Cytochrome P450 monooxygenases, one of the most important classes of heme-thiolate proteins, have attracted considerable interest in the biochemical community because of its catalytic versatility, substrate diversity and great number in the superfamily. Although P450s are capable of catalyzing numerous difficult oxidation reactions, the relatively low stability, low turnover rates and the need of electron-donating cofactors have limited their practical biotechnological and pharmaceutical applications as isolated enzymes. The goal of this study is to tailor such heme-thiolate proteins into efficient biocatalysts with high specificity and selectivity by protein engineering and to better understand the structure-function relationship in cytochromes P450. In the effort to engineer P450cam, the prototype member of the P450 superfamily, into an efficient peroxygenase that utilizes hydrogen peroxide via the “peroxide-shunt” pathway, site-directed mutagenesis has been used to elucidate the critical roles of hydrophobic residues in the active site. Various biophysical, biochemical and spectroscopic techniques have been utilized to investigate the wild-type and mutant proteins. Three important P450cam variants were obtained showing distinct structural and functional features. In P450camV247H mutant, which exhibited almost identical spectral properties with the wild-type, it is demonstrated that a single amino acid switch turned the monooxygenase into an efficient preoxidase by increasing the peroxidase activity nearly one thousand folds. In order to tune the distal pocket of P450cam with polar residues, Leu 246 was replaced with a basic residue, lysine, resulting in a mutant with spectral features identical to P420, the inactive species of P450. But this inactive-species-like mutant showed catalytic activities without the facilitation of any cofactors. By substituting Gly 248 with a histidine, a novel Cys-Fe-His ligation set was obtained in P450cam which represented the very rare case of His ligation in heme-thiolate proteins. In addition to serving as a convenient model for hemoprotein structural studies, the G248H mutant also provided evidence about the nature of the axial ligand in cytochrome P420 and other engineered hemoproteins with thiolate ligations. Furthermore, attempts have been made to replace the proximal ligand in sperm whale myoglobin to construct a heme-thiolate protein model by mimicking the protein environment of cytochrome P450cam and chloroperoxidase.

Immunotherapy has revolutionized the treatment of certain cancers by facilitating the antitumor immune response and represents today one of the mainstays of cancer therapy. However, only a subset of patients responds to immunotherapy, which can also lead to serious complications. The tumor microenvironment is composed of multiple and complex cellular and molecular interactions providing to cancer cells not only a supportive framework but promoting also many steps of immunosuppression and tumor progression. To date, the mechanisms that drive the acquisition of these immunosuppressive features are still poorly defined. Tumor-associated macrophages can be highly represented in the tumor microenvironment where they are shaped and become key players in the innate and adaptive immune escape of the tumor cells.Heme oxygenase-1 is the rate-limiting enzyme that catabolizes heme into three major biologically active byproducts which display cytoprotective, antioxidant and immunomodulatory effects. We hypothesized that tumor-associated macrophages might suppress anti-tumor T-cell response through heme oxygenase-1 induction in the tumor microenvironment and macrophage polarization. We showed that heme oxygenase-1 is highly expressed in tumor-associated macrophages. By using a subcutaneous EG7-OVA lymphoma model on genetically engineered mice with a conditional deletion of heme oxygenase-1 in macrophages, our data show that myeloid-restricted heme oxygenase-1 deficiency improves the effect of a therapeutic antitumor immunization by enhancing tumor-infiltrating antitumor CD8+ T-cell proliferation and cytotoxicity and represses tumor growth. Our data suggest a major role of myeloid heme oxygenase-1 in the differentiation and the phenotypic, functional, transcriptional and epigenetic reprograming of tumor-associated macrophages. Myeloid HO-1 inhibition might be considered as a new myeloid HO-1-mediated immune checkpoint blockade. Targeting myeloid compartment could reprogram the tumor microenvironment and synergize with other cancer therapies. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

