Electron paramagnetic resonance investigations of exogenous ligand complexes of low-spin ferric chloroperoxidase: further support for endogenous thiolate ligation to the heme iron.

Previous spectroscopic studies of chloroperoxidase have provided evidence for endogenous thiolate sulfur donor ligation to the central heme iron of the enzyme. This conclusion is further supported by recent DNA sequence data which revealed the existence of a third cysteine residue (in addition to a disulfide pair detected earlier) in the protein available for coordination to the heme iron. Thus, chloroperoxidase shares many spectroscopic properties with cytochrome P-450, the only other known thiolate-ligated heme protein. Surprisingly, a previous electron paramagnetic resonance (EPR) study of low-spin ferric chloroperoxidase-ligand complexes (Hollenberg, P.F., Hager, L.P., Blumberg, W.E. and Peisach, J. (1980) J. Biol. Chem. 255, 4801-4807) was unable to provide clear support for the presence of a thiolate ligand, although sulfur coordination was implicated. This was, in part, because an insufficient number of complexes was examined. In this work, we have significantly expanded upon the previous EPR study by using an extensive variety of over twenty exogenous ligands including carbon, nitrogen, oxygen, phosphorus and sulfur donors. Crystal field analysis, using the procedure of Blumberg and Peisach, of the present data in comparison with data for analogous complexes of cytochrome P-450-CAM, thiolate-ligated heme model systems, and myoglobin, is clearly indicative of endogenous thiolate ligation for chloroperoxidase. In addition, the UV-visible absorption and EPR spectral data suggest that a carboxylate ligand is a possible candidate for the endogenous sixth ligand to the heme iron that is responsible for the reversible conversion of ferric chloroperoxidase from high-spin to low-spin at low temperatures (less than 200 K).     

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.     

Novel heme ligation in a c-type cytochrome involved in thiosulfate oxidation: EPR and MCD of SoxAX from Rhodovulum sulfidophilum

The SoxAX complex of the bacterium Rhodovulum sulfidophilum is a heterodimeric c-type cytochrome that plays an essential role in photosynthetic thiosulfate and sulfide oxidation. The three heme sites of SoxAX have been analyzed using electronic absorption, electron paramagnetic resonance, and magnetic circular dichroism spectroscopies. Heme-3 in the ferric state is characterized by a Large g(max) EPR signal and has histidine and methionine axial heme iron ligands which are retained on reduction to the ferrous state. Hemes-1 and -2 both have thiolate plus nitrogenous ligand sets in the ferric state and give rise to rhombic EPR spectra. Heme-1, whose ligands derive from cysteinate and histidine residues, remains ferric in the presence of dithionite ion. Ferric heme-2 exists with a preparation-dependent mixture of two different ligand sets, one being cysteinate/histidine, the other an unidentified pair with a weaker crystal-field strength. Upon reduction of the SoxAX complex with dithionite, a change occurs in the ligands of heme-2 in which the thiolate is either protonated or replaced by an unidentified ligand. Sequence analysis places the histidine/methionine-coordinated heme in SoxX and the thiolate-liganded hemes in SoxA. SoxAX is the first naturally occurring c-type cytochrome in which a thiolate-coordinated heme has been identified.     

Characterization of the heme in human cystathionine beta-synthase by X-ray absorption and electron paramagnetic resonance spectroscopies

