Peroxynitrite-mediated oxidation of ferrous carbonylated myoglobin is limited by carbon monoxide dissociation

Peroxynitrite-mediated oxidation of ferrous nitrosylated myoglobin (Mb(II)-NO) involves the transient ferric nitrosylated species (Mb(III)-NO), followed by NO dissociation and formation of ferric myoglobin (Mb(III)). In contrast, peroxynitrite-mediated oxidation of ferrous oxygenated myoglobin (Mb(II)-O₂) involves the transient ferrous deoxygenated and ferryl derivatives (Mb(II) and Mb(IV)O, respectively), followed by Mb(III) formation. Here, kinetics of peroxynitrite-mediated oxidation of ferrous carbonylated horse heart myoglobin (Mb(II)-CO) is reported. Values of the first-order rate constant for peroxynitrite-mediated oxidation of Mb(II)-CO (i.e., for Mb(III) formation) and of the first-order rate constant for CO dissociation from Mb(II)-CO (i.e., for Mb(II) formation) are h = (1.2 ± 0.2) × 10⁻² s⁻¹ and k_off(CO) = (1.4 ± 0.2) × 10⁻² s⁻¹, respectively, at pH 7.2 and 20.0 °C. The coincidence of values of h and k_off(CO) indicates that CO dissociation represents the rate limiting step of peroxynitrite-mediated oxidation of Mb(II)-CO.     

Reductive dechlorination of trichloroethylene by the CO-reduced CO dehydrogenase enzyme complex from Methanosarcina thermophila

Trichloroethylene (TCE) was reductively dechlorinated to cis-dichloroethylene, trans-dichloroethylene, 1,1-dichloroethylene, vinyl chloride, and ethylene by the CO-reduced CO dehydrogenase enzyme complex from Methanosarcina thermophila; the apparent Km and Vmax values were 1.7 +/- 0.3 mM TCE and 26.2 +/- 1.7 mol TCE dechlorinated/min/mmol factor III. Factor III also catalysed the dechlorination of TCE when in the presence of titanium(III) citrate; the apparent Km and Vmax values were 1.2 +/- 0.3 mM TCE and 34.9 +/- 3.6 mol TCE dechlorinated/min/mmol factor III. The enzyme complex was resolved into the two-subunit nickel/iron-sulfur (Ni/Fe-S) component and the two-subunit factor III-containing corrinoid/iron-sulfur (Co/Fe-S) component. The Ni/Fe-S component was unable to dechlorinate TCE in the presence of CO; however, reconstitution with the Co/Fe-S component yielded the same dechlorinated products as with the CO dehydrogenase enzyme complex.     

Inhibition of the Fenton reaction by nitrogen monoxide

The toxicity of iron is believed to originate from the Fenton reaction which produces the hydroxyl radical and/or oxoiron(2+). The effect of nitrogen monoxide on the kinetics of the reaction of iron(II) bound to citrate, ethylenediamine-N,N′-diacetate (edda), ethylenediamine-N,N,N′,N′-tetraacetate (edta), (N-hydroxyethyl)amine-N,N′,N′-triacetate (hedta), and nitrilotriacetate (nta) with hydrogen peroxide was studied by stopped-flow spectrophotometry. Nitrogen monoxide inhibits the Fenton reaction to a large extent. For instance, hydrogen peroxide oxidizes iron(II) citrate with a rate constant of 5.8×10³ M⁻¹ s⁻¹, but in the presence of nitrogen monoxide, the rate constant is 2.9×10² M⁻¹ s⁻¹ . Similar to hydrogen peroxide, the reaction of tert-butyl hydroperoxide with iron(II) complexes is also efficiently inhibited by nitrogen monoxide. Generally, nitrogen monoxide binds rapidly to a coordination site of iron(II) occupied by water. The rate of oxidation is influenced by the rate of dissociation of the nitrogen monoxide from iron(II). Electronic Supplementary Material Supplementary material is available for this article at     

Carbon monoxide exposure in rat heart: glutathione depletion is prevented by antioxidants

Rat hearts were perfused for 15min with buffer equilibrated with 0.01% or 0.05% CO. The buffer was equilibrated with 21% O(2) throughout. The ventricular glutathione content decreased by 76% and 84%, 90min post-exposure to 0.01% and 0.05% CO, respectively, compared with 0% CO controls (0.45+/-0.01 micromol/g wet tissue; +/-SEM, n=3). Both reduced and oxidised glutathione contributed to this decline. When ascorbate and Trolox C were included during exposure to 0.05% CO the glutathione pool was partly protected; here the glutathione decrease was 46%. In most hearts additional creatine kinase activity in the perfusate indicated minor tissue injury occurring immediately after the start and/or about 10min after the end of exposure to 0.01% CO or 0.05% CO. Ventricle lactate levels were unaffected by exposure to 0.01% CO. This evidence supports a role for oxidative stress in CO cardiotoxicity.     