Nitric oxide reductase (NOR) from Paracoccus denitrificans catalyzes the two electronreduction of NO to N2O (2NO + 2H+ + 2e- → N2O + H2O) as part of the process ofdenitrification, the step-wise reduction of nitrate to dinitogen. The NOR-catalyzedreaction is central in the nitrogen cycle, since in this step the N=N double bond isformed. NOR is a deviant heme copper oxidase, located in the cytoplasmic membrane,containing four redox active cofactors. Like cytochrome c oxidase (CcO), NOR canreduce oxygen to water as a side reaction, but in contrast to CcO it does not contributeto the proton motive force that drives the conversion of ADP to ATP by ATP synthase.The active site in the catalytic subunit NorB consists of a non-heme iron FeB and a hemeb3 that are anti-ferromagnetically coupled. Additionally a low-spin heme b in NorB isinvolved in accepting electrons from heme c of NorC, a membrane anchored cytochromec, which is the second subunit of the purified NorBC heterodimer.We have studied the terminal region of the proton entry channel and possible ligands tothe binuclear active site of NOR using the flow-flash technique and could demonstratethat the putative proton channel residues Glu(E)198 and E267 in NorB are essential forproton uptake. We propose that they define the terminal proton channel region close tothe binuclear site. An alanine variant of the fully conserved amino acid residue E202 ofNOR that, according to the model of NOR (47), is located in the vicinity to the active site,is neither essential for catalytic activity nor integrity of the active site.Furthermore, we were able to demonstrate the [NO] dependency of NOR in the reactionbetween fully reduced protein and NO using the flow-flash technique (21, 24). Thebinding of NO to the fully reduced enzyme is clearly concentration dependent,inconsistent with a previously proposed obligatory binding of NO first to FeB before itligates to heme b3, where it, in the first turnover, is reduced by the electrons from theactive site. Further oxidation involves electron transfer from the low-spin hemes, which isaccelerated at lower [NO]. This acceleration at lower substrate concentration is evenlarger at decreased pH. We could demonstrate that substrate inhibition, observed insteady-state measurements, occurs already on oxidizing the fully reduced enzyme,indicating that NO binds to its inhibitory site before electrons can redistribute to theactive site from the low-spin hemes.

Variable-temperature stopped-flow (VT-SF) electronic spectroscopy (-85 to -50°C) was utilized to study the reactivity properties of a family of synthetic mononuclear and binuclear non-heme iron enzyme active site analogs. This technique was used to investigate the mechanisms of interactions of two diiron complexes, the diferrous [FeII2(H2Hbamb)2(NMI)2] and the mixed valent [FeII,FeIII(H2Hbamb)2]+, with either oxygen-atom donor (OAD) molecules or the mechanistic probe peroxide, 2-methyl-1-phenylprop-2-yl hydroperoxide (MPPH), and substrates containing weak C-H and O-H bonds. Single turnover studies with 9,10-dihydroanthracene (9,10-DHA) and the deuterated analog, d4-9,10-DHA allowed for the determination of kinetic isotope effects (KIE) which show an inverse KIE and evidence of a disproportionation mechanism. Previous investigations showed the rate of catalytic oxidation of cyclohexane to cyclohexanol by [FeII2(H2Hbamb)2(NMI)2] and MPPH decreased over time. Current VT-SF data show evidence of product inhibition by means of a pre-equilibrium process that inhibits the reaction of the oxidant with the [FeII,FeII] complex. Also examined is the ability of the [FeII,FeIII(H2Hbamb)2]+ complex to catalytically oxidize phenols to phenoxyl radicals via a putative [FeIV=O] species. The reactivity properties of substituted phenols that vary in their oxidation potentials and bond dissociation energies (BDE) was investigated by VT-SF electronic spectroscopic studies to gain insight into the mechanism of oxidation by the [FeII,FeIII] complex. Mechanistic studies were also performed utilizing a mononuclear non-heme iron complex [FeII(N2O1)(CH3OH)Cl2], which can bind alpha-keto acids (e.g. alpha-ketoglutarate, benzoylformate) in a bidentate fashion. Reactivity studies utilizing O2 shows coupled decarboxylation of the alpha-keto acid with catalytic oxidation of the methanol solvent formaldehyde (285 turnovers). In non-reactive solvents, the [FeII(N2O1)(alpha-KG)] adduct complex is capable of catalytically oxidizing a variety of substrates such as 9,10-dihydroanthracene, 2,4-di-tert-butyl phenol, cyclohexene, and cyclooctane at 25°C utilizing O2 as the oxidant. Investigations to the binding of alpha-keto acids to the mononuclear iron complex in the absence of O2 by VT-SF as well as binding studies with NO are discussed. Finally, VT-SF studies were performed to probe the reaction of O2 with [FeII(N2O1)(alpha-KG)(CH3OH)] and the proposed mechanism is discussed. The relevance of these data to non-heme iron enzymes like soluble Methane Monooxygenase, Ribonucleotide Reductase, and Taurine Dioxygenase is discussed.