Human cystathionine beta-synthase is one of two key enzymes involved in intracellular metabolism of homocysteine. It catalyzes a beta-replacement reaction in which the thiolate of homocysteine replaces the hydroxyl group of serine to give the product, cystathionine. The enzyme is unusual in its dependence on two cofactors: pyridoxal phosphate and heme. The requirement for pyridoxal phosphate is expected on the basis of the nature of the condensation reaction that is catalyzed; however the function of the heme in this protein is unknown. We have examined the spectroscopic properties of the heme in order to assign the axial ligands provided by the protein. The heme Soret peak of ferric cystathionine beta-synthase is at 428 nm and shifts to approximately 395 nm upon addition of the thiol chelator, mercuric chloride. This is indicative of 6-coordinate low-spin heme converting to a 5-coordinate high-spin heme. The enzyme as isolated exhibits a rhombic EPR signal with g values of 2.5, 2.3, and 1.86, which are similar to those of heme proteins and model complexes with imidazole/thiolate ligands. Mercuric chloride treatment of the enzyme results in conversion of the rhombic EPR signal to a g = 6 signal, consistent with formation of the high-spin ferric heme. The X-ray absorption data reveal that iron in ferric cystathionine beta-synthase is 6-coordinate, with 1 high-Z scatterer and 5 low-Z scatterers. This is consistent with the presence of 5 nitrogens and 1 sulfur ligand. Together, these data support assignment of the axial ligands as cysteinate and imidazole in ferric cystathionine beta-synthase.     

核雌激素受体配体结合域的晶体结构特性与功能

雌激素或类雌激素活性物质通过细胞核雌激素受体（nuclear estrogen receptor, nER）通路发挥相应的生理性作用。当这些配体被nER的配体结合域（ligand binding domain, LBD）识别后进入疏水性配体结合空腔内并引起受体构象发生改变,使得原先处于高度活动性的helix 12(H12)被固定从而进一步稳定空腔结构|同时nER也能通过招募一系列辅助调节因子及其他共调节蛋白质,最终调控基因转录。但是,由于不同的配体和受体结合形成的晶体结构并不完全相同,导致这些复合体具有不同的性质,从而影响基因的转录活性。本文综述了nER配体结合域及结合配体后形成的相应晶体结构与活性以及不同配体对受体结构和基因转录的影响。     

Replacement of the axial histidine heme ligand with cysteine in nitrophorin 1: spectroscopic and crystallographic characterization

To evaluate the potential of using heme-containing lipocalin nitrophorin 1 (NP1) as a template for protein engineering, we have replaced the native axial heme-coordinating histidine residue with glycine, alanine, and cysteine. We report here the characterization of the cysteine mutant H60C_NP1 by spectroscopic and crystallographic methods. The UV/vis, resonance Raman, and magnetic circular dichroism spectra suggest weak thiolate coordination of the ferric heme in the H60C_NP1 mutant. Reduction to the ferrous state resulted in loss of cysteine coordination, while addition of exogenous imidazole ligands gave coordination changes that varied with the ligand. Depending on the substitution of the imidazole, we could distinguish three heme coordination states: five-coordinate monoimidazole, six-coordinate bisimidazole, and six-coordinate imidazole/thiolate. Ligand binding affinities were measured and found to be generally 2–3 orders of magnitude lower for the H60C mutant relative to NP1. Two crystal structures of the H60C_NP1 in complex with imidazole and histamine were solved to 1.7- and 1.96-Å resolution, respectively. Both structures show that the H60C mutation is well tolerated by the protein scaffold and suggest that heme–thiolate coordination in H60C_NP1 requires some movement of the heme within its binding cavity. This adjustment may be responsible for the ease with which the engineered heme–thiolate coordination can be displaced by exogenous ligands.     

Spectral characterization of brain and macrophage nitric oxide synthases. Cytochrome P-450-like hemeproteins that contain a flavin semiquinone radical.

Nitric oxide (NO) is synthesized in mammals where it acts as a signal molecule for neurotransmission, vasorelaxation, and cytotoxicity. The NO synthases isolated from brain and cytokine-activated macrophages are FAD- and FMN-containing flavoproteins that display considerable sequence homology to NADPH-cytochrome P-450 reductase. However, the nature of their catalytic centers is unknown. We have found that both isoenzymes contain 2 mol of iron-protoporphyrin IX/mol of enzyme homodimer. The optical and EPR spectroscopic properties of the heme groups were found to be remarkably similar to those of high-spin cytochrome P-450. The heme iron in the resting NO synthase is ferric and five-coordinate with a cysteine thiolate as the proximal axial ligand. In addition, the EPR spectra of the resting NO synthases contained a free radical signal attributable to a bound flavin semiquinone that appeared to interact magnetically with the ferric heme iron. NO production was inhibited by carbon monoxide, implying a role for the heme groups in catalysis.     

