1-Aminobenzotriazole: A Mechanism-Based Cytochrome P450 Inhibitor and Probe of Cytochrome P450 Biology.

1-Aminobenzotriazole (1-ABT) is a pan-specific, mechanism-based inactivator of the xenobiotic metabolizing forms of cytochrome P450 in animals, plants, insects, and microorganisms. It has been widely used to investigate the biological roles of cytochrome P450 enzymes, their participation in the metabolism of both endobiotics and xenobiotics, and their contributions to the metabolism-dependent toxicity of drugs and chemicals. This review is a comprehensive evaluation of the chemistry, discovery, and use of 1-aminobenzotriazole in these contexts from its introduction in 1981 to the present.

Analysis of cytochrome P450 CYP119 ligand-dependent conformational dynamics by two-dimensional NMR and X-ray crystallography.

Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. We used two-dimensional (1)H,(15)N HSQC chemical shift perturbation mapping of (15)N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop with various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. The results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states.

Genetic and mass spectrometric tools for elucidating the physiological function(s) of cytochrome P450 enzymes from Mycobacterium tuberculosis.

Tuberculosis remains a leading cause of human mortality. The emergence of strains of Mycobacterium tuberculosis (Mtb), the causative agent, that are resistant to first- and second-line antitubercular drugs urges the development of new therapeutics. The genome of Mtb encodes 20 cytochrome P450 enzymes, at least some of which are potential candidates (CYP121, CYP125, and CYP128) for drug targeting. In this regard, we examined the specific role of CYP125 in the cholesterol degradation pathway, using genetic and mass spectrometric approaches. The analysis of lipid profiles from Mtb cells grown on cholesterol revealed that CYP125, by virtue of its C26-monooxygenase activity, is essential for cholesterol degradation, and, consequently, for the incorporation of side-chain carbon atoms into cellular lipids, as evidenced by an increase in the mass of the methyl-branched phthiocerol dimycocerosates (PDIM). Moreover, this work also led to the identification of cholest-4-en-3-one as a source of cellular toxicity. Herein, we describe the experimental procedures that led to elucidation of the physiological function of CYP125. A similar approach can be used to study other important Mtb P450 enzymes.

Expression in Escherichia coli of a cytochrome P450 enzyme with a cobalt protoporphyrin IX prosthetic group.

Unlike many hemoproteins, the prosthetic heme group of most cytochrome P450 enzymes cannot be extracted and replaced by modified heme groups. Here, we describe a procedure for generating a cytochrome P450 enzyme (CYP119) with cobalt protoporphyrin IX as its prosthetic group. This is achieved by expressing the protein in Escherichia coli in iron-limited medium and adding cobalt to the medium at the moment that inducible protein expression is initiated.

Solution NMR characterization of magnetic/electronic properties of azide and cyanide-inhibited substrate complexes of human heme oxygenase: implications for steric ligand tilt.

Solution 2D (1)H NMR was carried out on the azide-ligated substrate complex of human heme oxygenase, hHO, to provide information on the active site molecular structure, chromophore electronic/magnetic properties, and the distal H-bond network linked to the exogenous ligand by catalytically relevant oriented water molecules. While 2D NMR exhibited very similar patterns of two-dimensional nuclear Overhauser spectroscopy cross peaks of residues with substrate and among residues as the previously characterized cyanide complex, significant, broadly distributed chemical shift differences were observed for both labile and non-labile protons. The anisotropy and orientation of the paramagnetic susceptibility tensor, χ, were determined for both the azide and cyanide complexes. The most significant difference observed is the tilt of the major magnetic axes from the heme normal, which is only half as large for the azide than cyanide ligand, with each ligand tilted toward the catalytically cleaved α-meso position. The difference in chemical shifts is quantitatively correlated with differences in dipolar shifts in the respective complexes for all but the distal helix. The necessity of considering dipolar shifts, and hence determination of the orientation/anisotropy of χ, in comparing chemical shifts involving paramagnetic complexes, is emphasized. The analysis shows that the H-bond network cannot detect significant differences in H-bond acceptor properties of cyanide versus azide ligands. Lastly, significant retardation of distal helix labile proton exchange upon replacing cyanide with azide indicates that the dynamic stability of the distal helix is increased upon decreasing the steric interaction of the ligand with the distal helix.

