Spectroscopic study of in situ-formed metallocomplexes of proton pump inhibitors in water.

Proton pump inhibitors, such as omeprazole, pantoprazole and lansoprazole, are an important group of clinically used drugs. Generally, they are considered safe without direct toxicity. Nevertheless, their long-term use can be associated with a higher risk of some serious pathological states (e.g. amnesia and oncological and neurodegenerative states). It is well known that dysregulation of the metabolism of transition metals (especially iron ions) plays a significant role in these pathological states and that the above drugs can form complexes with metal ions. However, to the best of our knowledge, this phenomenon has not yet been described in water systems. Therefore, we studied the interaction between these drugs and transition metal ions in the surrounding water environment (water/DMSO, 99:1, v/v) by absorption spectroscopy. In the presence of Fe(III), a strong redshift was observed, and more importantly, the affinities of the drugs (represented as binding constants) were strong enough, especially in the case of omeprazole, so that the formation of a metallocomplex cannot be excluded during the explanation of their side effects.

Unique Electronic Structures of the Highly Ruffled Hemes in Heme-Degrading Enzymes of Staphylococcus aureus, IsdG and IsdI, by Resonance Raman and Electron Paramagnetic Resonance Spectroscopies.

Staphylococcus aureus uses IsdG and IsdI to convert heme into a mixture of staphylobilin isomers, 15-oxo-ß-bilirubin and 5-oxo-δ-bilirubin, formaldehyde, and iron. The highly ruffled heme found in the heme-IsdI and IsdG complexes has been proposed to be responsible for the unique heme degradation products. We employed resonance Raman (RR) and electron paramagnetic resonance (EPR) spectroscopies to examine the coordination and electronic structures of heme bound to IsdG and IsdI. Heme complexed to IsdG and IsdI is coordinated by a neutral histidine. The trans ligand is hydroxide in the ferric alkaline form of both proteins. In the ferric neutral form at pH 6.0, heme is six-coordinated with water as the sixth ligand for IsdG and is in the mixture of the five-coordinated and six-coordinated species for IsdI. In the ferrous CO-bound form, CO is strongly hydrogen bonded with a distal residue. The marker lines, ν2 and ν3, appear at frequencies that are distinct from other proteins having planar hemes. The EPR spectra for the ferric hydroxide and cyanide states might be explained by assuming the thermal mixing of the d-electron configurations, (dxy)2(dxz,dyz)3 and (dxz,dyz)4(dxy)1. The fraction for the latter becomes larger for the ferric cyanide form. In the ferric neutral state at pH 6.0, the quantum mechanical mixing of the high and intermediate spin configurations might explain the peculiar frequencies of ν2 and ν3 in the RR spectra. The heme ruffling imposed by IsdG and IsdI gives rise to unique electronic structures of heme, which are expected to modulate the first and subsequent steps of the heme oxygenation.

Hydrogen sulfide bypasses the rate-limiting oxygen activation of heme oxygenase.

Discovery of unidentified protein functions is of biological importance because it often provides new paradigms for many research areas. Mammalian heme oxygenase (HO) enzyme catalyzes the O2-dependent degradation of heme into carbon monoxide (CO), iron, and biliverdin through numerous reaction intermediates. Here, we report that H2S, a gaseous signaling molecule, is part of a novel reaction pathway that drastically alters HO's products, reaction mechanism, and catalytic properties. Our prediction of this interplay is based on the unique reactivity of H2S with one of the HO intermediates. We found that in the presence of H2S, HO produces new linear tetrapyrroles, which we identified as isomers of sulfur-containing biliverdin (SBV), and that only H2S, but not GSH, cysteine, and polysulfides, induces SBV formation. As BV is converted to bilirubin (BR), SBV is enzymatically reduced to sulfur-containing bilirubin (SBR), which shares similar properties such as antioxidative effects with normal BR. SBR was detected in culture media of mouse macrophages, confirming the existence of this H2S-induced reaction in mammalian cells. H2S reacted specifically with a ferric verdoheme intermediate of HO, and verdoheme cleavage proceeded through an O2-independent hydrolysis-like mechanism. This change in activation mode diminished O2 dependence of the overall HO activity, circumventing the rate-limiting O2 activation of HO. We propose that H2S could largely affect O2 sensing by mammalian HO, which is supposed to relay hypoxic signals by decreasing CO output to regulate cellular functions. Moreover, the novel H2S-induced reaction identified here helps sustain HO's heme-degrading and antioxidant-generating capacity under highly hypoxic conditions.

