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Heme Iron (heme + iron)
Selected AbstractsSynthesis and Bioaccessibility of Fe-Pheophytin Derivatives from Crude Spinach ExtractJOURNAL OF FOOD SCIENCE, Issue 5 2008R.E. Nelson ABSTRACT:, Heme iron is recognized as a highly bioavailable source of iron suitable for treatment of iron deficiency anemia. However, the animal origin of purified heme limits its broad applicability due to religious, personal, and food safety issues. Development of chlorophyll-derived heme mimetics offers opportunities to expand current iron fortification strategies. The objective of this study was the synthesis of Fe-pheophytin (FePhe) derivatives from natural chlorophyll and subsequent evaluation of their digestive behavior and bioaccessibility in vitro. FePhe a and a, were synthesized from crude spinach extracts by treatment with 1.3 M iron(II)chloride and 0.25 M Na-acetate dissolved in glacial acetic acid at 80 °C for 30 min. FePhe-rich extracts (approximately 1 mM) were formulated into corn starch based test meals (7.5% lipid) and subjected to a 2-step in vitro digestion designed to simulate in vivo gastric and small intestinal conditions. Recovery of FePhe following digestion and transfer of FePhe and pheophytins (Phe) from test meal matrix to mixed micelles was assessed by RP C18-HPLC to determine the digestive stability and micellarization efficiency (bioaccessibility). FePhe a and a, derivatives were moderately stable to digestive conditions with recoveries of 52.3% and 58.7%, respectively. Residual Phe a was stable to digestion. Micellarization efficiency of FePhe a (4%) and a, (3.4%) was significantly (P < 0.05) lower than Phe a (25.8%) from test meals. While digestive stability and micellarization efficiency are limiting, the presence of lipophilic FePhe derivatives in mixed micelles suggests that these compounds would be available for subsequent absorption in the intestinal tract. [source] Total Heme and Non-heme Iron in Raw and Cooked MeatsJOURNAL OF FOOD SCIENCE, Issue 5 2002G. Lombardi-Boccia ABSTRACT: This study provides data on the total heme and non-heme iron contents in poultry (chicken, turkey), beef, veal, lamb, horse, ostrich, rabbit, and pork meat cuts. The effect of cooking on heme iron content was also studied. Total iron and heme iron contents markedly differed between muscles in poultry. Heme iron in red meats ranged from 72 to 87%. Heme iron in rabbit and pork was 56 and 62% of total iron. Heating decreased heme iron, the severity of the losses depended on cooking methods: in poultry, losses ranged from 22 to 43%; less severe impact was detected in pan-cooked meat, where the losses ranged from 1 to 24%. [source] Proximal ligand motions in H93G myoglobinFEBS JOURNAL, Issue 19 2002Stefan Franzen Resonance Raman spectroscopy has been used to observe changes in the iron,ligand stretching frequency in photoproduct spectra of the proximal cavity mutant of myoglobin H93G. The measurements compare the deoxy ferrous state of the heme iron in H93G(L), where L is an exogenous imidazole ligand bound in the proximal cavity, to the photolyzed intermediate of H93G(L)*CO at 8 ns. There are significant differences in the frequencies of the iron,ligand axial out-of-plane mode ,(Fe,L) in the photoproduct spectra depending on the nature of L for a series of methyl-substituted imidazoles. Further comparison was made with the proximal cavity mutant of myoglobin in the absence of exogenous ligand (H93G) and the photoproduct of the carbonmonoxy adduct of H93G (H93G-*CO). For this case, it has been shown that H2O is the axial (fifth) ligand to the heme iron in the deoxy form of H93G. The photoproduct of H93G-*CO is consistent with a transiently bound ligand proposed to be a histidine. The data presented here further substantiate the conclusion that a conformationally driven ligand switch exists in photolyzed H93G-*CO. The results suggest that ligand conformational changes in response to dynamic motions of the globin on the nanosecond and longer time scales are a general feature of the H93G proximal cavity mutant. [source] Covalently crosslinked complexes of bovine adrenodoxin with adrenodoxin reductase and cytochrome P450sccFEBS JOURNAL, Issue 6 2001Edman degradation of complexes of the steroidogenic hydroxylase system, Mass spectrometry NADPH-dependent adrenodoxin reductase, adrenodoxin and several diverse cytochromes P450 constitute the mitochondrial steroid hydroxylase system of vertebrates. During the reaction cycle, adrenodoxin transfers electrons from the FAD of adrenodoxin reductase to the heme iron of the catalytically active cytochrome P450 (P450scc). A shuttle model for