The DosS-DosT/DosR Mycobacterial Sensor System.

DosS/DosR is a two-component regulatory system in which DosS, a heme-containing sensor also known as DevS, under certain conditions undergoes autophosphorylation and then transfers the phosphate to DosR, a DNA-binding protein that controls the entry of Mycobacterium tuberculosis and other mycobacteria into a latent, dormant state. DosT, a second sensor closely related to DosS, is present in M. tuberculosis and participates in the control of the dormancy response mediated by DosR. The binding of phosphorylated DosR to DNA initiates the expression of approximately fifty dormancy-linked genes. DosT is accepted to be a gas sensor that is activated in the ferrous state by the absence of an oxygen ligand or by the binding of NO or CO. DosS functions in a similar fashion as a gas sensor, but contradictory evidence has led to the suggestion that it also functions as a redox state sensor. This review focuses on the structure, biophysical properties, and function of the DosS/DosT heme sensors.

Photosystem I from plants as a bacterial cytochrome P450 surrogate electron donor: terminal hydroxylation of branched hydrocarbon chains.

The ability of cytochrome P450 enzymes to catalyze highly regio- and stereospecific hydroxylations makes them attractive alternatives to approaches based on chemical synthesis but they require expensive cofactors, e.g. NAD(P)H, which limits their commercial potential. Ferredoxin (Fdx) is a multifunctional electron carrier that in plants accepts electrons from photosystem I (PSI) and facilitates photoreduction of NADP(+) to NADPH mediated by ferredoxin-NAD(P)H oxidoreductase (FdR). In bacteria, the electron flow is reversed and Fdx accepts electrons from NADPH via FdR and serves as the direct electron donor to bacterial P450s. By combining the two systems, we demonstrate that irradiation of PSI can drive the activity of a bacterial P450, CYP124 from Mycobacterium tuberculosis. The substitution of the costly cofactor NADPH with sunlight illustrates the potential of the light-driven hydroxylation system for biotechnology applications.

Cholesterol catabolism as a therapeutic target in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that infects 10 million people worldwide and kills 2 million people every year. The uptake and utilization of nutrients by Mtb within the host cell is still poorly understood, although lipids play an important role in Mtb persistence. The recent identification of a large regulon of cholesterol catabolic genes suggests that Mtb can use host sterol for infection and persistence. In this review, we report on recent progress in elucidation of the Mtb cholesterol catabolic reactions and their potential utility as targets for tuberculosis therapeutic agents.

Active-site residues move independently from the rest of the protein in a 200 ns molecular dynamics simulation of cytochrome P450 CYP119.

The conformational dynamics of cytochrome P450 enzymes are critical to their catalytic activity. In this study, the correlated motion between residues in a 200 ns molecular dynamics trajectory of the thermophilic CYP119 was analyzed to parse out conformational relationships. Residues that are structurally related, for example residues within a helix, generally have highly correlated motion. In addition, clusters of non-adjacent residues that show correlated motion ("hot spots") are seen in various regions, including at the base of the F and G helices that make up the most dynamic region of the enzyme. A modified k-means algorithm that clusters residues based on their correlated motion indicates that functionally related residues are in the same cluster (e.g., the catalytic threonines and the heme). Tightly coupled clusters form a solvent-exposed "shell" around the enzyme, whereas less coupling between clusters is seen in regions that are critical to ligand interactions, redox partner interactions, and catalysis. Most notably, we find that residues in the active site move independently from the rest of the enzyme, effectively insulating the catalytic machinery from other regions of the protein.

