Heme Oxygenase-Like Metalloenzymes.

Heme oxygenase (HO) is an enzyme that catalyzes the regiospecific conversion of heme to biliverdin IXalpha, CO, and free iron. In mammals, HO has a variety of physiological functions, including heme catabolism, iron homeostasis, antioxidant defense, cellular signaling, and O(2) sensing. The enzyme is also found in plants (producing light-harvesting pigments) and in some pathogenic bacteria, where it acquires iron from the host heme. The HO-catalyzed heme conversion proceeds through three successive oxygenations, a process that has attracted considerable attention because of its reaction mechanism and physiological importance. The HO reaction is unique in that all three O(2) activations are affected by the substrate itself. The first step is the regiospecific self-hydroxylation of the porphyrin alpha-meso carbon atom. The resulting alpha-meso-hydroxyheme reacts in the second step with another O(2) to yield verdoheme and CO. The third O(2) activation, by verdoheme, cleaves its porphyrin macrocycle to release biliverdin and free ferrous iron. In this Account, we provide an overview of our current understanding of the structural and biochemical properties of the complex self-oxidation reactions in HO catalysis. The first meso-hydroxylation is of particular interest because of its distinct contrast with O(2) activation by cytochrome P450. Although most heme enzymes oxidize exogenous substrates by high-valent oxo intermediates, HO was proposed to utilize the Fe-OOH intermediate for the self-hydroxylation. We have succeeded in preparing and characterizing the Fe-OOH species of HO at low temperature, and an analysis of its reaction, together with mutational and crystallographic studies, reveals that protonation of Fe-OOH by a distal water molecule is critical in promoting the unique self-hydroxylation. The second oxygenation is a rapid, spontaneous auto-oxidation of the reactive alpha-meso-hydroxyheme; its mechanism remains elusive, but the HO enzyme has been shown not to play a critical role in it. Until recently, the means of the third O(2) activation had remained unclear as well, but we have recently untangled its mechanistic outline. Reaction analysis of the verdoheme-HO complex strongly suggests the Fe-OOH species as a key intermediate of the ring-opening reaction. This mechanism is very similar to that of the first meso-hydroxylation, including the critical roles of the distal water molecule. A comprehensive study of the three oxygenations of HO highlights the rational design of the enzyme architecture and its catalytic mechanism. Elucidation of the last oxygenation step has enabled a kinetic analysis of the rate-determining step, making it possible to discuss the HO reaction mechanism in relation to its physiological functions.

In mammals, catabolism of the heme group is indispensable for life. Heme is first cleaved by the enzyme Heme Oxygenase (HO) to the linear tetrapyrrole Biliverdin IXα (BV), and BV is then converted into bilirubin by Biliverdin Reductase (BVR). HO utilizes three Oxygen molecules (O2) and seven electrons supplied by NADPH-cytochrome P450 oxidoreductase (CPR) to open the heme ring and BVR reduces BV through the use of NAD(P)H. Structural studies of HOs, including substrate-bound, reaction intermediate-bound, and several specific inhibitor-bound forms, reveal details explaining substrate binding to HO and mechanisms underlying-specific HO reaction progression. Cryo-trapped structures and a time-resolved spectroscopic study examining photolysis of the bond between the distal ligand and heme iron demonstrate how CO, produced during the HO reaction, dissociates from the reaction site with a corresponding conformational change in HO. The complex structure containing HO and CPR provides details of how electrons are transferred to the heme-HO complex. Although the tertiary structure of BVR and its complex with NAD+ was determined more than 10 years ago, the catalytic residues and the reaction mechanism of BVR remain unknown. A recent crystallographic study examining cyanobacterial BVR in complex with NADP+ and substrate BV provided some clarification regarding these issues. Two BV molecules are bound to BVR in a stacked manner, and one BV may assist in the reductive catalysis of the other BV. In this review, recent advances illustrated by biochemical, spectroscopic, and crystallographic studies detailing the chemistry underlying the molecular mechanism of HO and BVR reactions are presented.