Biochemical and structural characterization of oxygen-sensitive 2-thiouridine synthesis catalyzed by an iron-sulfur protein TtuA.

Two-thiouridine (s2U) at position 54 of transfer RNA (tRNA) is a posttranscriptional modification that enables thermophilic bacteria to survive in high-temperature environments. s2U is produced by the combined action of two proteins, 2-thiouridine synthetase TtuA and 2-thiouridine synthesis sulfur carrier protein TtuB, which act as a sulfur (S) transfer enzyme and a ubiquitin-like S donor, respectively. Despite the accumulation of biochemical data in vivo, the enzymatic activity by TtuA/TtuB has rarely been observed in vitro, which has hindered examination of the molecular mechanism of S transfer. Here we demonstrate by spectroscopic, biochemical, and crystal structure analyses that TtuA requires oxygen-labile [4Fe-4S]-type iron (Fe)-S clusters for its enzymatic activity, which explains the previously observed inactivation of this enzyme in vitro. The [4Fe-4S] cluster was coordinated by three highly conserved cysteine residues, and one of the Fe atoms was exposed to the active site. Furthermore, the crystal structure of the TtuA-TtuB complex was determined at a resolution of 2.5 Å, which clearly shows the S transfer of TtuB to tRNA using its C-terminal thiocarboxylate group. The active site of TtuA is connected to the outside by two channels, one occupied by TtuB and the other used for tRNA binding. Based on these observations, we propose a molecular mechanism of S transfer by TtuA using the ubiquitin-like S donor and the [4Fe-4S] cluster.

Charge-state-distribution analysis of Bach2 intrinsically disordered heme binding region.

Bach2 is a transcriptional repressor that plays an important role in the differentiation of T-cells and B-cells. Bach2 is functionally regulated by heme binding, and possesses five Cys-Pro Cys-Pro (CP)-motifs as the heme binding site. To reveal the molecular mechanism of heme binding by Bach2, the intrinsically disordered heme binding region (a.a. 331-520; Bach2331-520) and its CP-motif mutant were prepared and characterized with and without heme, by UV-Vis spectroscopy and thermal profiles. In addition, the charge-state-distributions (CSDs) were assessed by electrospray ionization mass spectrometry. The UV-Vis spectroscopy revealed a lack of five-coordinated heme binding in the CP-motif mutant of Bach2331-520 The thermal profile and CSDs of Bach2331-520 indicated that heme binding induces the destabilization of Bach2331-520 The thermal profile revealed that the wild type Bach2331-520 was destabilized more than the CP-motif mutant. The shift in the CSDs by heme binding suggested that heme binding causes Bach2331-520 to adopt a more compact conformation. In addition, heme binding to the CP-motif could reduce the flexibility of Bach2331-520 Consequently, the five-coordinated heme binding destabilizes Bach2331-520, by reducing the flexibility of the polypeptide chain.

Unique coupling of mono- and dioxygenase chemistries in a single active site promotes heme degradation.

Bacterial pathogens must acquire host iron for survival and colonization. Because free iron is restricted in the host, numerous pathogens have evolved to overcome this limitation by using a family of monooxygenases that mediate the oxidative cleavage of heme into biliverdin, carbon monoxide, and iron. However, the etiological agent of tuberculosis, Mycobacterium tuberculosis, accomplishes this task without generating carbon monoxide, which potentially induces its latent state. Here we show that this unusual heme degradation reaction proceeds through sequential mono- and dioxygenation events within the single active center of MhuD, a mechanism unparalleled in enzyme catalysis. A key intermediate of the MhuD reaction is found to be meso-hydroxyheme, which reacts with O2 at an unusual position to completely suppress its monooxygenation but to allow ring cleavage through dioxygenation. This mechanistic change, possibly due to heavy steric deformation of hydroxyheme, rationally explains the unique heme catabolites of MhuD. Coexistence of mechanistically distinct functions is a previously unidentified strategy to expand the physiological outcome of enzymes, and may be applied to engineer unique biocatalysts.