Chicken breast muscle was cooked to an internal temperature of 165oF by four methods: boiling, baking, sautéing and deep-frying. All cooking methods led to a decrease in formation of dialyzable iron, formed by both extraction and digestion in vitro, compared to raw muscle. After cooking most of the dialyzable iron formed results from extraction and the formation of dialyzable iron by digestion is essentially eliminated. Cooking also decreased the levels of cysteine and histidine; these losses may contribute to the loss in dialyzable iron.

No description available.

Yes / Induction of the antioxidant enzyme heme oxygenase-1 (HO-1) affords cellular protection and suppresses proliferation of vascular smooth muscle cells (VSMCs) associated with a variety of pathological cardiovascular conditions including myocardial infarction and vascular injury. However, the underlying mechanisms are not fully understood. Over-expression of Cav3.2 T-type Ca2+ channels in HEK293 cells raised basal [Ca2+]i and increased proliferation as compared with non-transfected cells. Proliferation and [Ca2+]i levels were reduced to levels seen in non-transfected cells either by induction of HO-1 or exposure of cells to the HO-1 product, carbon monoxide (CO) (applied as the CO releasing molecule, CORM-3). In the aortic VSMC line A7r5, proliferation was also inhibited by induction of HO-1 or by exposure of cells to CO, and patch-clamp recordings indicated that CO inhibited T-type (as well as L-type) Ca2+ currents in these cells. Finally, in human saphenous vein smooth muscle cells, proliferation was reduced by T-type channel inhibition or by HO-1 induction or CO exposure. The effects of T-type channel blockade and HO-1 induction were non-additive. Collectively, these data indicate that HO-1 regulates proliferation via CO-mediated inhibition of T-type Ca2+ channels. This signalling pathway provides a novel means by which proliferation of VSMCs (and other cells) may be regulated therapeutically. / This work was supported by the British Heart Foundation.

Evidências clínicas e experimentais mostram que a isquemia e reperfusão intestinal (I/R-intestinal) induz lesão pulmonar aguda (LPA) que, em casos mais graves, pode evoluir para a síndrome do desconforto respiratório agudo (SDRA). A LPA se caracteriza pela liberação de amplo espectro de mediadores inflamatórios, infiltração de neutrófilos e aumento de permeabilidade vascular. Sabe-se que mediadores inflamatórios gerados no local da I/R-intestinal são transportados pelo sistema linfático mesentérico e, ao atingirem o pulmão, contribuem para a LPA. A enzima heme oxigenase 1 (HO-1), exerce importante função na homeostasia celular, devido à sua ação catabólica sobre o grupo heme das hemoproteínas, gerando como subprodutos ferro, biliverdina e monóxido de carbono. Esses subprodutos possuem ação antiinflamatória, antioxidante e antiapoptótica. Todavia, o papel da HO-1 no controle da LPA causada pela I/R-intestinal ainda não está totalmente esclarecido. No presente estudo investigamos a expressão da HO-1 e o efeito de sua indução sobre as repercussões pulmonares decorrentes da I/R-intestinal. Para tanto, ratos machos Wistar (220-250 g) foram submetidos a 45 min de isquemia intestinal pela obstrução da artéria mesentérica superior e a 2 h de reperfusão. O grupo controle consistiu de animais falsamente operados (Sham). Ainda, a indução da HO-1 foi realizada pelo tratamento dos animais com o composto Hemin (10 mg/kg) 48 e 24 h antes da indução da I/R-intestinal. A I/R-intestinal aumentou a atividade pulmonar da mieloperoxidase (MPO) e o extravasamento do corante azul de Evans (AE) no pulmão. Os níveis de IL-1<font face=\"Symbol\">b elevaram no explante pulmonar (24 h) enquanto os de IL-10 foram reduzidos após a I/R-intestinal. Ainda, a I/R-intestinal diminui a expressão pulmonar da SOD-1 e promoveu aumento da expressão da iNOS. Os resultados obtidos revelam que a I/R-intestinal por si só não induziu