Heme oxygenase (HO) catalyzes the regiospecific conversion of heme to biliverdin, CO, and free iron through three successive oxygenation reactions. HO catalysis is unique in that all three O(2) activations are performed by the substrate itself. This Forum Article overviews our current understanding on the structural and biochemical properties of HO catalysis, especially its first and third oxygenation steps. The HO first step, regiospecific hydroxylation of the porphyrin alpha-meso-carbon atom, is of particular interest because of its sharp contrast to O(2) activation by cytochrome P450. HO was proposed to utilize the FeOOH species but not conventional ferryl hemes as a reactive intermediate for self-hydroxylation. We have succeeded in preparing and characterizing the FeOOH species of HO at low temperature, and our analyses of its reaction, together with mutational and crystallographic studies, reveal that protonation of FeOOH by a distal water molecule is critical in promoting the unique self-hydroxylation. The second oxygenation is a rapid, spontaneous autooxidation of the reactive alpha-meso-hydroxyheme in which the HO enzyme does not play a critical role. Further O(2) activation by verdoheme cleaves its porphyrin macrocycle to form biliverdin and free ferrous iron. This third step has been considered to be a major rate-determining step of HO catalysis to regulate the enzyme activity. Our reaction analysis strongly supports the FeOOH verdoheme as the key intermediate of the ring-opening reaction. This mechanism is very similar to that of the first meso-hydroxylation, and the distal water is suggested to enhance the third step as expected from the similarity. The HO mechanistic studies highlight the catalytic importance of the distal hydrogen-bonding network, and this manuscript also involves our attempts to develop HO inhibitors targeting the unique distal structure.

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.

Heme oxygenase‐1 (HO‐1) catalyzes the oxidative degradation of heme. The catalytic mechanism of the HO‐1 reaction has been determined gradually by studies of its crystal structure and HO‐1 mutants. However, the neutralizing epitopes responsible for HO‐1 activity remain elusive. Screening of a phage display library revealed four epitopes that could interact with the polyclonal antibody prepared by immunizing rabbits with the purified HO‐1 protein. Two of these four epitopes are responsible for HO‐1 catalytic activity because their antibodies were able to neutralize HO‐1 activity. The results of the present study shed further light on the molecular character of HO‐1.

Advances in genome sequencing and analysis have afforded a trove of "orphan" bacterial biosynthetic pathways, many of which contain hypothetical proteins. Given the potential for these hypothetical proteins to carry out novel chemistry, orphan pathways serve as a rich reservoir for the discovery of new enzymes responsible for the production of metabolites with both fascinating chemistries and biological functions. We previously identified a rare hybrid nonribosomal peptide synthetase (NRPS)-carbohydrate genomic island in the entomopathogen Photorhabdus luminescens. Heterologous expression of the pathway led to the characterization of oligosaccharides harboring a 1,6-anhydro-β-d- N-acetyl-glucosamine moiety, but these new metabolites lacked modification by the NRPS machinery. Here, through the application of top-down protein mass spectrometry, pathway-targeted molecular networking, stable isotope labeling, and in vitro protein biochemistry, we complete the characterization of this biosynthetic pathway and identify the hybrid product of the pathway, a new "glycoamino acid" metabolite termed photolose. Intriguingly, a hypothetical protein served as a bridge to condense a glycyl unit derived from the NRPS machinery onto the free 1,6-anhydro-β-d- N-acetyl-glucosamine core. We further demonstrate that the gene cluster confers a growth advantage to antimicrobial peptide challenge.

38] Assay of microsomal heme oxygenase in liver and spleen

Herein, we report on our studies on the reaction of organoselenium compounds with triazoles under thermal conditions using simple Rh(ii) catalysts. These reactions do not provide the product of classic rearrangement reactions. Instead two different cascade reactions were uncovered. While allyl selenides react in a cascade of sigmatropic rearrangement and selenium-mediated radical cyclization reaction to give dihydropyrroles, cinnamyl selenides undergo a double rearrangement reaction cascade involving a final aza-Cope reaction to give the product of 1,3-difunctionalization. Theoretical and experimental studies were conducted to provide an understanding of the reaction mechanism of these cascade reactions. The former provide an important insight into fundamental question on the nature of the ylide intermediate in rearrangement reactions and reveal that organoselenium compounds take up multiple roles in rearrangement reactions and mediate a free ylide reaction mechanism.