adrenodoxin or an organized cluster model of all three components has been discussed to explain electron transfer from adrenodoxin reductase to P450. Here, we characterize new covalent, zero-length crosslinks mediated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide between bovine adrenodoxin and adrenodoxin reductase, and between adrenodoxin and P450scc, respectively, which allow to discriminate between the electron transfer models. Using Edman degradation, mass spectrometry and X-ray crystallography a crosslink between adrenodoxin reductase Lys27 and adrenodoxin Asp39 was detected, establishing a secondary polar interaction site between both molecules. No crosslink exists in the primary polar interaction site around the acidic residues Asp76 to Asp79 of adrenodoxin. However, in a covalent complex of adrenodoxin and P450scc, adrenodoxin Asp79 is involved in a crosslink to Lys403 of P450scc. No steroidogenic hydroxylase activity could be detected in an adrenodoxin ,P450scc complex/adrenodoxin reductase test system. Because the acidic residues Asp76 and Asp79 belong to the binding site of adrenodoxin to adrenodoxin reductase, as well as to the P450scc, the covalent bond within the adrenodoxin,P450scc complex prevents electron transfer by a putative shuttle mechanism. Thus, chemical crosslinking provides evidence favoring the shuttle model over the cluster model for the steroid hydroxylase system. [source] Searching for Neuroglobin's role in the brainIUBMB LIFE, Issue 8-9 2007Karin Nienhaus Abstract Neuroglobin is a small globin that plays an important role in the protection of brain neurons from ischemic and hypoxic injuries. The molecular mechanisms by which Ngb performs its physiological function are still under debate. Suggestions include oxygen storage and delivery, scavenging of NO and/or reactive oxygen species, oxygen sensing and signal transduction. In recent years, the molecular structures of Ngb with carbon monoxide bound to the heme iron and without an exogenous ligand have been solved, and interesting structural changes have been noticed upon ligand binding. Moreover, equilibrium and kinetic properties of the reactions with ligands have been examined in great detail. Here we summarize the molecular properties of Ngb and discuss them in relation to the potential physiological functions. [source] Differential mechanisms for the inhibition of human cytochrome P450 1A2 by apigenin and genisteinJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 4 2010Hideaki Shimada Abstract The inhibitory effects of flavonoids on the human cytochrome P450 1A2 (CYP1A2) were examined. Among flavonoids tested, galangin, kaempferol, chrysin, and apigenin were potent inhibitors. Although apigenin belonging to flavones and genistein belonging to isoflavones are similar in the chemical structures, the inhibitory potencies for CYP1A2 were distinguished markedly between these two flavonoids. In computer-docking simulation, apigenin interacted with the same mode of cocrystallized ,-naphthoflavone in the active site of CYP1A2, and then the B ring of apigenin was placed close to the heme iron of the enzyme with a single orientation. In contrast, the docked genistein conformation showed two different binding modes, and the A ring of genistein was oriented to the heme iron of CYP1A2. Furthermore, the binding free energy of apigenin was lower than that of genistein. These results demonstrate a possible mechanism that causes the differential inhibitory potencies of apigenin and genistein for CYP1A2. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:230,234, 2010; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.20328 [source] Haemopexin affects iron distribution and ferritin expression in mouse brainJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 10 2009Noemi Morello Abstract Haemopexin (Hx) is an acute phase plasma glycoprotein, mainly produced by the liver and released into plasma where it binds heme with high affinity and delivers it to the liver. This system provides protection against free heme-mediated oxidative stress, limits access by pathogens to heme and contributes to iron homeostasis by recycling heme iron. Hx protein has been found in the sciatic nerve, skeletal muscle, retina, brain and cerebrospinal fluid (CSF). Recently, a comparative proteomic analysis has shown an increase of Hx in CSF from patients with Alzheimer's disease, thus suggesting its involvement in heme detoxification in brain. Here, we report that Hx is synthesised in brain by the ventricular ependymal cells. To verify whether Hx is involved in heme scavenging in brain, and consequently, in the control of iron level, iron deposits