a expressão gênica da enzima HO-1, porém o tratamento dos animais com Hemin elevou a sua expressão, a qual foi acompanhada pela redução da atividade pulmonar de MPO e do extravasamento do corante AE. Os elevados níveis pulmonares de IL-1<font face=\"Symbol\">b foram reduzidos pelo tratamento dos animais com o Hemin e houve elevação da IL-10 e VEGF no mesmo tecidos. A indução da HO-1 preveniu o aumento dos níveis de IL-1<font face=\"Symbol\">b e IL-10 e promoveu aumento dos níveis de VEGF na linfa dos animais. Com respeito ao sistema antioxidante, nossos dados indicaram que a indução da HO-1, parece estar relacionada com a elevação da expressão de SOD-1, SOD-2 e redução da expressão de iNOS. Concluindo, os dados obtidos permitem sugerir que a indução prévia da expressão de HO-1 controla a magnitude da lesão pulmonar causada pela I/R-intestinal por mecanismos envolvendo o aumento da atividade de parcela do sistema antioxidante e regulação do balanço entre a geração de citocinas antiinflamatórias e pró-inflamatórias no pulmão. / Clinical and experimental evidences have reported that intestinal ischemia and reperfusion (I/R-intestinal) induces acute lung injury (ALI), which in severe cases can progress to acute respiratory distress syndrome (ARDS). The ALI is characterized by the release of a broad spectrum of inflammatory mediators, neutrophil infiltration and increased vascular permeability. It is known that inflammatory mediators generated at the site of I/R-intestinal are transported by the mesenteric lymphatic system and, on reaching the lung, contribute to the ALI. The enzyme heme oxygenase 1 (HO-1) plays an important role in cellular homeostasis, due to its catabolic action on heme group of hemoproteins, forming as by-products such as iron, biliverdin and carbon monoxide. It is known that these by-products have anti-inflammatory, antioxidant and antiapoptotic actions. However, the role of HO-1 in the control of ALI caused by I/R-intestinal is not yet fully understood. In the present study we investigated the expression of HO-1 and the effects of its induction on pulmonary complications resulting from I/R-intestinal. So, male Wistar rats (220-250 g) were subjected to 45 min of intestinal ischemia by occlusion of the superior mesenteric artery and 2 h of reperfusion. The control group consisted of animals falsely operated (Sham). Still, the induction of HO-1 was performed by treating animals with the compound Hemin (10 mg/kg) 48 and 24 h before the induction of I/R-intestinal. The I/R-intestinal increased the pulmonary activity of the myeloperoxidase (MPO) and the extravasation of Evans blue dye (EB) in the lung. Levels of IL-1<font face=\"Symbol\">b increased in lung explant (24 h) while the IL-10 were reduced after I/R-intestinal. Further, the I/R-intestinal reduces pulmonary expression of SOD-1 and promoted the increase of iNOS expression. The results indicate that the I/R-intestinal alone did not induce gene expression of HO-1 enzyme, but the treatment of animals with Hemin increased its expression which was accompanied by reduction of pulmonary activity of MPO and extravasation of the dye EB. The high pulmonary levels of IL-1 were reduced by treatment of animals with Hemin and there was an increase of IL-10 and VEGF in the same tissue. The Induction of HO-1 prevented the increased levels of IL-<font face=\"Symbol\">b 1 and IL-10 and promoted increasing of the VEGF levels in the animals lymph. With respect to the antioxidant system, our data indicate that induction of HO-1, seems to be related to the elevation of expression of SOD-1, SOD-2 and reduction of iNOS expression. In conclusion, our data may suggest that prior induction of HO-1 expression controls the magnitude of lung injury caused by I/R-intestinal by mechanisms involving increased activity of a portion of the antioxidant system and regulation of the balance between generation anti-inflammatory cytokines and pro-inflammatory in the lung.

