Powered by gas--a ligand for a fruit fly nuclear receptor

The difficulty in identifying ligands for nuclear hormone receptors remains an obstacle to understanding their function. For example, in the fruit fly Drosophila melanogaster, only one of its nuclear receptors has a known ligand. In this issue of Cell, report that the fruit fly E75 nuclear receptor contains heme in its ligand binding pocket and that the oxidation state of this molecule controls E75 activity. They also show that E75-heme responds to the small diatomic gases, nitric oxide and carbon monoxide. This study sheds light on how heme, gas signaling, and nuclear receptors interact to control metabolic and developmental pathways.     

Heme Ligand Binding Properties and Intradimer Interactions in the Full-length Sensor Protein Dos from Escherichia coli and Its Isolated Heme Domain

Dos from Escherichia coli is a bacterial gas sensor protein comprising a heme-containing gas sensor domain and a phosphodiesterase catalytic domain. Using a combination of static light scattering and gel filtration experiments, we established that, as are many other sensor proteins, the full-length protein is dimeric. The full-length dimer (association constant <10 nm) is more stable than the dimeric heme domain (association constant ∼1 μm), and the dimer interface presumably includes both sensor and catalytic domains. Ultrafast spectroscopic studies showed little influence of the catalytic domain on kinetic processes in the direct vicinity of the heme. By contrast, the properties of ligand (CO and O₂) binding to the heme in the sensor domain, occurring on a microsecond to second time scale, were found to be influenced by (i) the presence of the catalytic domain, (ii) the dimerization state, and in dimers, (iii) the ligation state of the other subunit. These results imply allosteric interactions within dimers. Steady-state titrations demonstrated marked cooperativity in oxygen binding to both the full-length protein and the isolated heme domain, a feature not reported to date for any dimeric sensor protein. Analysis of a variety of time-resolved experiments showed that Met-95 plays a major role in the intradimer interactions. The intrinsic binding and dissociation rates of Met-95 to the heme were modulated ∼10-fold by intradimer and sensor-catalytic domain interactions. Dimerization effects were also observed for cyanide binding to the ferric heme domains, suggesting a similar role for Met-95 in ferric proteins.     

Thermodynamics of heme binding to the HasA(SM) hemophore: effect of mutations at three key residues for heme uptake

HasA(SM) secreted by the Gram-negative bacterium Serratia marcescens belongs to the hemophore family. Its role is to take up heme from host heme carriers and to shuttle it to specific receptors. Heme is linked to the HasA(SM) protein by an unusual axial ligand pair: His32 and Tyr75. The nucleophilic nature of the tyrosine is enhanced by the hydrogen bonding of the tyrosinate to a neighboring histidine in the binding site: His83. We used isothermal titration microcalorimetry to examine the thermodynamics of heme binding to HasA(SM) and showed that binding is strongly exothermic and enthalpy driven: DeltaH = -105.4 kJ x mol(-1) and TDeltaS = -44.3 kJ x mol(-1). We used displacement experiments to determine the affinity constant of HasA(SM) for heme (K(a) = 5.3 x 10(10) M(-1)). This is the first time that this has been reported for a hemophore. We also analyzed the thermodynamics of the interaction between heme and a panel of single, double, and triple mutants of the two axial ligands His32 and Tyr75 and of His83 to assess the implication of each of these three residues in heme binding. We demonstrated that, in contrast to His32, His83 is essential for the binding of heme to HasA(SM), even though it is not directly coordinated to iron, and that the Tyr75/His83 pair plays a key role in the interaction.     

Ultrafast heme-residue bond formation in six-coordinate heme proteins: implications for functional ligand exchange

A survey is presented of picosecond kinetics of heme-residue bond formation after photolysis of histidine, methionine, or cysteine, in a broad range of ferrous six-coordinate heme proteins. These include human neuroglobin, a bacterial heme-binding superoxide dismutase (SOD), plant cytochrome b 559, the insect nuclear receptor E75, horse heart cytochrome c and the heme domain of the bacterial sensor protein Dos. We demonstrate that the fastest and dominant phase of binding of amino acid residues to domed heme invariably takes place with a time constant in the narrow range of 5-7 ps. Remarkably, this is also the case in the heme-binding SOD, where the heme is solvent-exposed. We reason that this fast phase corresponds to barrierless formation of the heme-residue bond from a configuration close to the bound state. Only in proteins where functional ligand exchange occurs, additional slower rebinding takes place on the time scale of tens of picoseconds after residue dissociation. We propose that the presence of these slower phases reflects flexibility in the heme environment that allows external ligands (O2, CO, NO, . . .) to functionally replace the internal residue after thermal dissociation of the heme-residue bond.     

