Initial O2 Insertion Step of the Tryptophan Dioxygenase Reaction Proposed by a Heme-Modification Study.

L-Tryptophan 2,3-dioxygenase (TDO) is a protoheme-containing enzyme that catalyzes the production of N-formylkynurenine by inserting O2 into the pyrrole ring of L-tryptophan. Although a ferrous-oxy form (Fe²âº-O2) has been established to be an obligate intermediate in the reaction, details of the ring opening reaction remain elusive. In this study, the O2 insertion reaction catalyzed by Pseudomonas TDO (PaTDO) was examined using a heme-modification approach, which allowed us to draw a quantitative correlation between the inductive electronic effects of the heme substituents and the substituent-induced changes in the functional behaviors of the ferrous-oxy form. We succeeded in preparing reconstituted PaTDO with synthetic hemes, which were different with respect to the inductive electron-withdrawing nature of the heme substituents at positions 2 and 4. An increase in the electron-withdrawing power of the heme substituents elevated the redox potential of reconstituted PaTDO, showing that the stronger the electron-withdrawing ability of the heme substituents, the lower the electron density on the heme iron. The decrease in the electron density of the heme iron resulted in a higher frequency shift of the C-O stretch of the heme-bound CO and enhanced the dissociation of O2 from the ferrous-oxy intermediate. This result was interpreted as being due to weaker π back-donation from the heme iron to the bound CO or O2. More importantly, the reaction rates of the ferrous-oxy intermediate to oxidize L-Trp were increased with the electron-withdrawing ability of the heme substituents, implying that the more electron-deficient ferrous-oxy heme is favored for the PaTDO-catalyzed oxygenation. On the basis of these results, we propose that the initial step of the dioxygen activation by PaTDO is a direct electrophilic addition of the heme-bound O2 to the indole ring of L-Trp.

The undifferentiated cell zone is a stem cell zone in adult rat adrenal cortex.

The adrenal cortex of mammals has been known to consist of three morphologically and functionally distinct zones, i.e. the zona glomerulosa (zG), the zona fasciculata (zF) and the zona reticularis (zR), each of which secretes a specific corticosteroid different from those produced by the other two zones. We found previously, however, that an additional zone existed between zG and zF of adult rat adrenal cortex and that the cells in that zone were in a functionally undifferentiated state as an adrenocortical cell [Endocrinology 135, (1994) 431]: they were incapable of synthesizing highly active forms of corticosteroids, such as aldosterone and corticosterone, although they could produce their precursors. Hence, we named the zone as the undifferentiated cell zone (zU) of the adrenal cortex. Here we show that zU and its surroundings, i.e. the innermost portion of zG and the outermost portion of zF are the sites for cell replication in adult rat adrenal cortex and that the cells raised there migrate to other regions. Such cell replications in this region occur regardless of physiological conditions, such as the rise and fall of hormonal stimuli and circadian fluctuation of adrenocortical activities. On the bases of these and other findings previously described, we propose that zU is the stem cell zone of the adult rat adrenal cortex. Our recent success in isolating novel cell lines, which display an undifferentiated phenotype similar to that of zU cells, could facilitate the exploration of molecular mechanisms for the differentiation and development of the adrenocortical cells.

Oxygen metabolism by endothelial nitric-oxide synthase.

Nitric-oxide synthase (NOS) catalyzes both coupled and uncoupled reactions that generate nitric oxide and reactive oxygen species. Oxygen is often the overlooked substrate, and the oxygen metabolism catalyzed by NOS has been poorly defined. In this paper we focus on the oxygen stoichiometry and effects of substrate/cofactor binding on the endothelial NOS isoform (eNOS). In the presence of both L-arginine and tetrahydrobiopterin, eNOS is highly coupled (>90%), and the measured stoichiometry of O(2)/NADPH is very close to the theoretical value. We report for the first time that the presence of L-arginine stimulates oxygen uptake by eNOS. The fact that nonhydrolyzable L-arginine analogs are not stimulatory indicates that the occurrence of the coupled reaction, rather than the accelerated uncoupled reaction, is responsible for the L-arginine-dependent stimulation. The presence of 5,6,7,8-tetrahydrobiopterin quenched the uncoupled reactions and resulted in much less reactive oxygen species formation, whereas the presence of redox-incompetent 7,8-dihydrobiopterin demonstrates little quenching effect. These results reveal different mechanisms for oxygen metabolism for eNOS as opposed to nNOS and, perhaps, partially explain their functional differences.