and ferritin expression were analysed in cerebral regions known for iron accumulation. We show a twofold increase in the number of iron-loaded oligodendrocytes in the basal ganglia and thalamus of Hx-null mice compared to wild-type controls. Interestingly, there was no increase in H- and L-ferritin expression in these regions. This condition is common to several human neurological disorders such as Alzheimer's disease and Parkinson's disease in which iron loading is not associated with an adequate increase in ferritin expression. However, a strong reduction in the number of ferritin-positive cells was observed in the cerebral cortex of Hx-null animals. Consistent with increased iron deposits and inadequate ferritin expression, malondialdehyde level and Cu,Zn superoxide dismutase-1 expression were higher in the brain of Hx-null mice than in that of wild-type controls. These data demonstrate that Hx plays an important role in controlling iron distribution within brain, thus suggesting its involvement in iron-related neurodegenerative diseases. [source] Total Heme and Non-heme Iron in Raw and Cooked MeatsJOURNAL OF FOOD SCIENCE, Issue 5 2002G. Lombardi-Boccia ABSTRACT: This study provides data on the total heme and non-heme iron contents in poultry (chicken, turkey), beef, veal, lamb, horse, ostrich, rabbit, and pork meat cuts. The effect of cooking on heme iron content was also studied. Total iron and heme iron contents markedly differed between muscles in poultry. Heme iron in red meats ranged from 72 to 87%. Heme iron in rabbit and pork was 56 and 62% of total iron. Heating decreased heme iron, the severity of the losses depended on cooking methods: in poultry, losses ranged from 22 to 43%; less severe impact was detected in pan-cooked meat, where the losses ranged from 1 to 24%. [source] Ferroportin q248h, Dietary Iron, and Serum Ferritin in Community African-Americans With Low to High Alcohol ConsumptionALCOHOLISM, Issue 11 2008Victor R. Gordeuk Background:, Alcohol consumption is associated with increased iron stores. In sub-Saharan Africa, high dietary ionic iron and the ferroportin Q248H allele have also been implicated in iron accumulation. We examined the associations of ferroportin Q248H, alcohol and dietary iron with serum ferritin, aspartate aminotransaminase (AST) and alanine aminotransaminase (ALT) concentrations in African-Americans. Methods:, Inner-city African-Americans (103 men, 40 women) were recruited from the community according to reported ingestion of >4 alcoholic drinks/d or <2/wk. Typical daily heme iron, nonheme iron and alcohol were estimated using University of Hawaii's multiethnic dietary questionnaire. Based on dietary questionnaire estimates we established categories of < versus ,56 g alcohol/d, equivalent to 4 alcoholic drinks/d assuming 14 g alcohol per drink. Results:, Among 143 participants, 77% drank <56 g alcohol/d and 23%,56 g/d as estimated by the questionnaire. The prevalence of ferroportin Q248H was 23.3% with alcohol >56 g/d versus 7.5% with lower amounts (p = 0.014). Among subjects with no history of HIV disease, serum ferritin concentration had positive relationships with male gender (p = 0.041), alcohol consumption (p = 0.021) and ALT concentration (p = 0.0001) but not with dietary iron intake or ferroportin Q248H. Serum AST and ALT concentrations had significant positive associations with male gender and hepatitis C seropositivity but not with alcohol or dietary iron intake or ferroportin Q248H. Conclusions:, Our findings suggest a higher prevalence of ferroportin Q248H with greater alcohol consumption, and this higher prevalence raises the possibility that the allele might ameliorate the toxicity of alcohol. Our results suggest that alcohol but not dietary iron contributes to higher body iron stores in African-Americans. Studies with larger numbers of participants are needed to further clarify the relationship of ferroportin Q248H with the toxicity of alcohol consumption. [source] Cryoradiolytic reduction of crystalline heme proteins: analysis by UV-Vis spectroscopy and X-ray crystallographyJOURNAL OF SYNCHROTRON RADIATION, Issue 1 2007Thorsten Beitlich The X-ray crystallographic analysis of redox-active systems may be complicated by photoreduction. Although radiolytic reduction by the probing X-ray beam may be exploited to generate otherwise short-lived reaction intermediates of metalloproteins, it is generally an undesired feature. Here, the X-ray-induced reduction of the three heme proteins myoglobin, cytochrome P450cam and chloroperoxidase has been followed by on-line UV-Vis absorption spectroscopy. All three systems showed a very rapid reduction of the heme iron. In