Chemoreceptor discharges and cytochrome redox changes of the rat carotid body: role of heme ligands.
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In superfused in vitro rat carotid body, we recorded chemoreceptor discharges and the redox state of cytochromes simultaneously to identify the primary oxygen-sensing protein controlling transmitter release and electrical activity of the carotid sinus nerve. These parameters were tested under the influence of heme ligands such as oxygen, cyanide, 4-(2-aminoethyl)-benzenesulfonyl fluoride, and CO. During stimulation, there was an initial increase in discharge frequency followed by a decline or suppression of activity. Photometric changes lagged and were maintained as nerve activity decreased. Reducing mitochondrial cytochromes by cyanide or prolonged severe hypoxia, suppressed the chemoreceptor discharge. 4-(2-Aminoethyl)-benzenesulfonyl fluoride, a specific inhibitor of the phagocytic cytochrome b(558), also silenced the chemoreceptors after an initial excitation. CO increased the chemoreceptor discharge under normoxia, an effect inhibited by light, when the cytochromes were not reduced. When the discharges were depressed by severe hypoxia, exposure to light excited the chemoreceptors and the cytochromes were reduced. The rapidity of the chemosensory responses to light and lack of effect on dopamine release from type I cells led us to hypothesize that carotid body type I cells and the apposed nerve endings use different mechanisms for oxygen sensing: the nerve endings generate action potentials in association with membrane heme proteins whereas cytosolic heme proteins signal the redox state, releasing modulators or transmitters from type I cells. (+info)
Proton nuclear magnetic resonance study of the binary complex of cytochrome P450cam and putidaredoxin: interaction and electron transfer rate analysis.
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A 1H nuclear magnetic resonance study of the complex of cytochrome P450cam-putidaredoxin has been performed. Isocyanide is bound to cytochrome P450cam in order to increase the stability of the protein both in the reduced and the oxidized state. Diprotein complex formation was detected through variation of the heme methyl proton resonances which have been assigned in the two redox states. The electron transfer rate at equilibrium was determinated by magnetization transfer experiments. The observed rate of oxidation of reduced cytochrome P450 by the oxidized putidaredoxin is 27 (+/- 7) per s. (+info)
Iron coordination structures of oxygen sensor FixL characterized by Fe K-edge extended x-ray absorption fine structure and resonance raman spectroscopy.
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FixL is a heme-based O(2) sensor protein involved in a two-component system of a symbiotic bacterium. In the present study, the iron coordination structure in the heme domain of Rhizobium meliloti FixLT (RmFixLT, a soluble truncated FixL) was examined using Fe K-edge extended x-ray absorption fine structure (EXAFS) and resonance Raman spectroscopic techniques. In the EXAFS analyses, the interatomic distances and angles of the Fe-ligand bond and the iron displacement from the heme plane were obtained for RmFixLT in the Fe(2+), Fe(2+)O(2), Fe(2+)CO, Fe(3+), Fe(3+)F(-), and Fe(3+)CN(-) states. An apparent correlation was found between the heme-nitrogen (proximal His-194) distance in the heme domain and the phosphorylation activity of the histidine kinase domain. Comparison of the Fe-CO coordination geometry between RmFixLT and RmFixLH (heme domain of RmFixL), based on the EXAFS and Raman results, has suggested that the kinase domain directly or indirectly influences steric interaction between the iron-bound ligand and the heme pocket. Referring to the crystal structure of the heme domain of Bradyrhizobium japonicum FixL (Gong, W., Hao, B., Mansy, S. S., Gonzalez, G., Gilles-Gonzalez, M. A., and Chan, M. K. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 15177-15182), we discussed details of the iron coordination structure of RmFixLT and RmFixLH in relation to an intramolecular signal transduction mechanism in its O(2) sensing. (+info)
Hemoglobinase activity of the lysine gingipain protease (Kgp) of Porphyromonas gingivalis W83.
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Porphyromonas gingivalis, an important periodontal disease pathogen, forms black-pigmented colonies on blood agar. Pigmentation is believed to result from accumulation of iron protoporphyrin IX (FePPIX) derived from erythrocytic hemoglobin. The Lys-X (Lys-gingipain) and Arg-X (Arg-gingipain) cysteine proteases of P. gingivalis bind and degrade erythrocytes. We have observed that mutations abolishing activity of the Lys-X-specific cysteine protease, Kgp, resulted in loss of black pigmentation of P. gingivalis W83. Because the hemagglutinating and hemolytic potentials of mutant strains were reduced but not eliminated, we hypothesized that this protease played a role in acquisition of FePPIX from hemoglobin. In contrast to Arg-gingipain, Lys-gingipain was not inhibited by hemin, suggesting that this protease played a role near the cell surface where high concentrations of hemin confer the black pigmentation. Human hemoglobin contains 11 Lys residues in the alpha chain and 10 Lys residues in the beta chain. In contrast, there are only three Arg residues in each of the alpha and beta chains. These observations are consistent with human hemoglobin being a preferred substrate for Lys-gingipain but not Arg-gingipain. The ability of the Lys-gingipain to cleave human hemoglobin at Lys residues was confirmed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry of hemoglobin fragments resulting from digestion with the purified protease. We were able to detect several of the predicted hemoglobin fragments rendered by digestion with purified Lys-gingipain. Thus, we postulate that the Lys-gingipain of P. gingivalis is a hemoglobinase which plays a role in heme and iron uptake by effecting the accumulation of FePPIX on the bacterial cell surface. (+info)
Carbon monoxide as a novel mediator of the febrile response in the central nervous system.