Protein oxidation mediated by heme-induced active site conversion specific for heme-regulated transcription factor, iron response regulator.

The Bradyrhizobium japonicum transcriptional regulator Irr (iron response regulator) is a key regulator of the iron homeostasis, which is degraded in response to heme binding via a mechanism that involves oxidative modification of the protein. Here, we show that heme-bound Irr activates O2 to form highly reactive oxygen species (ROS) with the "active site conversion" from heme iron to non-heme iron to degrade itself. In the presence of heme and reductant, the ROS scavenging experiments show that Irr generates H2O2 from O2 as found for other hemoproteins, but H2O2 is less effective in oxidizing the peptide, and further activation of H2O2 is suggested. Interestingly, we find a time-dependent decrease of the intensity of the Soret band and appearance of the characteristic EPR signal at g = 4.3 during the oxidation, showing the heme degradation and the successive formation of a non-heme iron site. Together with the mutational studies, we here propose a novel "two-step self-oxidative modification" mechanism, during which O2 is activated to form H2O2 at the heme regulatory motif (HRM) site and the generated H2O2 is further converted into more reactive species such as ·OH at the non-heme iron site in the His-cluster region formed by the active site conversion.

Artificial Hydrogenases Based on Cobaloximes and Heme Oxygenase.

The insertion of cobaloxime catalysts in the heme-binding pocket of heme oxygenase (HO) yields artificial hydrogenases active for H2 evolution in neutral aqueous solutions. These novel biohybrids have been purified and characterized by using UV/visible and EPR spectroscopy. These analyses revealed the presence of two distinct binding conformations, thereby providing the cobaloxime with hydrophobic and hydrophilic environments, respectively. Quantum chemical/molecular mechanical docking calculations found open and closed conformations of the binding pocket owing to mobile amino acid residues. HO-based biohybrids incorporating a {Co(dmgH)2 } (dmgH2 =dimethylglyoxime) catalytic center displayed up to threefold increased turnover numbers with respect to the cobaloxime alone or to analogous sperm whale myoglobin adducts. This study thus provides a strong basis for further improvement of such biohybrids, using well-designed modifications of the second and outer coordination spheres, through site-directed mutagenesis of the host protein.

Heme binds to an intrinsically disordered region of Bach2 and alters its conformation.

The transcriptional repressor Bach2 regulates humoral and cellular immunity, including antibody class switching. It possesses a basic leucine zipper domain that mediates DNA binding. Heme inhibits the DNA-binding activity of Bach2 in vitro and induces the degradation of Bach2 in B cells. However, the structural basis of the heme-Bach2 interaction has not been identified. Spectroscopic analyses revealed that Bach2(331-520) is the heme-binding domain, as it includes three Cys-Pro motifs known to be important for heme binding. Heme-titration experiments demonstrated the presence of 5- and 6-coordinated heme-binding modes. Circular dichroism measurements indicated that Bach2(331-520) exists mostly in a random-coil conformation. However, dynamic light scattering analyses showed that, upon heme binding to Bach2(331-520), this region becomes denatured at a lower temperature, as compared with unbound Bach2(331-520). In addition, small-angle X-ray scattering and chemical modification analyses revealed that heme binding induces conformational alterations within the unstructured region. A GAL4-based luciferase assay in 293T cells showed that heme alters the protein interactions mediated by Bach2(331-520). These observations suggested that the unstructured region of Bach2 is important for heme binding, and consequently for its functional regulation.

Heme utilization by pathogenic bacteria: not all pathways lead to biliverdin.