Flavocytochrome P450 BM3 mutant A264E undergoes substrate-dependent formation of a novel heme iron ligand set

A conserved glutamate covalently attaches the heme to the protein backbone of eukaryotic CYP4 P450 enzymes. In the related Bacillus megaterium P450 BM3, the corresponding residue is Ala264. The A264E mutant was generated and characterized by kinetic and spectroscopic methods. A264E has an altered absorption spectrum compared with the wild-type enzyme (Soret maximum at approximately 420.5 nm). Fatty acid substrates produced an inhibitor-like spectral change, with the Soret band shifting to 426 nm. Optical titrations with long-chain fatty acids indicated higher affinity for A264E over the wild-type enzyme. The heme iron midpoint reduction potential in substrate-free A264E is more positive than that in wild-type P450 BM3 and was not changed upon substrate binding. EPR, resonance Raman, and magnetic CD spectroscopies indicated that A264E remains in the low-spin state upon substrate binding, unlike wild-type P450 BM3. EPR spectroscopy showed two major species in substrate-free A264E. The first has normal Cys-aqua iron ligation. The second resembles formate-ligated P450cam. Saturation with fatty acid increased the population of the latter species, suggesting that substrate forces on the glutamate to promote a Cys-Glu ligand set, present in lower amounts in the substrate-free enzyme. A novel charge-transfer transition in the near-infrared magnetic CD spectrum provides a spectroscopic signature characteristic of the new A264E heme iron ligation state. A264E retains oxygenase activity, despite glutamate coordination of the iron, indicating that structural rearrangements occur following heme iron reduction to allow dioxygen binding. Glutamate coordination of the heme iron is confirmed by structural studies of the A264E mutant (Joyce, M. G., Girvan, H. M., Munro, A. W., and Leys, D. (2004) J. Biol. Chem. 279, 23287-23293).     

Probing conformational changes in the estrogen receptor: evidence for a partially unfolded intermediate facilitating ligand binding and release

Because the ligand bound to the ligand-binding domain (LBD) of nuclear hormone receptors is completely enveloped by protein, it is thought that the process of ligand binding or unbinding must involve a significant conformational change of this domain. We have used the intrinsic tryptophan fluorescence of the estrogen receptor-alpha (ERalpha) or estrogen receptor-beta (ERbeta) LBD, as well as bis-anilinonaphthalenesulfonate (bis-ANS), a probe for accessible interior regions of protein, to follow the guanidine-hydrochloride (Gua-HCl)-induced unfolding of this domain. In both cases, we find that the ER-LBD unfolding follows a two-phase process. At low Gua-HCl, the ER-LBD undergoes partial unfolding, whereas at high Gua-HCl, this domain undergoes a global unfolding, with bis-ANS binding preferentially to the partially unfolded state. The partially unfolded state of the ERalpha-LBD induced by denaturant does not bind ligand stably, but it may resemble an intermediate that this domain accesses transiently under native conditions that allow ligands to enter or exit the ligand-binding pocket.     