Efficient hypoxic activation of the anticancer agent AQ4N by CYP2S1 and CYP2W1.

AQ4N [1,4-bis{[2-(dimethylamino-N-oxide)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione], a prodrug with two dimethylamino N-oxide groups, is converted to the topoisomerase II inhibitor AQ4 [1,4-bis{[2-(dimethylamino)ethyl]amino}-5,8-dihydroxy-anthracene-9,10-dione] by reduction of the N-oxides to dimethylamino substituents. Earlier studies showed that several drug-metabolizing cytochrome P450 (P450) enzymes can catalyze this reductive reaction under hypoxic conditions comparable with those in solid tumors. CYP2S1 and CYP2W1, two extrahepatic P450 enzymes identified from the human genome whose functions are unknown, are expressed in hypoxic tumor cells at much higher levels than in normal tissue. Here, we demonstrate that CYP2S1, contrary to a published report (Mol Pharmacol 76:1031-1043, 2009), is efficiently reduced by NADPH-P450 reductase. Most importantly, both CYP2S1 and CYP2W1 are better catalysts for the reductive activation of AQ4N to AQ4 than all previously examined P450 enzymes. The overexpression of CYP2S1 and CYP2W1 in tumor tissues, together with their high catalytic activities for AQ4N activation, suggests that they may be exploited for the localized activation of anticancer prodrugs.

Mycobacterium tuberculosis CYP125A1, a steroid C27 monooxygenase that detoxifies intracellularly generated cholest-4-en-3-one.

The infectivity and persistence of Mycobacterium tuberculosis requires the utilization of host cell cholesterol. We have examined the specific role of cytochrome P450 CYP125A1 in the cholesterol degradation pathway using genetic, biochemical and high-resolution mass spectrometric approaches. The analysis of lipid profiles from cells grown on cholesterol revealed that CYP125A1 is required to incorporate the cholesterol side-chain carbon atoms into cellular lipids, as evidenced by an increase in the mass of the methyl-branched phthiocerol dimycocerosates. We observed that cholesterol-exposed cells lacking CYP125A1 accumulate cholest-4-en-3-one, suggesting that this is a physiological substrate for this enzyme. Reconstitution of enzymatic activity with spinach ferredoxin and ferredoxin reductase revealed that recombinant CYP125A1 indeed binds both cholest-4-en-3-one and cholesterol, efficiently hydroxylates both of them at C-27, and then further oxidizes 27-hydroxycholest-4-en-3-one to cholest-4-en-3-one-27-oic acid. We determined the X-ray structure of cholest-4-en-3-one-bound CYP125A1 at a resolution of 1.58 A. CYP125A1 is essential for growth of CDC1551 in media containing cholesterol or cholest-4-en-3-one. In its absence, the latter compound is toxic for both CDC1551 and H37Rv when added with glycerol as a second carbon source. CYP125A1 is a key enzyme in cholesterol metabolism and plays a crucial role in circumventing the deleterious effect of cholest-4-en-3-one.

Two-dimensional NMR and all-atom molecular dynamics of cytochrome P450 CYP119 reveal hidden conformational substates.

Cytochrome P450 enzymes are versatile catalysts involved in a wide variety of biological processes from hormonal regulation and antibiotic synthesis to drug metabolism. A hallmark of their versatility is their promiscuous nature, allowing them to recognize a wide variety of chemically diverse substrates. However, the molecular details of this promiscuity have remained elusive. Here, we have utilized two-dimensional heteronuclear single quantum coherence NMR spectroscopy to examine a series of mutants site-specific labeled with the unnatural amino acid, [(13)C]p-methoxyphenylalanine, in conjunction with all-atom molecular dynamics simulations to examine substrate and inhibitor binding to CYP119, a P450 from Sulfolobus acidocaldarius. The results suggest that tight binding hydrophobic ligands tend to lock the enzyme into a single conformational substate, whereas weak binding low affinity ligands bind loosely in the active site, resulting in a distribution of localized conformers. Furthermore, the molecular dynamics simulations suggest that the ligand-free enzyme samples ligand-bound conformations of the enzyme and, therefore, that ligand binding may proceed largely through a process of conformational selection rather than induced fit.