Asp274 and his346 are essential for heme binding and catalytic function of human indoleamine 2,3-dioxygenase

L-Tryptophan is the least abundant essential amino acid in humans. Indoleamine 2,3-dioxgyenase (IDO) is a cytosolic heme protein which, together with the hepatic enzyme tryptophan 2,3-dioxygenase, catalyzes the first and rate-limiting step in the major pathway of tryptophan metabolism, the kynurenine pathway. The physiological role of IDO is not fully understood but is of great interest, because IDO is widely distributed in human tissues, can be up-regulated via cytokines such as interferon-gamma, and can thereby modulate the levels of tryptophan, which is vital for cell growth. To identify which amino acid residues are important in substrate or heme binding in IDO, site-directed mutagenesis of conserved residues in the IDO gene was undertaken. Because it had been proposed that a histidine residue might be the proximal heme ligand in IDO, mutation to alanine of the three highly conserved histidines His16, His303, and His346 was conducted. Of these, only His346 was shown to be essential for heme binding, indicating that this histidine residue may be the proximal ligand and suggesting that neither His303 nor His16 act as the proximal ligand. Site-directed mutagenesis of Asp274 also compromised the ability of IDO to bind heme. This observation indicates that Asp274 may coordinate to heme directly as the distal ligand or is essential in maintaining the conformation of the heme pocket.     

Role of arginine 220 in the oxygen sensor FixL from Bradyrhizobium japonicum

In the heme-based oxygen sensor protein FixL, conformational changes induced by oxygen binding to the heme sensor domain regulate the activity of a neighboring histidine kinase, eventually restricting expression of specific genes to hypoxic conditions. The conserved arginine 220 residue is suggested to play a key role in the signal transduction mechanism. To obtain detailed insights into the role of this residue, we replaced Arg(220) by histidine (R220H), glutamine (R220Q), glutamate (R220E), and isoleucine (R220I) in the heme domain FixLH from Bradyrhizobium japonicum. These mutations resulted in dramatic changes in the O(2) affinity with K(d) values in the order R220I < R220Q < wild type < R220H. For the R220H and R220Q mutants, residue 220 interacts with the bound O(2) or CO ligands, as seen by resonance Raman spectroscopy. For the oxy-adducts, this H-bond modifies the pi acidity of the O(2) ligand, and its strength is correlated with the back-bonding-sensitive nu(4) frequency, the k(off) value for O(2) dissociation, and heme core-size conformational changes. This effect is especially strong for the wild-type protein where Arg(220) is, in addition, positively charged. These observations strongly suggest that neither strong ligand fixation nor the displacement of residue 220 into the heme distal pocket are solely responsible for the reported heme conformational changes associated with kinase activity regulation, but that a significant decrease of the heme pi(*) electron density because of strong back-bonding toward the oxygen ligand also plays a key role.     

Identification of crucial histidines for heme binding in the N-terminal domain of the heme-regulated eIF2alpha kinase

The heme-regulated eukaryotic initiation factor-2alpha (eIF2alpha) kinase (HRI) regulates the initiation of protein synthesis in reticulocytes. The binding of NO to the N-terminal heme-binding domain (NTD) of HRI positively modulates its kinase activity. By utilizing UV-visible absorption, resonance Raman, EPR and CD spectroscopies, two histidine residues have been identified that are crucial for the binding of heme to the NTD. The UV-visible absorption and resonance Raman spectra of all the histidine to alanine mutants constructed were similar to those of the unmutated NTD. However, the change in the CD spectra of the NTD construct containing mutation of His78 to Ala (H78A) indicated loss of the specific binding of heme. The EPR spectrum for the ferric H78A mutant was also substantially perturbed. Thus, His78 is one of the axial ligands for the NTD of HRI. Significant changes in the EPR spectrum of the H123A mutant were also observed, and heme readily dissociated from both the H123A and the H78A NTD mutants, suggesting that His123 was also an axial heme ligand. However, the CD spectrum for the Soret region of the H123A mutant indicated that this mutant still bound heme specifically. Thus, while both His78 and His123 are crucial for stable heme binding, the effects of their mutations on the structure of the NTD differed. His78 appears to play the primary role in the specific binding of heme to the NTD, acting analogously to the "proximal histidine" ligand of globins, while His123 appears to act as the "distal" heme ligand.     

Comparative analysis of structural and dynamic properties of the loaded and unloaded hemophore HasA: functional implications

A heme-acquisition system present in several Gram-negative bacteria requires the secretion of hemophores. These extracellular carrier proteins capture heme and deliver it to specific outer membrane receptors. The Serratia marcescens HasA hemophore is a monodomain protein that binds heme with a very high affinity. Its α/β structure, as that of its binding pocket, has no common features with other iron- or heme-binding proteins. Heme is held by two loops L1 and L2 and coordinated to iron by an unusual ligand pair, H32/Y75. Two independent regions of the hemophore β-sheet are involved in HasA-HasR receptor interaction. Here, we report the 3-D NMR structure of apoHasA and the backbone dynamics of both loaded and unloaded hemophore. While the overall structure of HasA is very similar in the apo and holo forms, the hemophore presents a transition from an open to a closed form upon ligand binding, through a large movement, of up to 30 Å, of loop L1 bearing H32. Comparison of loaded and unloaded HasA dynamics on different time scales reveals striking flexibility changes in the binding pocket. We propose a mechanism by which these structural and dynamic features provide the dual function of heme binding and release to the HasR receptor.