The heme of cystathionine beta-synthase likely undergoes a thermally induced redox-mediated ligand switch

Cystathionine beta-synthase (CBS) is a pyridoxal-5'-dependent enzyme that catalyzes the condensation of homocysteine and serine to form cystathionine. Human CBS is unique in that heme is also required for maximal activity, although the function of heme in this enzyme is presently unclear. The study presented herein reveals that the heme of human CBS undergoes a coordination change upon reduction at elevated temperatures. We have termed this new species "CBS424" and demonstrate that its formation is likely irreversible when pH 9 Fe(III) CBS is reduced at moderately elevated temperatures (approximately 40 degrees C and higher) or when pH 9 Fe(II) CBS is heated to similar temperatures. Spectroscopic techniques, including resonance Raman, electronic absorption, and variable temperature/variable field magnetic circular dichroism spectroscopy, provide strong evidence that CBS424 is coordinated by two neutral donor ligands. It appears likely that the native cysteine(thiolate) heme ligand is displaced by an endogenous neutral donor upon conversion to CBS424. This behavior is consistent with other six-coordinate, cysteine(thiolate)-ligated heme centers, which seek to avoid this coordination structure in the Fe(II) state. Functional assays show that CBS424 is inactive and suggest that the ligand switch is responsible for eliminating enzyme activity. When this investigation is taken together with other functional studies of CBS, it provides strong evidence that coordination of Cys52 to the heme iron is crucial for full activity in this enzyme. We hypothesize that cysteine displacement may serve as a mechanism for CBS inactivation and that second-sphere interactions of the Cys52 thiolate with surrounding residues are responsible for communicating the heme ligand displacement to the CBS active site.     

The structure of murine neuroglobin: Novel pathways for ligand migration and binding

Neuroglobin, a recently discovered globin predominantly expressed in neuronal tissue of vertebrates, binds small, gaseous ligands at the sixth coordination position of the heme iron. In the absence of an exogenous ligand, the distal histidine (His64) binds to the heme iron in the ferrous and ferric states. The crystal structure of murine ferric (met) neuroglobin at 1.5 A reveals interesting features relevant to the ligand binding mechanism. Only weak selectivity is observed for the two possible heme orientations, the occupancy ratio being 70:30. Two small internal cavities are present on the heme distal side, which enable the His64(E7) side chain to move out of the way upon exogenous ligand binding. Moreover, a third, huge cavity (volume approximately 290 A3) connecting both sides of the heme, is open towards the exterior and provides a potential passageway for ligands. The CD and EF corners exhibit substantial flexibility, which may assist ligands in entering the protein and accessing the active site. Based on this high-resolution structure, further structure-function studies can be planned to elucidate the role of neuroglobin in physiological responses to hypoxia.     

Spin, oxidation, and ligand states of p-nitrothiophenolatoiron(III) complex of protoporphyrin-IX-dimethylester in the presence of 1-MeIm: magnetic circular dichroism and 1H nuclear magnetic resonance spectroscopic studies

Complex formation of 5-coordinated iron(III) heme containing thiolate anion (p-nitrothiophenol) with imidazole (1-methylimidazole) showed very interesting features depending on the nature of the solvent and the ratio of the ligand to heme. The complexes formed under different conditions were not only low spin iron(III) complexes with a thiolate anion and an imidazole or with two imidazoles, but also reduced (iron(II] complexes with a thiolate and an imidazole or with two imidazoles. Absorption, magnetic circular dichroism, and 1H NMR spectroscopies could identify the complex formed when they were used concurrently. The dependence of polarity of the solvents used on the resultant chemical species was ascribed to the stability of Fe(III) or Fe(II) complex in the different solvents. The iron(III) complex with a thiolate anion and an imidazole was found to be reduced automatically to the iron(II) complex with a thiolate and an imidazole which exchanged ligand to the iron(II) bisimidazoles in the presence of excess imidazole. This study showed that the ligands of heme are easily exchanged and that the heme iron(III) is automatically reduced in several conditions. Possible significance with respect to biological systems containing a sulfur ligand is discussed.     