The eukaryotic heme oxygenases (HOs) (E.C. 1.14.99.3) convert heme to biliverdin, iron, and carbon monoxide (CO) in three successive oxygenation steps. Pathogenic bacteria require iron for survival and infection. Extracellular heme uptake from the host plays a critical role in iron acquisition and virulence. In the past decade, several HOs required for the release of iron from extracellular heme have been identified in pathogenic bacteria, including Corynebacterium diphtheriae, Neisseriae meningitides, and Pseudomonas aeruginosa. The bacterial enzymes were shown to be structurally and mechanistically similar to those of the canonical eukaryotic HO enzymes. However, the recent discovery of the structurally and mechanistically distinct noncanonical heme oxygenases of Staphylococcus aureus and Mycobacterium tuberculosis has expanded the reaction manifold of heme degradation. The distinct ferredoxin-like structural fold and extreme heme ruffling are proposed to give rise to the alternate heme degradation products in the S. aureus and M. tuberculosis enzymes. In addition, several "heme-degrading factors" with no structural homology to either class of HOs have recently been reported. The identification of these "heme-degrading proteins" has largely been determined on the basis of in vitro heme degradation assays. Many of these proteins were reported to produce biliverdin, although no extensive characterization of the products was performed. Prior to the characterization of the canonical HO enzymes, the nonenzymatic degradation of heme and heme proteins in the presence of a reductant such as ascorbate or hydrazine, a reaction termed "coupled oxidation", served as a model for biological heme degradation. However, it was recognized that there were important mechanistic differences between the so-called coupled oxidation of heme proteins and enzymatic heme oxygenation. In the coupled oxidation reaction, the final product, verdoheme, can readily be converted to biliverdin under hydrolytic conditions. The differences between heme oxygenation by the canonical and noncanonical HOs and coupled oxidation will be discussed in the context of the stabilization of the reactive Fe(III)-OOH intermediate and regioselective heme hydroxylation. Thus, in the determination of heme oxygenase activity in vitro, it is important to ensure that the reaction proceeds through successive oxygenation steps. We further suggest that when bacterial heme degradation is being characterized, a systems biology approach combining genetics, mechanistic enzymology, and metabolite profiling should be undertaken.

Structures of the substrate-free and product-bound forms of HmuO, a heme oxygenase from corynebacterium diphtheriae: x-ray crystallography and molecular dynamics investigation.

Heme oxygenase catalyzes the degradation of heme to biliverdin, iron, and carbon monoxide. Here, we present crystal structures of the substrate-free, Fe(3+)-biliverdin-bound, and biliverdin-bound forms of HmuO, a heme oxygenase from Corynebacterium diphtheriae, refined to 1.80, 1.90, and 1.85 Å resolution, respectively. In the substrate-free structure, the proximal and distal helices, which tightly bracket the substrate heme in the substrate-bound heme complex, move apart, and the proximal helix is partially unwound. These features are supported by the molecular dynamic simulations. The structure implies that the heme binding fixes the enzyme active site structure, including the water hydrogen bond network critical for heme degradation. The biliverdin groups assume the helical conformation and are located in the heme pocket in the crystal structures of the Fe(3+)-biliverdin-bound and the biliverdin-bound HmuO, prepared by in situ heme oxygenase reaction from the heme complex crystals. The proximal His serves as the Fe(3+)-biliverdin axial ligand in the former complex and forms a hydrogen bond through a bridging water molecule with the biliverdin pyrrole nitrogen atoms in the latter complex. In both structures, salt bridges between one of the biliverdin propionate groups and the Arg and Lys residues further stabilize biliverdin at the HmuO heme pocket. Additionally, the crystal structure of a mixture of two intermediates between the Fe(3+)-biliverdin and biliverdin complexes has been determined at 1.70 Å resolution, implying a possible route for iron exit.

Heme degradation by Staphylococcus aureus IsdG and IsdI liberates formaldehyde rather than carbon monoxide.

IsdG and IsdI from Staphylococcus aureus are novel heme-degrading enzymes containing unusually nonplanar (ruffled) heme. While canonical heme-degrading enzymes, heme oxygenases, catalyze heme degradation coupled with the release of CO, in this study we demonstrate that the primary C1 product of the S. aureus enzymes is formaldehyde. This finding clearly reveals that both IsdG and IsdI degrade heme by an unusual mechanism distinct from the well-characterized heme oxygenase mechanism as recently proposed for MhuD from Mycobacterium tuberculosis. We conclude that heme ruffling is critical for the drastic mechanistic change for these novel bacterial enzymes.

A new way to degrade heme: the Mycobacterium tuberculosis enzyme MhuD catalyzes heme degradation without generating CO.