chloroperoxidase the change of the ionization state from ferric to ferrous heme is associated with a movement of the heme-coordinating water molecule. The influence of the energy of the incident X-ray photons and of the presence of scavengers on the apparent reduction rate of ferric myoglobin crystals was analyzed. [source] Real time monitoring of drug metabolic enzyme response inside human hepatoma GS-3A4-HepG2 cells by means of electrochemical impedance measurementPOLYMERS FOR ADVANCED TECHNOLOGIES, Issue 5 2004Masaaki Kobayashi Abstract Cytochrome P-450s (CYPs) are important biopolymers for the maintenance of cellular function. If metabolic activity of the CYP in the cells can be estimated, so can the function of metabolism, which is closer to the organism. In this research, the method of measuring the drug metabolic activity inside the cell by making use of an electrochemical technique was examined. Human hepatoma GS-3A4-HepG2 cells of which the cytochrome P-4503A4 (CYP3A4) drug metabolic activity is found to be the same as that of primary hepatocytes were used in the experiment. The GS-3A4-HepG2 cells were cultured on an indium-tin oxide (ITO) electrode until they became confluent. Substrate testosterone and inhibitor ketoconazole of CYP3A4 were exposed to cells cultured on an ITO electrode, and the reaction was observed by noting the electrochemical impedance measurement. Impedance was decomposed into the resistance component and the reactance component, and each was examined in detail. As a result, according to testosterone concentration change, there was a remarkable time change in the reactance component. A similar impedance measurement was done by using human hepatoma HepG2 cells in which the drug metabolic activity had extremely decreased. Nevertheless, no time change in the reactance component that was noticed in GS-3A4-HepG2 cells was observed. Next, the amount of metabolite in the solution after impedance measurement was measured by means of liquid chromatography-tandem mass spectroscopy (LC-MS/MS). In the experiment with GS-3A4-HepG2 cells, a testosterone concentration-dependent correlation was observed between the reactance component change and the amount of metabolite. But, in the impedance measurement by ketoconazole, the change in reactance components was not observed in either the GS-3A4-HepG2 cells or the HepG2 cells. Ketoconazole and the heme iron in CYP3A4 effect the coordination bond, but ketoconazole was not metabolized by CYP3A4. It was confirmed that the time change in the reactance component which was caused by the testosterone was detected neither in the cells that take up the substrate, nor in the coordination bond between the CYP enzyme and the drug. Therefore, the time change in the remarkable reactance component observed by this electrochemical impedance measurement is dependent on drug metabolic activity. An electrochemical drug metabolic activity measuring method with the human hepatoma GS-3A4-HepG2 cells was able to be established. Copyright © 2004 John Wiley & Sons, Ltd. [source] Cleavage of the iron-methionine bond in c-type cytochromes: Crystal structure of oxidized and reduced cytochrome c2 from Rhodopseudomonas palustris and its ammonia complexPROTEIN SCIENCE, Issue 1 2002Silvano Geremia Abstract The three-dimensional structures of the native cytochrome c2 from Rhodopseudomonas palustris and of its ammonia complex have been obtained at pH 4.4 and pH 8.5, respectively. The structure of the native form has been refined in the oxidized state at 1.70 Å and in the reduced state at 1.95 Å resolution. These are the first high-resolution crystal structures in both oxidation states of a cytochrome c2 with relatively high redox potential (+350 mV). The differences between the two oxidation states of the native form, including the position of internal water molecules, are small. The unusual six-residue insertion Gly82-Ala87, which precedes the heme binding Met93, forms an isolated 310 -helix secondary structural element not previously observed in other c-type cytochromes. Furthermore, this cytochrome shows an external methionine residue involved in a strained folding near the exposed edge of the heme. The structural comparison of the present cytochrome c2 with other c-type cytochromes has revealed that the presence of such a residue, with torsion angles , and , of approximately ,140 and ,130°, respectively, is a typical feature of this family of proteins. The refined crystal structure of the ammonia complex, obtained at 1.15 Å resolution, shows that the sulphur atom of the Met93 axial ligand does not coordinate the heme iron atom, but is replaced by an exogenous ammonia molecule. This