A malária é uma doença causada pelo parasita do gênero Plasmodium e que foi responsável por cerca de 440.000 mortes em 2015. A síndrome do desconforto respiratório agudo (SDRA) é uma das principais complicações clínicas da malária. O modelo murino DBA/2 reproduz os sinais clínicos da SDRA observados em humanos, quando infectado com o Plasmodium berghei ANKA. Além disso, altos níveis da enzima heme oxigenase 1 (HO-1) foram observados em casos de malária cerebral e em SDRA em humanos. Os nossos dados indicam que os níveis da HO-1 estão aumentados em camundongos que desenvolvem SDRA associada à malária (SDRA-AM). Adicionalmente, a droga indutora de HO-1 (hemina) aumentou a sobrevivência e preveniu a SDRA-AM. Verificou-se também uma redução na permeabilidade pulmonar e nos níveis de VEGF, além de uma melhoria nos parâmetros respiratórios em animais tratados com hemina. Assim sendo, a indução da HO-1 antes do desenvolvimento da SDRA-AM é protetora e assim, a HO-1 pode ser um alvo de novos fármacos, como forma de prevenir o desenvolvimento da SDRA-AM em humanos. / Malaria is a serious disease, caused by the parasite of the genus Plasmodium, which was responsible to 440,000 deaths in 2015. Acute lung injury/ acute respiratory distress syndrome (ALI/ARDS) is one of the main clinical complications in severe malaria. The murine model DBA/2 reproduces the clinical signs of ALI/ARDS observed in humans, when infected with Plasmodium berghei ANKA. Additionally, high levels heme oxygenase 1 (HO-1) were reported in cases of cerebral malaria and in ALI/ARDS in humans. Our data have indicated that the HO-1 levels are increased in mice that develop malaria associated ALI/ARDS (MA-ALI/ARDS). Additionally, a HO-1 inducing drug (hemin) increased the survival rate and prevented mice from developing MA-ALI/ARDS in treated mice. Also, there was a decrease in the lung permeability and in lung VEGF levels, and an amelioration of respiratory parameters. Therefore, the induction of HO-1 before the development of MA-ALI/ARDS is protective, making this enzyme a possible target of new drugs to prevent the development of MA-ALI/ARDS in humans.

O gás composto monóxido de carbono (CO), está envolvido na modulação de diferentes funções orgânicas, tais como a regulação cardiovascular, a temperatura corporal e a nocicepção. A participação do CO nos processos fisiológicos ocorre por meio da atividade da enzima heme-oxigenase (HO), e seu produto CO, o qual por sua vez aumenta a produção de guanosina monofosfato ciclíco intracelular (GMPc). De particular interesse, o locus coeruleus possui elevada expressão da enzima HO-2 sugerindo o envolvimento do gasotransmissor CO na modulação das funções executadas por esta estrutura encefálica. O objetivo deste trabalho foi avaliar o envolvimento da via HO-CO do LC na modulação da ansiedade, avaliada pelo teste de labirinto em cruz elevado e teste claro-escuro; nocicepção aguda, avaliada pelo teste de retirada de cauda e a nocicepção inflamatória, avaliada pelo teste de formalina em ratos. Para atingir estes objetivos, ratos (± 250grs; Wistar) foram anestesiados (ketamina 75 mg/kg e xilasina 10 mg/kg i.m.) e submetidos à cirurgia estereotáxica para implante unilateral de cânulas-guias direcionadas para o LC, e para o ventrículo lateral. Após o período de recuperação, os ratos foram divididos em distintos grupos experimentais para administração intra-LC do ZnDPBG (inibidor inespecífico da enzima HO, nas doses 5,50 ou 200 nmol/0,1 µl) ou seu veículo, Na2CO3 (50 mmol/0,1 µl); do Heme-lisinato (150, 300 ou 600 nmol/0,1 µl) ou seu veículo, L-lisina (14,2 µmol/0,1 µl); do ODQ i.c.v. (inibidor específico da enzima guanilase ciclase solúvel, 1,3 nmol/1,0 µl) ou seu veículo (DMSO 1%, 1,0 µl) e após 15 min o Heme-lisinato (600 nmol/0,1 µl) ou seu veículo (L-lisina, 14,2 µmol/0,1 µl), intra-LC. Após o tempo de 15 min, os ratos foram avaliados no teste de LCE ou no TCE por 5 minutos, no teste de retirada de cauda por 120 minutos e no teste de formalina intra-podal por 45 minutos. Os resultados obtidos