Interaction of Mycobacterium tuberculosis CYP130 with heterocyclic arylamines.

The Mycobacterium tuberculosis P450 enzymes are of interest for their pharmacological development potential, as evidenced by their susceptibility to inhibition by antifungal azole drugs that normally target sterol 14alpha-demethylase (CYP51). Although antifungal azoles show promise, direct screening of compounds against M. tuberculosis P450 enzymes may identify novel, more potent, and selective inhibitory scaffolds. Here we report that CYP130 from M. tuberculosis has a natural propensity to bind primary arylamines with particular chemical architectures. These compounds were identified via a high throughput screen of CYP130 with a library of synthetic organic molecules. As revealed by subsequent x-ray structure analysis, selected compounds bind in the active site by Fe-coordination and hydrogen bonding of the arylamine group to the carbonyl oxygen of Gly(243). As evidenced by the binding of structural analogs, the primary arylamine group is indispensable, but synergism due to hydrophobic contacts between the rest of the molecule and protein amino acid residues is responsible for a binding affinity comparable with that of the antifungal azole drugs. The topology of the CYP130 active site favors angular coordination of the arylamine group over the orthogonal coordination of azoles. Upon substitution of Gly(243) by an alanine, the binding mode of azoles and some arylamines reverted from type II to type I because of hydrophobic and steric interactions with the alanine side chain. We suggest a role for the conserved Ala(Gly)(243)-Gly(244) motif in the I-helix in modulating both the binding affinity of the axial water ligand and the ligand selectivity of cytochrome P450 enzymes.

Spin scavenging analysis of myoglobin protein-centered radicals using stable nitroxide radicals: characterization of oxoammonium cation-induced modifications.

Spin scavenging combined with chromatographic and mass spectrometric procedures can, in principle, be employed to detect and identify protein-based radicals within complex biological matrices. This approach is based on the well-known ability of stable synthetic nitroxide radicals to scavenge carbon-centered radicals, forming stable diamagnetic addition products. Hence, characterization of these addition products would allow for the identification of specific free radicals. In the present work, we have explored the use of the stable nitroxide radical 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL) in scavenging protein-based radicals generated in a horse heart metmyoglobin/hydrogen peroxide (metMb/H(2)O(2)) system. Inclusion of a substoichiometric amount of TEMPOL in the metMb/H(2)O(2) system resulted in a complete loss of peroxyl and tyrosyl radical signals and effectively inhibited the formation of oxidatively damaged heme species, as monitored by electron paramagnetic resonance and reversed-phase liquid chromatography. Scavenging of globin radicals by TEMPOL did not lead to the formation of stable diamagnetic addition adducts; in fact, reversed-phase liquid chromatographic studies and oxygen electrode measurements indicated that TEMPOL acts as a catalyst and is recycled in this system. The oxoammonium cation generated in the course of this reaction initiated secondary reactions resulting in the formation of a free carbonyl on the N-terminal Gly-residue of the protein. This oxidative deamination was confirmed through the combined use of reversed-phase liquid chromatographic purification, tandem MS experiments, and chemical analysis (e.g., by use of 2,4-dinitrophenyl hydrazine). The results reveal the pitfalls inherent in using stable nitroxide radicals such as TEMPOL to identify sites of radical formation on hemoproteins.

Human flavin-containing monooxygenase 2.1 catalyzes oxygenation of the antitubercular drugs thiacetazone and ethionamide.