Ligand and halide binding properties of chloroperoxidase: peroxidase-type active site heme environment with cytochrome P-450 type endogenous axial ligand and spectroscopic properties

Equilibrium binding studies of exogenous ligands and halides to the active site heme iron of chloroperoxidase have been carried out from pH 2 to 7. Over twenty ligands have been studied including C, N, O, P, and S donors and the four halides. As judged from changes in the optical absorption spectra, direct binding of the ligands to the heme iron of ferric or ferrous chloroperoxidase occurs in all cases; this has been ascertained for the ferric enzyme in several cases through competition experiments with cyanide. All of the ligands except for the halides, nitrate, and acetate form exclusively low-spin complexes in analogy to results obtained with the spectroscopically related protein, cytochrome P-450-CAM [Sono, M., & Dawson, J.H. (1982) J. Biol. Chem. 257, 5496-5502]. The titration results show that, for the ferric enzyme, (i) weakly acidic ligands (pKa greater than 3) bind to the enzyme in their neutral (protonated) form, followed by deprotonation upon ligation to the heme iron. In contrast, (ii) strongly acidic ligands (pKa less than 0) including SCN-, NO3-, and the halides except for F- likely bind in their anionic (deprotonated) form to the acid form of the enzyme: a single ionizable group on the protein with a pKa less than 2 is involved in this binding. For the ferrous enzyme, (iii) a single ionizable group with the pKa value of 5.5 affects ligand binding. These results reveal that chloroperoxidase, in spite of the previously established close spectroscopic and heme iron coordination structure similarities to the P-450 enzymes, clearly belongs to the hydroperoxidases in terms of its ligand binding properties and active site heme environment. Magnetic circular dichroism studies indicate that the alkaline form (pH 9.5) of ferric chloroperoxidase has an RS-ferric heme-N donor ligand coordination structure with the N donor likely derived from histidine imidazole.     

Leghemoglobin. An electron paramagnetic resonance and optical spectral study of the free protein and its complexes with nicotinate and acetate.

Electron paramagnetic resonance (EPR) and optical spectra are used as probes of the heme and its ligands in ferric and ferrous leghemoglobin. The proximal ligand to the heme iron atom of ferric soybean leghemoglobin is identified as imidazole by comparison of the EPR of leghemoglobin hydroxide, azide, and cyanide with the corresponding derivatives of human hemoglobin. Optical spectra show that ferric soybean leghemoglobin near room temperature is almost entirely in the high spin state. At 77 K the optical spectrum is that of a low spin compound, while at 1.6 K the EPR is that of a low spin form resembling bis-imidazole heme. Acetate binds to ferric leghemoglobin to form a high spin complex as judged from the optical spectrum. The EPR of this complex is that of high spin ferric heme in a nearly axial environment. The complexes of ferrous leghemoglobin with substituted pyridines exhibit optical absorption maxima near 685 nm, whose absorption maxima and extinctions are strongly dependent on the nature of the substitutents of the pyridine ring; electron withdrawing groups on the pyridine ring shift the absorption maxima to lower energy. A crystal field analysis of the EPR of nicotinate derivatives of ferric leghemoblobin demonstrates that the pyridine nitrogen is also bound to the heme iron in the ferric state. These findings lead us to picture leghemoglobin as a somewhat flexible molecule in which the transition region between the E and F helices may act as a hinge, opening a small amount at higher temperature to a stable configuration in which the protein is high spin and can accommodate exogenous ligand molecules and closing at low temperature to a second stable configuration in which the protein is low spin and in which close approach of the E helix permits the distal histidine to become the principal sixth ligand.     

Structure-function relationships in the growing hexa-coordinate hemoglobin sub-family

Hemoglobin and related heme proteins, generally referred to as 'globins', reversibly bind gaseous diatomic ligands (O2, NO, and CO) to a penta-coordinate heme iron atom, the ligand filling the sixth coordination site. Over the last decade, several new globins have been reported to display a functionally-relevant hexa-coordinate heme iron atom, whose sixth coordination site is taken by an endogenous protein ligand. The reversible intramolecular hexa- to penta-coordination process at the heme-Fe atom modulates exogenous ligand binding properties of hexa-coordinate globins. Here, we review current knowledge on hexa-coordinate globins in terms of their structural and functional properties.