Oxygen metabolism by neuronal nitric-oxide synthase.

Nitric-oxide synthases (NOS) catalyze nitric oxide (NO) formation from the amino acid L-arginine. NOS is known to catalyze more than one reaction: the NO-producing reaction is considered to be the coupled reaction, and the uncoupled reactions are those that produce reactive (reduced) oxygen species (ROS), such as superoxide anion (O-2.) and/or hydrogen peroxide (H2O2). As an oxygenase, NOS has been known for more than two decades, yet there is no complete description of oxygen stoichiometry. The present paper is focused on oxygen stoichiometry and the effects of cofactor binding on the neuronal isoform (nNOS) on oxygen uptake and product formation. Products of the uncoupled reactions are analyzed using diacetyldeuteroheme-substituted horseradish peroxidase as a trapping agent for both O-2. and H2O2. The addition of calmodulin not only stimulated the oxygen uptake rate but also changed the product of the uncoupled reaction, supporting the possibility of two different sites for electron leakage to molecular oxygen. Quantitative analysis of the uncoupled (substrate-free) reaction revealed a stoichiometry close to the theoretical value, and adding L-arginine not only initiates the coupled reaction, but also inhibits oxygen uptake. The presence of tetrahydrobiopterin affects oxygen metabolism by lowering the apparent Km value of nNOS for oxygen in the uncoupled reaction.

Detection of nitrous oxide in the neuronal nitric oxide synthase reaction by gas chromatography-mass spectrometry.

Using headspace gas chromatography-mass spectrometry, we detected significant amounts of nitrous oxide in the reaction products of the monooxygenase reaction catalyzed by neuronal nitric oxide synthase. Nitrous oxide is a dimerization product of nitroxyl anion; its presence in the reaction products indicates that the nitroxyl anion is a product of the neuronal nitric oxide synthase-catalyzed reaction.

An inverse correlation between expression of a preprocathepsin B-related protein with cysteine-rich sequences and steroid 11beta -hydroxylase in adrenocortical cells.

A cDNA encoding a secretory protein hitherto unknown was cloned from mouse adrenocortical cells by subtractive hybridization between the cells without and with expressing steroid 11beta-hydroxylase (Cyp11b-1), a marker for the functional differentiation of cells in the zonae fasciculata reticularis (zFR). The deduced protein consisting of 466 amino acids contained a secretory signal, epidermal growth factor-like repeats, and a proteolytically inactive cathepsin B-related sequence. The amino acid sequence was 89% identical with that of human tubulointerstitial nephritis antigen-related protein. Among the mouse organs examined, adrenal glands prominently expressed its mRNA. The mRNA and its encoded protein were detected in the outer adrenocortical zones that do not express Cyp11b-1, i.e. the zona glomerulosa and the undifferentiated cell zone, while being undetectable in zFR that express Cyp11b-1. The new protein was designated as adrenocortical zonation factor 1 (AZ-1). Clonal lines with different levels of AZ-1 expression were established from Y-1 adrenocortical cells that originally express Cyp11b-1 but little AZ-1. Analyses of the clonal lines revealed that Cyp11b-1 is detected in the clonal lines maintaining little AZ-1 expression and becomes undetectable in those expressing AZ-1. On the other hand, irrespective of the AZ-1 expression, all clones expressed cholesterol side-chain cleavage enzyme, which occurs throughout the cortical zones. These results demonstrated that adrenocortical cells expressing AZ-1 do not express Cyp11b-1, whereas those with little AZ-1 express this zFR marker in vitro and in vivo, implying a putative role of AZ-1 in determining the zonal differentiation of adrenocortical cells.

Refolding processes of cytochrome P450cam from ferric and ferrous acid forms to the native conformation. Formations of folding intermediates with non-native heme coordination state.