MhuD is an oxygen-dependent heme-degrading enzyme from Mycobacterium tuberculosis with high sequence similarity (∼45%) to Staphylococcus aureus IsdG and IsdI. Spectroscopic and mutagenesis studies indicate that the catalytically active 1:1 heme-MhuD complex has an active site structure similar to those of IsdG and IsdI, including the nonplanarity (ruffling) of the heme group bound to the enzyme. Distinct from the canonical heme degradation, we have found that the MhuD catalysis does not generate CO. Product analyses by electrospray ionization-MS and NMR show that MhuD cleaves heme at the α-meso position but retains the meso-carbon atom at the cleavage site, which is removed by canonical heme oxygenases. The novel tetrapyrrole product of MhuD, termed "mycobilin," has an aldehyde group at the cleavage site and a carbonyl group at either the ß-meso or the δ-meso position. Consequently, MhuD catalysis does not involve verdoheme, the key intermediate of ring cleavage by canonical heme oxygenase enzymes. Ruffled heme is apparently responsible for the heme degradation mechanism unique to MhuD. In addition, MhuD heme degradation without CO liberation is biologically significant as one of the signals of M. tuberculosis transition to dormancy is mediated by the production of host CO.

Crystallographic studies of heme oxygenase complexed with an unstable reaction intermediate, verdoheme.

This article discusses the accuracy of X-ray structural studies of heme oxygenase (HO) in complex with an unstable intermediate, verdoheme. Heme degradation by HO proceeds through three successive steps of O(2) activation. The mechanism of the third step, the ring opening of verdoheme, has been the least understood. Recent structural studies of the verdoheme-HO complex provide detailed information concerning this mechanism. Due to X-ray-induced photoreduction and the instability of verdoheme, it has been difficult to obtain an accurate structure for the ferrous verdoheme-HO complex. Therefore, accurate structural studies, including analysis of the electronic state of the verdoheme-HO complex, are needed to elucidate the proper reaction mechanism.

A heme degradation enzyme, HutZ, from Vibrio cholerae.

HutZ, one of the crucial proteins of the iron uptake system in Vibrio cholerae, was purified, which binds to heme at a stoichiometry of 1 : 1. In the presence of ascorbic acid, the HutZ-bound heme degrades via the same intermediates observed in heme oxygenase, suggesting that HutZ works as a heme degradation enzyme.

Binding and selectivity of the marine toxin neodysiherbaine A and its synthetic analogues to GluK1 and GluK2 kainate receptors.

Dysiherbaine (DH) and neodysiherbaine A (NDH) selectively bind and activate two kainate-type ionotropic glutamate receptors, GluK1 and GluK2. The ligand-binding domains of human GluK1 and GluK2 were crystallized as bound forms with a series of DH analogues including DH, NDH, 8-deoxy-NDH, 9-deoxy-NDH and 8,9-dideoxy-NDH (MSVIII-19), isolated from natural sources or prepared by total synthesis. Since the DH analogues exhibit a wide range of binding affinities and agonist efficacies, it follows that the detailed analysis of crystal structure would provide us with a significant opportunity to elucidate structural factors responsible for selective binding and some aspects of gating efficacy. We found that differences in three amino acids (Thr503, Ser706 and Ser726 in GluK1 and Ala487, Asn690 and Thr710 in GluK2) in the ligand-binding pocket generate differences in the binding modes of NDH to GluK1 and GluK2. Furthermore, deletion of the C(9) hydroxy group in NDH alters the ligand conformation such that it is no longer suited for binding to the GluK1 ligand-binding pocket. In GluK2, NDH pushes and rotates the side chain of Asn690 (substituted for Ser706 in GluK1) and disrupts an interdomain hydrogen bond with Glu409. The present data support the idea that receptor selectivities of DH analogues resulted from the differences in the binding modes of the ligands in GluK1/GluK2 and the steric repulsion of Asn690 in GluK2. All ligands, regardless of agonist efficacy, induced full domain closure. Consequently, ligand efficacy and domain closure did not directly coincide with DH analogues and the kainate receptors.

Heme regulates B-cell differentiation, antibody class switch, and heme oxygenase-1 expression in B cells as a ligand of Bach2.