mostram que o aumento da produção do neuromodulador gasoso CO no LC, pela ativação da via HO-CO-GMPc com Heme-lisinato, promove efeito ansiolítico avaliado no teste do LCE e no TCE, evidenciado pelo aumento do tempo de permanência e pelo aumento do número de entradas nos braços abertos do LCE, e pelo aumento tempo de permanência no compartimento claro do TCE. Este efeito ansiolítico é dependente da atividade de GMPc intracelular, desde que o tratamento i.c.v. com inibidor específico da enzima GCs bloqueou os efeitos do Heme-lisinato no LCE e no TCE. Ainda, a ativação da via HO-CO-GMPc por meio da administração intra-LC do Heme-lisinato promoveu efeito antinociceptivo frente estímulo térmico agudo (teste de retirada de cauda em ratos), sendo este efeito dependente da atividade do GMPc, desde que o pré-tratamento com o inibidor da enzima guanilase ciclase solúvel, ODQ, bloqueou o aumento do IARC. O bloqueio da via HO-CO promove efeito hipernociceptivo em modelo de dor inflamatória, desde que o tratamento intra-LC com inibidor inespecífico da HO, ZnDPBG aumenta o número de sacudidas no teste de formalina intra-podal. Assim, este estudo é pioneiro em demonstrar que o neuromodulador CO do LC modula a ansiedade e a nocicepção aguda térmica e inflamatória. / The gas composed carbon monoxide (CO) is involved in the modulation of various physiological functions such as cardiovascular regulation, nociception and body temperature. CO participation in physiological processes occurs through the activity of the enzyme heme oxygenase (HO), and its product CO, which in turn increases the production of intracellular cyclic guanosine monophosphate (cGMP). In particular interest, the locus coeruleus (LC) has a high HO-2 enzyme expression suggesting the involvement of CO in the modulation of the functions performed by this brain structure. The aim of this study was to evaluate the involvement of HO-CO pathway of LC in modulating anxiety, assessed by elevated plus maze test and light-dark box test. Additionally, acute nociception, as assessed by the tail flick test and inflammatory nociception, as assessed by formalin test in rats were analyzed after HO-CO pathway activation. Rats (±250 grs; Wistar) were anesthetized (ketamine 75 mg/kg and xylazine 10 mg/kg im) and underwent stereotactic surgery for cannulas guides unilateral implantation directed to the LC, and to the lateral ventricle. After the recovery period, rats were divided into distinct experimental groups for intra-LC ZnDPBG (nonspecific enzyme inhibitor HO doses 5, 50 or 200 nmol/0.l µl) or its vehicle, Na2CO3 (50 mmol/0.l µl); Heme-lysinate (150, 300 or 600 nmol/0.l µl) or its vehicle, L-lysine (14.2 nmol/0.1 µl), the ODQ i.c.v. (specific inhibitor of the enzyme guanilase soluble cyclase, 1.3 nmol/1.0 µl) or its vehicle (1% DMSO, 1.0 µl) and after 15 min the Heme-lysinate (600 nmol/0.1 µl), or its vehicle (L-lysine, 14.2 mmol/0.1 µl), intra-LC. After time 15 min, rats were evaluated in the EPM test or LDB for 5 minutes and in the tail flick test for 120 minutes and in the formalin test for 45 minutes. The results show that CO increased production in LC, by HO-CO-cGMP pathway activation, promotes anxiolytic effect evaluated in the EPM test and LDB. The anxiolytic effect is dependent on the activity of intracellular cGMP, since treatment i.c.v. with enzyme sGC inhibitor blocked the effects of Heme-lysinate. Moreover, the activation of the HO-CO-cGMP pathway into the LC promoted antinociceptive effect in the tail flick test, this effect being dependent on the activity of cGMP, since pre-treatment with the guanilase cyclase soluble inhibitor, ODQ, blocked the increase in analgesic index. Furthermore, the block of the HO-CO pathway intra-LC promoted hypernociception in a model of inflammatory pain, since treatment with nonspecific inhibitor HO, ZnDPBG, increases the nociceptive behavior in the formalin test. Thus, this study is the first to demonstrate that the CO neuromodulator into LC modulates anxiety and acute thermal and inflammatory nociception.