Changes in heme coordination state and protein conformation of cytochrome P450(cam) (P450(cam)), a b-type heme protein, were investigated by employing pH jump experiments coupled with time-resolved optical absorption, fluorescence, circular dichroism, and resonance Raman techniques. We found a partially unfolded form (acid form) of ferric P450(cam) at pH 2.5, in which a Cys(-)-heme coordination bond in the native conformation was ruptured. When the pH was raised to pH 7.5, the acid form refolded to the native conformation through a distinctive intermediate. Formations of similar acid and intermediate forms were also observed for ferrous P450(cam). Both the ferric and ferrous forms of the intermediate were found to have an unidentified axial ligand of the heme at the 6th coordination sphere, which is vacant in the high spin ferric and ferrous forms at the native conformation. For the ferrous form, it was also indicated that the 5th axial ligand is different from the native cysteinate. The folding intermediates identified in this study demonstrate occurrences of non-native coordination state of heme during the refolding processes of the large b-type heme protein, being akin to the well known folding intermediates of cytochromes c, in which c-type heme is covalently attached to a smaller protein.

The low-spin heme of cytochrome c oxidase as the driving element of the proton-pumping process.

Mitochondrial cytochrome c oxidase plays an essential role in aerobic cellular respiration, reducing dioxygen to water in a process coupled with the pumping of protons across the mitochondrial inner membrane. An aspartate residue, Asp-51, located near the enzyme surface, undergoes a redox-coupled x-ray structural change, which is suggestive of a role for this residue in redox-driven proton pumping. However, functional or mechanistic evidence for the involvement of this residue in proton pumping has not yet been obtained. We report that the Asp-51 --> Asn mutation of the bovine enzyme abolishes its proton-pumping function without impairment of the dioxygen reduction activity. Improved x-ray structures (at 1.8/1.9-A resolution in the fully oxidized/reduced states) show that the net positive charge created upon oxidation of the low-spin heme of the enzyme drives the active proton transport from the interior of the mitochondria to Asp-51 across the enzyme via a water channel and a hydrogen-bond network, located in tandem, and that the enzyme reduction induces proton ejection from the aspartate to the mitochondrial exterior. A peptide bond in the hydrogen-bond network critically inhibits reverse proton transfer through the network. A redox-coupled change in the capacity of the water channel, induced by the hydroxyfarnesylethyl group of the low-spin heme, suggests that the channel functions as an effective proton-collecting region. Infrared results indicate that the conformation of Asp-51 is controlled only by the oxidation state of the low-spin heme. These results indicate that the low-spin heme drives the proton-pumping process.

Infrared spectroscopic and mutational studies on putidaredoxin-induced conformational changes in ferrous CO-P450cam.

Ferrous-carbon monoxide bound form of cytochrome P450cam (CO-P450cam) has two infrared (IR) CO stretching bands at 1940 and 1932 cm(-1). The former band is dominant (>95% in area) for CO-P450cam free of putidaredoxin (Pdx), while the latter band is dominant (>95% in area) in the complex of CO-P450cam with reduced Pdx. The binding of Pdx to CO-P450cam thus evokes a conformational change in the heme active site. To study the mechanism involved in the conformational change, surface amino acid residues Arg79, Arg109, and Arg112 in P450cam were replaced with Lys, Gln, and Met. IR spectroscopic and kinetic analyses of the mutants revealed that an enzyme that has a larger 1932 cm(-1) band area upon Pdx-binding has a larger catalytic activity. Examination of the crystal structures of R109K and R112K suggested that the interaction between the guanidium group of Arg112 and Pdx is important for the conformational change. The mutations did not change a coupling ratio between the hydroxylation product and oxygen consumed. We interpret these findings to mean that the interaction of P450cam with Pdx through Arg112 enhances electron donation from the proximal ligand (Cys357) to the O-O bond of iron-bound O(2) and, possibly, promotes electron transfer from reduced Pdx to oxyP450cam, thereby facilitating the O-O bond splitting.

Kinetic and spectroscopic characterization of a hydroperoxy compound in the reaction of native myoglobin with hydrogen peroxide.