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Heme oxygenase catalyzes the metabolism of heme to biliverdin, free iron, and carbon monoxide (CO), which has been shown to be an important neuromodulatory agent. Recently, it has been demonstrated that lipopolysaccharide (LPS) can induce the enzyme heme oxygenase in glial cells. Therefore, the present study was designed to test the hypothesis that central CO plays a role in LPS-induced fever. Colonic body temperature (T(b)) was measured in awake, unrestrained rats (basal T(b) = 36.8 +/- 0.2 degrees C). Intracerebroventricular injection of zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG; 75 nmol), a heme oxygenase inhibitor, caused no significant change in T(b), indicating that the central heme oxygenase pathway plays no tonic role in T(b) under the experimental conditions used. Intraperitoneal injections of LPS (50-100 microgram/kg) evoked dose-dependent increases in T(b). Intracerebroventricular injection of ZnDPBG in febrile rats attenuated LPS-induced fever (thermal index with ZnDPBG = 1.1 +/- 0. 2 degrees C, thermal index with vehicle = 2.3 +/- 0.4 degrees C), suggesting that the central heme oxygenase pathway plays a role in fever generation. The antipyretic effect of ZnDPBG could be reversed by intracerebroventricular administration of heme-lysinate or CO-saturated saline. Collectively, our data indicate that CO arising from heme oxygenase may play an important role in fever generation by acting on the central nervous system. (+info)
Characterization of the Asp94 and Glu242 mutants in myeloperoxidase, the residues linking the heme group via ester bonds.
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The heme group of all mammalian peroxidases is covalently linked to the protein matrix via two esterbonds, as we have recently shown by Fourier transform infrared (FTIR) difference spectroscopy [Kooter, I. M., Pierik, A.J., Merkx, M., Averill, B.A., Moguilevsky, N., Bollen, A. & Wever, R. (1997) J. Am. Chem. Soc. 119, 11542-11543]. We have examined the effects of mutation of Asp94 and Glu242, responsible for those ester bonds in myeloperoxidase, on the spectroscopic properties and catalytic activity of this enzyme. Mutation of Asp94 in myeloperoxidase results in two species. The first species has spectroscopic characteristics similar to that of wild-type myeloperoxidase. The second species has spectroscopic characteristics similar to that of Met243-->Gln mutant, and it is therefore concluded that, besides loss of the ester bond involving Asp94, this species also has lost the sulfonium ion linkage that is also characteristic of myeloperoxidase. The Asp94-->Asn mutant still has about 30% residual peroxidase activity while for the Asp94-->Val mutant only a few percentage activity is left. When Glu242 is mutated the sulfonium ion linkage is not affected, but this residue together with its neighbouring residue Met243, according to resonance Raman spectra, is responsible for the low symmetry of the heme group. Mutation of either of these residues results in loss of the bowed distortion from the planar conformation, and in a heme group with higher symmetry. For the Glu242-->Gln mutant 8% residual peroxidase activity is found. (+info)
Redox-linked transient deprotonation at the binuclear site in the aa(3)-type quinol oxidase from Acidianus ambivalens: implications for proton translocation.
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The hyperthermophilic archaeon Acidianus ambivalens expresses a membrane-bound aa(3)-type quinol oxidase, when grown aerobically, that we have studied by resonance Raman spectroscopy. The purified aa(3) oxidase, which does not contain bound quinol, undergoes a reversible slow conformational change at heme a(3) upon reduction, as indicated by a change in the frequency of its heme formyl stretching mode, from 1,660 cm(-1) to 1,667 cm(-1). In contrast, upon reduction of the integral membrane enzyme or the purified enzyme preincubated with decylubiquinol, this mode appears at 1,667 cm(-1) much more rapidly, suggesting a role of the bound quinol in controlling the redox-linked conformational changes. The shift of the formyl mode to higher frequency is attributed to a loss of hydrogen bonding that is associated with a group having a pKa of approximately 3.8. Based on these observations, a crucial element for proton translocation involving a redox-linked conformational change near the heme a(3) formyl group is postulated. (+info)
Dual role for Hsc70 in the biogenesis and regulation of the heme-regulated kinase of the alpha subunit of eukaryotic translation initiation factor 2.
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The heme-regulated kinase of the alpha subunit of eukaryotic initiation factor 2 (HRI) is activated in rabbit reticulocyte lysate (RRL) in response to a number of environmental conditions, including heme deficiency, heat shock, and oxidative stress. Activation of HRI causes an arrest of initiation of protein synthesis. Recently, we have demonstrated that the heat shock cognate protein Hsc70 negatively modulates the activation of HRI in RRL in response to these environmental conditions. Hsc70 is also known to be a critical component of the Hsp90 chaperone machinery in RRL, which plays an obligatory role for HRI to acquire and maintain a conformation that is competent to activate. Using de novo-synthesized HRI in synchronized pulse-chase translations, we have examined the role of Hsc70 in the regulation of HRI biogenesis and activation. Like Hsp90, Hsc70 interacted with nascent HRI and HRI that was matured to a state which was competent to undergo stimulus-induced activation (mature-competent HRI). Interaction of HRI with Hsc70 was required for the transformation of HRI, as the Hsc70 antagonist clofibric acid inhibited the folding of HRI into a mature-competent conformation. Unlike Hsp90, Hsc70 also interacted with transformed HRI. Clofibric acid disrupted the interaction of Hsc70 with transformed HRI that had been matured and transformed in the absence of the drug. Disruption of Hsc70 interaction with transformed HRI in heme-deficient RRL resulted in its hyperactivation. Furthermore, activation of HRI in response to heat shock or denatured proteins also resulted in a similar blockage of Hsc70 interaction with transformed HRI. These results indicate that Hsc70 is required for the folding and transformation of HRI into an active kinase but is subsequently required to negatively attenuate the activation of transformed HRI. (+info)