The second-line antitubercular drugs thiacetazone (TAZ) and ethionamide (ETA) are bioactivated by the mycobacterial enzyme EtaA. We report here that human flavin-containing monooxygenase 2.1 (FMO2.1), which is expressed predominantly in the lung, catalyzes oxygenation of TAZ. The metabolites generated, the sulfenic acid, sulfinic acid, and carbodiimide derivatives, are the same as those produced by EtaA and human FMO1 and FMO3. Two of the metabolites, the sulfenic acid and carbodiimide, are known to be harmful to mammalian cells. FMO2.1 also catalyzes oxygenation of ETA, producing the S-oxide. We have developed a novel spectrophotometric assay for TAZ oxygenation. The assay was used to determine kinetic parameters for TAZ oxygenation catalyzed by human FMO1, FMO2.1, and FMO3 and by EtaA. Although the K(M) values for the four enzyme-catalyzed reactions are similar, k(cat) and, consequently, k(cat)/K(M) (the specificity constant) for FMO2.1-catalyzed TAZ oxygenation are much higher than those of FMO1, FMO3, or EtaA. This indicates that FMO2.1 is more effective in catalyzing TAZ oxygenation than are the other three enzymes and thus is likely to contribute substantially to the metabolism of TAZ, decreasing the availability of the prodrug to mycobacteria and producing toxic metabolites. Because of a genetic polymorphism, Europeans and Asians lack FMO2.1. However, in sub-Saharan Africa, a region in which tuberculosis is a major health problem, a substantial proportion of individuals express FMO2.1. Thus, our results may explain some of the observed interindividual differences in response to TAZ and ETA and have implications for the treatment of tuberculosis in sub-Saharan Africa.

A distal tyrosine residue is required for ligand discrimination in DevS from Mycobacterium tuberculosis.

DevS is a heme-based sensor kinase required for sensing environmental conditions leading to nonreplicating persistence in Mycobacterium tuberculosis. Kinase activity is observed when the heme is a ferrous five-coordinate high-spin or six-coordinate low-spin CO or NO complex but is strongly inhibited in the oxy complex. Discrimination between these exogenous ligands has been proposed to depend on a specific hydrogen bond network with bound oxygen. Here we report resonance Raman data and autophosphorylation assays of wild-type and Y171F DevS in various heme complexes. The Y171F mutation eliminates ligand discrimination for CO, NO, and O2, resulting in equally inactive complexes. In contrast, the ferrous-deoxy Y171F variant exhibits autokinase activity equivalent to that of the wild type. Raman spectra of the oxy complex of Y171F indicate that the environment of the oxy group is significantly altered from that in the wild type. They also suggest that a solvent molecule in the distal pocket substitutes for the Tyr hydroxyl group to act as a poorer hydrogen bond donor to the oxy group. The wild-type CO and NO complexes exist as a major population in which the CO or NO groups are free of hydrogen bonds, while the Y171F mutation results in a mild increase in the distal pocket polarity. The Y171F mutation has no impact on the proximal environment of the heme, and the activity observed with the five-coordinate ferrous-deoxy wild type is conserved in the Y171F variant. Thus, while the absence of an exogenous ligand in the ferrous-deoxy proteins leads to a moderate kinase activity, interactions between Tyr171 and distal diatomic ligands turn the kinase activity on and off. The Y171F mutation disrupts the on-off switch and renders all states with a distal ligand inactive. This mechanistic model is consistent with Tyr171 being required for distal ligand discrimination, but nonessential for autophosphorylation activity.

Crystal structure and properties of CYP231A2 from the thermoacidophilic archaeon Picrophilus torridus.