Heme oxygenase (HO) plays a central role in heme catabolism, which is involved in keeping iron homeostasis, defense for oxidative stress, and signal transduction in mammals. HO catalyzes the site-specific cleavage of heme to produce biliverdin IXα, CO, and iron using reducing equivalents and O2. To address the reaction mechanism of HO, I determined the crystal structures of rat HO-1 in substrate-free form, heme-bound form, O2-analog bound forms, and biliverdiniron chelate bound form. These structures revealed that the narrow distal pocket of HO limits the direction of distal ligand by which the site-specific cleavage is allowed. Moreover, I discuss how HO incorporates heme and dissociates iron and biliverdin as well. I also determined the crystal structure of rat heme-HO-1 complex in CO bound form and succeeded in trapping an intermediate structure during the CO releasing step. Discrimination mechanism between O2 and CO by HO is discussed based on these structures. Finally I refer to a possibility of development of novel inhibitors to HO using imidazole-dioxolane compounds.

Many effector functions of nitrogen monoxide (NO) and carbon monoxide (CO) are mediated through their high-affinity for iron (Fe). In this review, the roles of NO and CO are examined in terms of their effects on the molecular and cellular mechanisms involved in Fe metabolism. Both NO and CO avidly form complexes with a plethora of Fe-containing molecules. The generation of NO and CO is mediated by the nitric oxide synthase and haem oxygenase (HO) families of enzymes respectively. The effects of NO on Fe metabolism have been well characterized, whereas knowledge of the effects of CO remains within its infancy. In terms of the role of NO in Fe metabolism, one of the best characterized interactions includes its effect on the iron regulatory proteins. These molecules are mRNA-binding proteins that control the expression of the transferrin receptor 1 and ferritin, molecules that are involved in Fe uptake and storage respectively. Apart from this, activated macrophages impart their cytotoxic activity by generating NO, which results in marked Fe mobilization from tumour-cell targets. This deprives the cell of the Fe that is required for DNA synthesis and energy production. Considering that HO degrades haem, resulting in the release of CO, Fe(II) and biliverdin, it is suggested that a CO-Fe complex will form. This may account for the rapid Fe mobilization observed from macrophages after haemoglobin catabolism. Intriguingly, overexpression of HO results in cellular Fe mobilization, suggesting that CO has a similar effect to NO on Fe trafficking. Preliminary evidence suggests that, like NO, CO plays important roles in Fe metabolism.

The heme-based oxygen-sensor phosphodiesterase from Escherichia coli (Ec DOS), is composed of an N-terminal heme-bound oxygen sensing domain and a C-terminal catalytic domain. Oxygen (O2) binding to the heme Fe(II) complex in Ec DOS substantially enhances catalysis. Addition of hydrogen sulfide (H2S) to the heme Fe(III) complex in Ec DOS also remarkably stimulates catalysis in part due to the heme Fe(III)-SH and heme Fe(II)-O2 complexes formed by H2S. In this study, we examined the roles of the heme distal amino acids, M95 (the axial ligand of the heme Fe(II) complex) and R97 (the O2 binding site in the heme Fe(II)-O2 complex) of the isolated heme-binding domain of Ec DOS (Ec DOS-PAS) in the binding of H2S under aerobic conditions. Interestingly, R97A and R97I mutant proteins formed an oxygen-incorporated modified heme, verdoheme, following addition of H2S combined with H2O2 generated by the reactions. Time-dependent mass spectroscopic data corroborated the findings. In contrast, H2S did not interact with the heme Fe(III) complex of M95H and R97E mutants. Thus, M95 and/or R97 on the heme distal side in Ec DOS-PAS significantly contribute to the interaction of H2S with the Fe(III) heme complex and also to the modification of the heme Fe(III) complex with reactive oxygen species. Importantly, mutations of the O2 binding site of the heme protein converted its function from oxygen sensor to that of a heme oxygenase. This study establishes the novel role of H2S in modifying the heme iron complex to form verdoheme with the aid of reactive oxygen species.