O2-mediated oxidation of ferrous nitrosylated human serum heme-albumin is limited by nitrogen monoxide dissociation

Human serum heme–albumin (HSA-heme-Fe) displays globin-like properties. Here, kinetics of O₂-mediated oxidation of ferrous nitrosylated HSA-heme-Fe (HSA-heme-Fe(II)-NO) is reported. Values of the first-order rate constants for O₂-mediated oxidation of HSA-heme-Fe(II)-NO (i.e., for ferric HSA-heme-Fe formation) and for NO dissociation from HSA-heme-Fe(II)-NO (i.e., for NO replacement by CO) are k = 9.8 × 10⁻⁵ and 8.3 × 10⁻⁴ s⁻¹, and h = 1.3 × 10⁻⁴ and 8.5 × 10⁻⁴ s⁻¹, in the absence and presence of rifampicin, respectively, at pH = 7.0 and T = 20.0 °C. The coincidence of values of k and h indicates that NO dissociation represents the rate limiting step of O₂-mediated oxidation of HSA-heme-Fe(II)-NO. Mixing HSA-heme-Fe(II)-NO with O₂ does not lead to the formation of the transient adduct(s), but leads to the final ferric HSA-heme-Fe derivative. These results reflect the fast O₂-mediated oxidation of ferrous HSA-heme-Fe and highlight the role of drugs in modulating allosterically the heme-Fe-atom reactivity.     

Processivity of cellobiohydrolases is limited by the substrate

Processive cellobiohydrolases (CBHs) are the key components of fungal cellulase systems. Despite the wealth of structural data confirming the processive mode of action, little quantitative information on the processivity of CBHs is available. Here, we developed a method for measuring cellulase processivity. Sensitive fluorescence detection of enzyme-generated insoluble reducing groups on cellulose after labeling with diaminopyridine enabled quantification of the number of reducing-end exo-mode and endo-mode initiations. Both CBHs TrCel7A from Trichoderma reesei and PcCel7D from Phanerochaete chrysosporium employed reducing-end exo- and endo-mode initiation in parallel. Processivity values measured for TrCel7A and PcCel7D on cellulose hydrolysis were more than an order of magnitude lower than the values of intrinsic processivity that were found from the ratio of catalytic constant (k(cat)) and dissociation rate constant (k(off)). We propose that the length of the obstacle-free path available for a processive run on cellulose chain limits the processivity of CBHs on cellulose. TrCel7A and PcCel7D differed in their k(off) values, whereas the k(cat) values were similar. Furthermore, the k(off) values for endoglucanases (EGs) were much higher than the k(off) values for CBHs, whereas the k(cat) values for EGs and CBHs were within the same order of magnitude. These results suggest that the value of k(off) may be the primary target for the selection of cellulases.     

The inhibition of mitochondrial cytochrome oxidase by the gases carbon monoxide, nitric oxide, hydrogen cyanide and hydrogen sulfide: chemical mechanism and physiological significance

The four gases, nitric oxide (NO), carbon monoxide (CO), hydrogen sulfide (H₂S) and hydrogen cyanide (HCN) all readily inhibit oxygen consumption by mitochondrial cytochrome oxidase. This inhibition is responsible for much of their toxicity when they are applied externally to the body. However, recently these gases have all been implicated, to greater or lesser extents, in normal cellular signalling events. In this review we analyse the chemistry of this inhibition, comparing and contrasting mechanism and discussing physiological consequences. The inhibition by NO and CO is dependent on oxygen concentration, but that of HCN and H₂S is not. NO and H₂S are readily metabolised by oxidative processes within cytochrome oxidase. In these cases the enzyme may act as a physiological detoxifier of these gases. CO oxidation is much slower and unlikely to be as physiologically important. The evidence for normal physiological levels of these gases interacting with cytochrome oxidase is equivocal, in part because there is little robust data about their steady state concentrations. A reasonable case can be made for NO, and perhaps CO and H₂S, inhibiting cytochrome oxidase in vivo, but endogenous levels of HCN seem unlikely to be high enough.     

Free-ligand accelerated dissociation of insulin-like growth factor 1 (IGF-1) from the type I IGF receptor is reduced by insulin-like growth factor binding protein 3

Insulin-like growth factor binding proteins (IGFBP) can inhibit or accentuate the mitogenic activities of insulin-like growth factor 1 (IGF-1) depending upon the experimental model employed. Inhibitory effects may be attributed to sequestration of IGF-1 onto IGFBP rather than the type I IGF receptor. We have demonstrated that the presence of IGFBP in a simple equilibrium binding assay significantly reduces the total amount of IGF-1 bound to the type I IGF receptor and increases the IC₅₀ for IGF-1 binding. On the basis of such an experiment, performed at equilibrium, IGFBP should reduce the mitogenic activity of IGF-1. Recent work has demonstrated an inverse correlation between the dissociation rate of insulin-like molecules from their receptors and their mitogenic activity. It has also been suggested that the increased rate of dissociation of insulin and IGF-1 from their receptors at increased ligand concentrations serves as a ‘dampening’ mechanism to decrease mitogenic signalling. We have demonstrated increased rates of dissociation of IGF-1 from the type I IGF receptor with increasing concentrations of IGF-1. Furthermore, IGFBP-3 inhibits the acceleration of dissociation rates due to increased IGF-1 levels. Thus, under receptor saturating conditions IGFBP-3 may act to increase mitogenesis by increasing the residence time of individual molecules of IGF-1 upon the type I IGF receptor.     