The crystal structure of a cytochrome P450 from the thermoacidophile Picrophilus torridus, CYP231A2 (PTO1399), has been solved. This structure reveals a wide open substrate access channel. To better understand ligand-induced structural transitions in CYP231A2, protein-ligand interactions were investigated using 4-phenylimidazole. Comparison of the ligand-free and -bound CYP231A2 structures shows conformational changes where the F and G helices swing as a single rigid body about a pivot point at the N-terminal end of the F helix, allowing the F helix region to dip toward the heme, resulting in closer contacts with the ligand. Thermal melting data illustrate that the melting temperature for CYP231A2 increases nearly 10 degrees C upon ligand binding, thus illustrating that the closed conformation is substantially more stable. Furthermore, spectroscopic data indicate that the active site is stable at pH 4.5, although, unusually, the thiolate ligand to the iron can be reversibly protonated. CYP231A2 does not exhibit structural features normally associated with thermophilic proteins such as an increase in salt bridge networks or extensive aromatic clustering. The increase in thermal stability instead is best correlated with the smaller size and shorter loops in CYP231A2 compared to other P450s.

Mycobacterium tuberculosis CYP130: crystal structure, biophysical characterization, and interactions with antifungal azole drugs.

CYP130 is one of the 20 Mycobacterium tuberculosis cytochrome P450 enzymes, only two of which, CYP51 and CYP121, have so far been studied as individually expressed proteins. Here we characterize a third heterologously expressed M. tuberculosis cytochrome P450, CYP130, by UV-visible spectroscopy, isothermal titration calorimetry, and x-ray crystallography, including determination of the crystal structures of ligand-free and econazole-bound CYP130 at a resolution of 1.46 and 3.0A(,) respectively. Ligand-free CYP130 crystallizes in an "open" conformation as a monomer, whereas the econazole-bound form crystallizes in a "closed" conformation as a dimer. Conformational changes enabling the "open-closed" transition involve repositioning of the BC-loop and the F and G helices that envelop the inhibitor in the binding site and reshape the protein surface. Crystal structure analysis shows that the portion of the BC-loop relocates as much as 18A between the open and closed conformations. Binding of econazole to CYP130 involves a conformational change and is mediated by both a set of hydrophobic interactions with amino acid residues in the active site and coordination of the heme iron. CYP130 also binds miconazole with virtually the same binding affinity as econazole and clotrimazole and ketoconazole with somewhat lower affinities, which makes it a plausible target for this class of therapeutic drugs. Overall, binding of the azole inhibitors is a sequential two-step, entropy-driven endothermic process. Binding of econazole and clotrimazole exhibits positive cooperativity that may reflect a propensity of CYP130 to associate into a dimeric structure.

Implication for using heme methyl hyperfine shifts as indicators of heme seating as related to stereoselectivity in the catabolism of heme by heme oxygenase: in-plane heme versus axial his rotation.

The triple mutant of the solubilized, 265-residue construct of human heme oxygenase, K18E/E29K/R183E-hHO, has been shown to redirect the exclusive alpha-regioselectivity of wild-type hHO to primarily beta,delta-selectivity in the cleavage of heme (Wang, J., Evans, J. P., Ogura, H., La Mar, G. N., and Ortiz de Montellano, P. R. (2006) Biochemistry 45, 61-73). The 1H NMR hyperfine shift pattern for the substrate and axial His CbetaH's and the substrate-protein contacts of the cyanide-inhibited protohemin and 2,4-dimethyldeuterohemin complexes of the triple mutant have been analyzed in detail and compared to data for the WT complex. It is shown that protein contacts for the major solution isomers for both substrates in the mutant dictate approximately 90 degrees in-plane clockwise rotation relative to that in the WT. The conventional interpretation of the pattern of substrate methyl hyperfine shifts, however, indicates substrate rotations of only approximately 50 degrees . This paradox is resolved by demonstrating that the axial His25 imidazole ring also rotates counterclockwise with respect to the protein matrix in the mutant relative to that in the WT. The axial His25 CbetaH hyperfine shifts are shown to serve as independent probes of the imidazole plane orientation relative to the protein matrix. The analysis indicates that the pattern of heme methyl hyperfine shifts cannot be used alone to determine the in-plane orientation of the substrate as it relates to the stereospecificity of heme cleavage, without explicit consideration of the orientation of the axial His imidazole plane relative to the protein matrix.

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.