is the only example so far reported of an X-ray structure with the heme iron coordinated by an ammonia molecule. The detachment of Met93 is accompanied by a very localized change in backbone conformation, involving mainly the residues Lys92, Met93, and Thr94. Previous studies under typical denaturing conditions, including high-pH values and the presence of exogenous ligands, have shown that the detachment of the Met axial ligand is a basic step in the folding/unfolding process of c-type cytochromes. The ammonia adduct represents a structural model for this important step of the unfolding pathway. Factors proposed to be important for the methionine dissociation are the strength of the H-bond between the Met93 and Tyr66 residues that stabilizes the native form, and the presence in this bacterial cytochrome c2 of the rare six-residue insertion in the helix 310 conformation that increases Met loop flexibility. [source] X-ray structure of the metcyano form of dehaloperoxidase from Amphitrite ornata: evidence for photoreductive dissociation of the iron,cyanide bondACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2010V. S. De Serrano X-ray crystal structures of the metcyano form of dehaloperoxidase-hemoglobin (DHP A) from Amphitrite ornata (DHPCN) and the C73S mutant of DHP A (C73SCN) were determined using synchrotron radiation in order to further investigate the geometry of diatomic ligands coordinated to the heme iron. The DHPCN structure was also determined using a rotating-anode source. The structures show evidence of photoreduction of the iron accompanied by dissociation of bound cyanide ion (CN,) that depend on the intensity of the X-ray radiation and the exposure time. The electron density is consistent with diatomic molecules located in two sites in the distal pocket of DHPCN. However, the identities of the diatomic ligands at these two sites are not uniquely determined by the electron-density map. Consequently, density functional theory calculations were conducted in order to determine whether the bond lengths, angles and dissociation energies are consistent with bound CN, or O2 in the iron-bound site. In addition, molecular-dynamics simulations were carried out in order to determine whether the dynamics are consistent with trapped CN, or O2 in the second site of the distal pocket. Based on these calculations and comparison with a previously determined X-ray crystal structure of the C73S,O2 form of DHP [de Serrano et al. (2007), Acta Cryst. D63, 1094,1101], it is concluded that CN, is gradually replaced by O2 as crystalline DHP is photoreduced at 100,K. The ease of photoreduction of DHP A is consistent with the reduction potential, but suggests an alternative activation mechanism for DHP A compared with other peroxidases, which typically have reduction potentials that are 0.5,V more negative. The lability of CN, at 100,K suggests that the distal pocket of DHP A has greater flexibility than most other hemoglobins. [source] Distal histidine conformational flexibility in dehaloperoxidase from Amphitrite ornataACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2009Zuxu Chen The enzyme dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is a heme protein which has a globin fold but can function as both a hemoglobin and a peroxidase. As a peroxidase, DHP is capable of converting 2,4,6-trihalophenols to the corresponding 2,6-dihaloquinones in the presence of hydrogen peroxide. As a hemoglobin, DHP cycles between the oxy and deoxy states as it reversibly binds oxygen for storage. Here, it is reported that the distal histidine, His55, exhibits conformational flexibility in the deoxy form and is consequently observed in two solvent-exposed conformations more than 9.5,Å away from the heme. These conformations are analogous to the open conformation of sperm whale myoglobin. The heme iron in deoxy ferrous DHP is five-coordinate and has an out-of-plane displacement of 0.25,Å from the heme plane. The observation of five-coordinate heme iron with His55 in a remote solvent-exposed conformation is consistent with the hypothesis that His55 interacts with heme iron ligands through hydrogen bonding in the closed conformation. Since His55 is also displaced by the binding of 4-iodophenol in an internal pocket, these results provide new insight into the correlation between heme iron ligation, molecular binding in the distal pocket and the conformation of the distal histidine in DHP. [source] Toward understanding the inactivation mechanism of monooxygenase P450 BM-3 by organic cosolvents: A molecular dynamics simulation studyBIOPOLYMERS, Issue 5 2006Danilo Roccatano Abstract Cytochrome P450 BM-3 from Bacillus