynamic consequences of b heme binding for monomeric apoglobins and other apoproteins

The binding of a cofactor to a protein matrix often involves a reorganization of the polypeptide structure. b Hemoproteins provide multiple examples of this behavior. In this minireview, selected monomeric and single b heme proteins endowed with distinct topological properties are inspected for the extent of induced refolding upon heme binding. To complement the data reported in the literature, original results are presented on a two-on-two globin of cyanobacterial origin (Synechococcus sp. PCC 7002 GlbN) and on the heme-containing module of FixL, an oxygen-sensing protein with the mixed alpha/beta topology of PAS domains. GlbN had a stable apoprotein that was further stabilized and locally refolded by heme binding; in contrast, apoFixLH presented features of a molten globule. Sequence analyses (helicity, disorder, and polarity) and solvent accessibility calculations were performed to identify trends in the architecture of b hemoproteins. In several cases, the primary structure appeared biased toward a partially disordered binding pocket in the absence of the cofactor.     

Interdomain interactions within the two-component heme-based sensor DevS from Mycobacterium tuberculosis

DevS is the sensor of the DevS-DevR two-component regulatory system of Mycobacterium tuberculosis. This system is thought to be responsible for initiating entrance of this bacterium into the nonreplicating persistent state in response to NO and anaerobiosis. DevS is modular in nature and consists of two N-terminal GAF domains and C-terminal histidine kinase and ATPase domains. The first GAF domain (GAF A) binds heme, and this cofactor is thought to be responsible for sensing environmental stimuli, but the function of the second GAF domain (GAF B) is unknown. Here we report the RR characterization of full-length DevS (FL DevS) as well as truncated proteins consisting of the single GAF A domain (GAF A DevS) and both GAF domains (GAF A/B) in both oxidation states and bound to the exogenous ligands CO, NO, and O2. The results indicate that the GAF B domain increases the specificity with which the distal heme pocket of the GAF A domain interacts with CO and NO as opposed to O2. Specifically, while two comparable populations of CO and NO adducts are observed in GAF A DevS, only one of these two conformers is present in significant concentration in the GAF A/B and FL DevS proteins. In contrast, hydrogen bond interactions at the bound oxygen in the oxy complexes are conserved in all DevS constructs. The comparison of the data obtained with the O2 complexes with those of the CO and NO complexes suggests a model for ligand discrimination which relies on a specific hydrogen-bonding network with bound O2. It also suggests that interactions between the two GAF domains are responsible for transduction of structural changes at the heme domain that accompany ligand binding/dissociation to modulate activity at the kinase domain.