Periostin expression by epicardium-derived cells is involved in the development of the atrioventricular valves and fibrous heart skeleton

Abstract The epicardium is embryologically formed by outgrowth of proepicardial cells over the naked heart tube. Epicardium-derived cells (EPDCs) migrate into the myocardium, contributing to myocardial architecture, valve development, and the coronary vasculature. Defective EPDC formation causes valve malformations, myocardial thinning, and coronary defects. In the atrioventricular (AV) valves and the fibrous heart skeleton isolating atrial from ventricular myocardium, EPDCs colocalize with periostin, a matrix molecule involved in remodeling. We investigated whether proepicardial outgrowth inhibition affected periostin expression and how this related to development of the AV valves and fibrous heart skeleton.
Periostin expression by epicardium and EPDCs was confirmed in vitro in primary cultures of human and quail EPDCs. Disturbing EPDC formation in quail embryos reduced periostin expression in the endocardial cushions and AV junction. Disturbed fibrous tissue development resulted in AV myocardial connections reflected by preexcitation electrocardiographic patterns.
We conclude that EPDCs are local producers of periostin. Disturbance of EPDC formation results in decreased cardiac periostin levels and hampers the development of fibrous tissue in AV junction and the developing AV valves. The resulting cardiac anomalies might link to Wolff–Parkinson White syndrome with persistent AV myocardial connections.     

Identification of a specific protein in the mitochondrial fraction of the rat heart whose phosphorylation is inhibited by taurine

Summary It was previously reported that the mitochondrial fraction of the rat heart contained a specific protein with a molecular weight of approximately 44kDa whose phosphorylation was inhibited by taurine (Lombardini,1994a). Isolation of the 44kDa phosphoprotein on a 1-dimensional polyacrylamide gel using traditional glycine buffers followed by re-electrophoresing the cut out proportion of the gel which corresponds to the 44kDa protein on a tricine-buffered gel resulted in sufficient pure protein for sequence analysis. The results indicate that the 44kDa phosphoprotein is pyruvate dehydrogenase.     

Dual probe fluorescence monitoring of intracellular free calcium during ischemia in mouse heart by using continuous compensation for pH dependence of the dissociation constant of Fura-2, and the interference of myoglobin

Mitochondrial damage is the main source of cellular injury upon ischemia-reperfusion, and calcium loading has been implicated in this phenomenon. The use of optical probes for calcium monitoring of the intact heart is hampered by internal filter effects of intracellular hemoproteins, endogenous fluorescence, and their sensitivity to pH. We describe here a method for measurement of intracellular free calcium in isolated myoglobin-deficient perfused mouse hearts under conditions of large intracellular pH fluctuations by simultaneous fluorescence monitoring of the calcium-probe Fura-2 and the pH probe BCECF through dual wavelength excitation of both probes. In myoglobin-containing mouse heart endogenous chromophores interfere with Fura-2 fluorometry. It is shown that a paradoxical decrease in Fura-2 fluorescence occurs during ischemia in isolated mouse hearts. Simultaneous recording of BCECF fluorescence (calibrated against pH measurement with phosphorus NMR) and data reduction based on continual recalculation of the apparent dissociation constant of the calcium-probe complex revealed that a marked increase in intracellular free calcium occurs, and that the Fura-2 fluorescence decrease was caused by an increase in dissociation constant due to intracellular acidification. Intracellular free calcium rose almost linearly during a 20-min period of ischemia and returned to basal values rapidly upon the commencement of perfusion.     

The distribution of residues in a polypeptide sequence is a determinant of aggregation optimized by evolution

It has been shown that the propensity of a protein to form amyloid-like fibrils can be predicted with high accuracy from the knowledge of its amino acid sequence. It has also been suggested, however, that some regions of the sequences are more important than others in determining the aggregation process. Here, we have addressed this issue by constructing a set of “sequence scrambled” variants of the first 29 residues of horse heart apomyoglobin (apoMb_1-29), in which the sequence was modified while maintaining the same amino acid composition. The clustering of the most amyloidogenic residues in one region of the sequence was found to cause a marked increase of the elongation rate (k_agg) and a remarkable shortening of the lag phase (t_lag) of the fibril growth, as determined by far-UV circular dichroism and thioflavin T fluorescence. We also show that taking explicitly into consideration the presence of aggregation-promoting regions in the predictive methods results in a quantitative agreement between the theoretical and observed k_agg and t_lag values of the apoMb_1-29 variants. These results, together with a comparison between homologous segments from the family of globins, indicate the existence of a negative selection against the clustering of highly amyloidogenic residues in one or few regions of polypeptide sequences.