megaterium is an extensively studied enzyme for industrial applications. A major focus of current protein engineering research is directed to improving the catalytic performance of P450 BM-3 toward nonnatural substrates of industrial importance in the presence of organic solvents or cosolvents. For the latter reason, it is important to study the effect of organic cosolvent molecules on the structure and dynamics of the enzyme, in particular, the effect of cosolvent molecules on the active site's structure and dynamics. In this paper, we have studied, using molecular dynamics (MD) simulations, the F87A mutant of P450 BM-3 in the presence of DMSO as cosolvent, to understand the role of the F87A substitution for its catalytic activity. This mutant exhibits an altered regioselectivity and substrate specificity compared with wild-type; however, it has lower tolerance toward DMSO. The simulation results offer an explanation for the DMSO sensitivity of the F87A mutant. Our simulation results show that the F87 side chain prevents the disturbance of the water molecule bound to the heme iron by DMSO molecules. The absence of the phenyl ring in F87A mutant promotes interactions of the DMSO molecule with the heme iron resulting in water displacement by DMSO at the catalytic heme center. © 2006 Wiley Periodicals, Inc. Biopolymers 83: 467,476, 2006 This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] H93G myoglobin cavity mutant as versatile template for modeling heme proteins: Magnetic circular dichroism studies of thiolate- and imidazole-ligated complexesBIOPOLYMERS, Issue 4-5 2002John H. Dawson Abstract Recent ligand binding and spectroscopic investigations of the myoglobin H93G cavity mutant are reviewed, revealing it to be a versatile template for the preparation of model heme complexes of defined structure. The H93G myoglobin cavity mutant is shown to be capable of forming mixed ligand adducts because of the difference in accessibility of the two sides of the ferric heme iron. With imidazole bound in the proximal cavity, H93G myoglobin also forms reasonably stable oxyferrous and oxoferryl derivatives, thereby providing a potential system to use for the study of such complexes with proximal ligands other than imidazole. In addition, thiolate-ligated ferric H93G derivatives are described that serve as spectroscopic models for the high-spin ferric state of cytochrome P450. All of the complexes described are characterized with magnetic circular dichroism spectroscopy, and they are compared to the appropriate derivatives of native myoglobin and P450. © 2002 Wiley Periodicals, Inc. Biopolymers (Biospectroscopy) 67: 200,206, 2002 [source] Time-resolved resonance Raman study on ultrafast structural relaxation and vibrational cooling of photodissociated carbonmonoxy myoglobinBIOPOLYMERS, Issue 4-5 2002Teizo Kitagawa Abstract A localized small structural change is converted to a higher order conformational change of protein and extends to a mesoscopic scale to induce a physiological function. To understand such features of protein, ultrafast dynamics of myoglobin (Mb) following photolysis of carbon monoxide were investigated. Recent results are summarized here with a stress on structural and vibrational energy relaxation. The core expansion of heme takes place within 2 ps but the out of plane displacement of the heme iron and the accompanying protein conformational change occur in 10 and 100 s of the picosecond regimes, respectively. Unexpectedly, it was found from UV resonance Raman spectra that Trp7 in the N-terminal region and Tyr151 in the C-terminal region undergo appreciable structural changes upon ligand binding,dissociation while Tyr104, Tyr146, and Trp14 do not. Because of the communication between the movements of these surface residues and the heme iron, the rate of spectral change of the iron-histidine (Fe- His) stretching band after CO photodissociation is influenced by the viscosity of solvent. Temporal changes of the anti-Stokes Raman intensity demonstrated immediate generation of vibrationally excited heme upon photodissociation and its decay with a time constant of 1,2 ps. © 2002 Wiley Periodicals, Inc. Biopolymers (Biospectroscopy) 67: 207,213, 2002 [source] A Macrophage Cell Model for Selective Metalloproteinase Inhibitor DesignCHEMBIOCHEM, Issue 13 2008Faith E. Jacobsen Abstract The desire to inhibit zinc-dependent matrix metalloproteinases (MMPs) has, over the course of the last 30 years, led to the development of a plethora of MMP inhibitors that bind directly to the active-site metal. With one exception, all of these drugs have failed in clinical trials, due to many factors, including an apparent lack of