The heme peroxidase and heme oxygenase enzymes share a common heme prosthetic group but catalyze fundamentally different reactions, the first being H(2)O(2)-dependent oxidation of substrate using an oxidized Compound I intermediate, and the second O(2)-dependent degradation of heme. It has been proposed that these enzymes utilize a common reaction intermediate, a ferric hydroperoxide species, that sits at a crossroads in the mechanism and beyond which there are two mutually exclusive mechanistic pathways. Here, we present evidence to support this proposal in a heme peroxidase. Hence, we describe kinetic data for a variant of ascorbate peroxidase (W41A) which reacts slowly with tert-butyl hydroperoxide and does not form the usual peroxidase Compound I intermediate; instead, structural data show that a product is formed in which the heme has been cleaved at the alpha-meso position, analogous to the heme oxygenase mechanism. We interpret this to mean that the Compound I (peroxidase) pathway is shut down, so that instead the reaction intermediate diverts through the alternative (heme oxygenase) route. A mechanism for formation of the product is proposed and discussed in the light of what is known about the heme oxygenase reaction mechanism.

Despite the essential biological importance of reactions that involve heme, mechanisms of heme reactions in enzymes like nitric oxide synthase (NOS), heme oxygenase (HO), and cytochrome P450s (CYP450s) are still not well-understood. This Perspective on NOS, HO, and CYP450 mechanisms is written from the point of view of the heme chemistry. Steps in the classical heme catalytic cycle are discussed based on the specific environment within each of these enzymes. Elucidation of the mechanisms of NOS inactivation by some substrate analogues provides important mechanistic clues to the NOS catalytic mechanism. On the basis of mechanistic studies of NOS inactivation by amidine analogues of l-arginine and other previous mechanistic results, a new mechanism for NOS-catalyzed l-arginine NG-hydroxylation (the first half of the catalytic reaction) is proposed in this Perspective. The key step in the second half of the NOS catalytic reaction, the internal electron transfer between the substrate and heme, is discussed on the basis of mechanistic results of NOS inactivation by NG-allyl-l-arginine and the structures of the substrate intermediates. Elucidation of the mechanism of NOS inactivation by amidines, which leads to heme degradation, also provides important mechanistic implications for heme oxygenase-catalyzed heme catabolism. Focusing on the meso-hydroxylation step during inactivation of NOS by amidines as well as the HO-catalyzed reaction, the essential nature of the heme-oxygen species responsible for porphyrin meso-hydroxylation is discussed. Finally, on the basis of the proposed heme degradation mechanism during NOS inactivation and the HO-catalyzed reaction, the mechanism for the formation of the monooxygenated heme species in P450-catalyzed reactions is discussed.

DDAP is a tool for predicting the biosynthetic pathways of the products of type I modular polyketide synthase (PKS) with the focus on providing a more accurate prediction of the ordering of proteins and substrates in the pathway. In this study, the module docking domain (DD) affinity prediction performance on a hold-out testing dataset reached 0.88 as measured by the area under the receiver operating characteristic (ROC) curve (AUC); the Mean Reciprocal Ranking (MRR) of pathway prediction reached 0.67. DDAP has advantages compared to previous informatics tools in several aspects: (i) it does not rely on large databases, making it a high efficiency tool, (ii) the predicted DD affinity is represented by a probability (0-1), which is more intuitive than raw scores, (iii) its performance is competitive compared to the current popular rule-based algorithm. DDAP is so far the first machine learning based algorithm for type I PKS DD affinity and pathway prediction. We also established the first database of type I modular PKSs, featuring a comprehensive annotation of available docking domains information in bacterial biosynthetic pathways. The DDAP database is available at https://tylii.github.io/ddap. The prediction algorithm DDAP is freely available on GitHub (https://github.com/tylii/ddap) and released under the MIT license. Supplementary data are available at Bioinformatics online.

Synthesis of Alginate in Bacteria