Heme binds to proteins to modulate their function, thereby functioning as a signaling molecule in a variety of biologic events. We found that heme bound to Bach2, a transcription factor essential for humoral immunity, including antibody class switch. Heme inhibited the DNA binding activity of Bach2 in vitro and reduced its half-life in B cells. When added to B-cell primary cultures, heme enhanced the transcription of Blimp-1, the master regulator of plasma cells, and skewed plasma cell differentiation toward the IgM isotype, decreasing the IgG levels in vitro. Intraperitoneal injection of heme in mice inhibited the production of antigen-specific IgM when heme was administered simultaneously with the antigen but not when it was administered after antigen exposure, suggesting that heme also modulates the early phase of B-cell responses to antigen. Heme oxygenase-1, which is known to be regulated by heme, was repressed by both Bach2 and Bach1 in B cells. Furthermore, the expression of genes for heme uptake changed in response to B-cell activation and heme administration. Our results reveal a new function for heme as a ligand of Bach2 and as a modulatory signal involved in plasma cell differentiation.

Enzymatic ring-opening mechanism of verdoheme by the heme oxygenase: a combined X-ray crystallography and QM/MM study.

The least understood mechanism during heme degradation by the enzyme heme oxygenase (HO) is the third step of ring opening of verdoheme to biliverdin, a process which maintains iron homeostasis. In response to this mechanistic uncertainty, we launched a combined study of X-ray crystallography and theoretical QM/MM calculations, designed to elucidate the mechanism. The air-sensitive ferrous verdoheme complex of HmuO, a heme oxygenase from Corynebacterium diphtheriae, was crystallized under anaerobic conditions. Spectral analysis of the azide-bound verdoheme-HmuO complex crystals assures that the verdoheme group remains intact during the crystallization and X-ray diffraction measurement. The structure offers the first solid evidence for the presence of a water cluster in the distal pocket of this catalytically critical intermediate. The subsequent QM/MM calculations based on this crystal structure explore the reaction mechanisms starting from the FeOOH-verdoheme and FeHOOH-verdoheme complexes, which mimic, respectively, the O(2)- and H(2)O(2)-supported degradations. In both mechanisms, the rate-determining step is the initial O-O bond breaking step, which is either homolytic (for FeHOOH-verdoheme) or coupled to electron and proton transfers (in FeOOH-verdoheme). Additionally, the calculations indicate that the FeHOOH-verdoheme complex is more reactive than the FeOOH-verdoheme complex in accord with experimental findings. QM energies with embedded MM charges are close to and yield the same conclusions as full QM/MM energies. Finally, the calculations highlight the dominant influence of the distal water cluster which acts as a biocatalyst for the conversion of verdoheme to biliverdin in the two processes, by fixing the departing OH and directing it to the requisite site of attack, and by acting as a proton shuttle and a haven for the highly reactive OH(-) nucleophile.

Solution 1H NMR characterization of substrate-free C. diphtheriae heme oxygenase: pertinence for determining magnetic axes in paramagnetic substrate complexes.

Proton 2D NMR was used to confirm in solution a highly conserved portion of the molecular structure upon substrate loss for the heme oxygenase from the pathogenic bacterium Corynebacterium diphtheriae, HmuO. The chemical shifts for the conserved portion of the structure are assessed as references for the dipolar shifts needed to determine the orientation of the paramagnetic susceptibility tensor, chi, in paramagnetic substrate complexes of HmuO. It is shown that the chemical shifts for the structurally conserved portion of substrate-free HmuO serve as excellent references for residues with only small to moderate sized dipolar shifts in the cyanide-inhibited substrate complex of HmuO, yielding an orientation of chi that is essentially the same as conventionally obtained from large dipolar shifts based on empirical estimates of the diamagnetic reference. The implications of these diamagnetic chemical shifts for characterizing the hydrogen bonding in the physiologically relevant, resting-state, high-spin aquo complex are discussed. The pattern of labile proton exchange in the distal H-bond network of substrate-free HmuO allowed comparison of changes in dynamic stability of tertiary contacts in the substrate-free and substrate-bound HmuO and with the same complexes of human heme oxygenase.