specificity for MMPs. To address the question of whether these inhibitors are selective for MMPs in a biological setting, a cell-based screening method is presented to compare the relative activities of zinc, heme iron, and non-heme iron enzymes in the presence of these compounds using the RAW264.7 macrophage cell line. We screened nine different zinc-binding groups (ZBGs), four established MMP inhibitors (MMPis), and two novel MMP inhibitors developed in our laboratory to determine their selectivities against five different metalloenzymes. Using this model, we identified two nitrogen donor compounds,2,2,-dipyridylamine (DPA) and triazacyclononane (TACN),as the most selective ZBGs for zinc metalloenzyme inhibitor development. We also demonstrated that the model could predict known nonspecific interactions of some of the most commonly used MMPis, and could also give cross-reactivity information for newly developed MMPis. This work demonstrates the utility of cell-based assays in both the design and the screening of novel metalloenzyme inhibitors. [source] Structural Identification of Spectroscopic Substates in NeuroglobinCHEMPHYSCHEM, Issue 1 2010Karin Nienhaus Dr. Abstract The structural origins of infrared absorptions of photodissociated CO in murine neuroglobin (Ngb) are determined by combining Fourier transform infrared (FTIR) spectroscopy and molecular dynamics (MD) simulations. Such an approach allows to identify and characterize both the different conformations of the Ngb active site and the transient ligand docking sites. To capture the influence of the protein environment on the spectroscopy and dynamics, experiments and simulations are carried out for the wild type protein and its F28L and F28W mutants. It is found that a voluminous side chain at position 28 divides site B into two subsites, B' and B". At low temperatures, CO in wt Ngb only migrates to site B' from where it can rebind, and B" is not populated. The spectra of CO in site B' for wt Ngb from simulations and experiments are very similar in spectral shift and shape. They both show doublets, red-shifted with respect to gas-phase CO and split by,8 cm,1. The FTIR spectra of the F28L mutant show additional bands which are also found in the simulations and can be attributed to CO located in substate B". The different bands are mainly related to different orientations of the His64 side chain with respect to the CO ligand. Large red-shifts arise from strong interactions between the HistidineNH and the CO oxygen. After dissociation from the heme iron, the CO ligand visits multiple docking sites. The locations of the primary docking site B and a secondary site C, which corresponds to the Mb Xe4 cavity, could be identified unambiguously. Finally, by comparing experiment and simulations it is also possible to identify protonation of its , position (His,64 NgbCO) as the preferred heme-bound conformation in the wild type protein with a signal at 1935 cm,1. [source] Study on the Gas Phase Stability of Heme-binding Pocket in Cytochrome Tb5 and Its Mutants by Electrospray Mass SpectrometryCHINESE JOURNAL OF CHEMISTRY, Issue 12 2002Chong-Tian Yu Abstract To elucidate the effect of various amino add residues on the heme-binding pocket in cytochrome Tb5, several residues were chosen for replacement by means of site-directed mutagenesis. Comparison of the mass spectrum between the F35Y mutant and the wild type shows that the relative abundance of holoprotein ion of F35Y is lower than that of the wild type in gas phase. It is concluded that mutation from Phe35 residue to tyrosine decreases the hydrophobic character of cytochrome Tb5 heme pocket, which decreases the stability of heme-binding pocket. ESI-MS spectra of the mutants V61E, V61K, V61H and V61Y show various contribution of amino acid to the stability of heme-binding pocket. The small and non-polar residue Val61 was replaced with large or polar residues, resulting in enhancing the trend of heme leaving from the pocket. In addition, comparison of the mass relative abundance of holo-proteins among all the Vakil-mutants, shows mat their stability in gas phase appropriately submit the following order: wild type > V61H > V61E > V61K , V61Y. The extra great stability of quadruple sites mutant E44/48/56A/D60A shows that reduction of electrostatic or hydrogen bond interactions among the residues locating in the outside region of the heme edge remarkably affect the stability of heme. The results of analyzing the oxidation states of heme iron in Tb5 and its mutants by insource-CAD experiment suggest that the charge states of heme iron Maintain inflexible in mutation process. [source] | ||||||||||||||||||||||||||||