The reaction of metmyoglobin with H2O2 was investigated in a pH range between 8.5 and 6.0 with the aid of stopped flow-rapid scan and rapid freezing-EPR techniques. Singular value decomposition analyses of the stopped flow data at pH 8.5 revealed that a spectral species previously unknown accumulated during the reaction and exhibited a Soret absorption maximum at >/=423 nm. In the EPR experiments, the new species exhibited a set of g values at 2.32, 2.19, and 1.94, indicating that the species was assignable to a ferric hydroperoxy (Fe(III)[O-O-H]-) compound. In contrast, the hydroperoxy compound scarcely accumulated in the reaction at pH 6.0, and the dominant intermediate species accumulated was compound I, which was derived from the oxygen-oxygen bond cleavage of the hydroperoxy compound. The accumulated amount of the hydroperoxy compound relative to compound I showed a pH dependence with an apparent pKa (pKaapp) from 6.95 to 7.27 depending on the metmyoglobins examined. This variation in pKaapp paralleled that in pKa of the acid-alkaline transition (pKaAB) of metmyoglobins, suggesting that the accumulation of hydroperoxy compound is controlled by the distal histidine. We propose that the H2O2 activation by metmyoglobin is promoted at the acidic condition due to the imidazolium form of the distal histidine, and we further propose that the controlled protonation state of the distal histidine is important for the facile O-O bond cleavage in heme peroxidases.

Stabilization of mast cells by heme oxygenase-1: an anti-inflammatory role.

This study examined the role of bilirubin in heme oxygenase (HO)-1-mediated amelioration of mast cell (MC)-elicited inflammatory responses. Pretreatment of rats with an intraperitoneal injection of hemin, an inducer of HO-1, evolved a marked induction of the enzyme in MCs. Intravital videomicroscopy revealed that hemin pretreatment attenuated compound 48/80-elicited degranulation of MCs and resultant leukocyte adhesion in venules. Superfusion with biliverdin or bilirubin, but not with carbon monoxide (CO), another product of the HO reaction, mimicked suppressive actions of the HO-1 induction on both the cell degranulation and leukocyte adhesion elicited by the stimulus, suggesting a requirement of the enzyme reaction to generate bilirubin in the inhibitory mechanisms. Such MC-desensitizing actions of bilirubin were observed in primary-cultured MCs and reproduced irrespective of the choice of stimuli, such as compound 48/80, calcium ionophore, and anti-IgE serum. Furthermore, MC-stabilizing effects of HO-1 were reproduced by the gene transfection of the enzyme into mastocytoma cell line RBL2H3. These results suggest that bilirubin generated through HO-1 serves as an anti-inflammatory substance that desensitizes MCs and ameliorates leukocyte recruitment.

Conditionally immortalized adrenocortical cell lines at undifferentiated states exhibit inducible expression of glucocorticoid-synthesizing genes.

To facilitate studies on differentiation of adrenocortical cells and regulation of steroidogenic genes, we established cell lines from adrenals of adult transgenic mice harboring a temperature-sensitive large T-antigen gene of simian virus 40. Adrenal glands of the mice exhibited normal cortical zonation including a functionally undifferentiated cell-layer between the aldosterone-synthesizing zona glomerulosa cells and the corticosterone-synthesizing zona fasciculata cells. At a permissive temperature (33 degrees C), established cell lines AcA201, AcE60 and AcA101 expressed steroidogenic genes encoding steroidogenic factor-1, cholesterol side-chain cleavage P450scc, and steroidogenic acute regulatory protein, which are expressed throughout adrenal cortices and gonads. Genes encoding 3 beta-hydroxysteroid dehydrogenase and steroid 21-hydroxylase P450c21, which catalyze the intermediate steps for syntheses of both aldosterone and corticosterone, were inducible in the three cell lines in temperature- and/or dibutyryl cAMP-dependent manners. Notably, these cell lines displayed distinct expression patterns of the steroid 11 beta-hydroxylase P45011 beta gene responsible for the zone-specific synthesis of corticosterone. AcA201 cells expressed the P45011 beta gene at 33 degrees C, showing the property of the zona fasciculata cells, while AcE60 cells expressed it upon a shift to a nonpermissive temperature (39 degrees C). On the other hand, AcA101 expressed the P45011 beta gene at 39 degrees C synergistically with exposure to dibutyryl cAMP. None of these clones express the zona glomerulosa-specific aldosterone synthase P450aldo gene under the conditions we tested. These results show that AcE60 and AcA101 cells display a pattern of the steroidogenic gene expression similar to that of the undifferentiated cell-layer and are capable of differentiating into the zona fasciculata-like cells in vitro.