Haemozoin formation in the midgut of the blood-sucking insect Rhodnius prolixus.
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Malaria parasites digest haemoglobin and detoxify the free haem by its sequestration into an insoluble dark-brown pigment known as haemozoin (Hz). Until recently, this pigment could be found only in Plasmodium parasites. However, we have shown that Hz is also present in the midgut of the blood-sucking insect Rhodnius prolixus [Oliveira et al. (1999) Nature 400, 517-518]. Here we show that Hz synthesis in the midgut of this insect is promoted by a particulate fraction from intestine lumen. Haem aggregation activity is heat-labile and is inhibited in vitro by chloroquine (CLQ). Inhibition of Hz formation in vivo by feeding insects with CLQ leads to increased levels of haem in the haemolymph of the insect, which resulted in increased lipid peroxidation. Taken together, these results indicate that a factor capable of promoting Hz crystallisation is present in R. prolixus midgut and that this activity represents an important physiological defence of this insect against haem toxicity. (+info)
Flavohemoglobin Hmp affords inducible protection for Escherichia coli respiration, catalyzed by cytochromes bo' or bd, from nitric oxide.
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Respiration of Escherichia coli catalyzed either by cytochrome bo' or bd is sensitive to micromolar extracellular NO; extensive, transient inhibition of respiration increases as dissolved oxygen tension in the medium decreases. At low oxygen concentrations (25-33 microm), the duration of inhibition of respiration by 9 microm NO is increased by mutation of either oxidase. Respiration of an hmp mutant defective in flavohemoglobin (Hmp) synthesis is extremely NO-sensitive (I(50) about 0.8 microm); conversely, cells pre-grown with sodium nitroprusside or overexpressing plasmid-borne hmp(+) are insensitive to 60 microm NO and have elevated levels of immunologically detectable Hmp. Purified Hmp consumes O(2) at a rate that is instantaneously and extensively (>10-fold) stimulated by NO due to NO oxygenase activity but, in the absence of NO, Hmp does not contribute measurably to cell oxygen consumption. Cyanide binds to Hmp (K(d) 3 microm). Concentrations of KCN (100 microm) that do not significantly inhibit cell respiration markedly suppress the protection of respiration from NO afforded by Hmp and abolish NO oxygenase activity of purified Hmp. The results demonstrate the role of Hmp in protecting respiration from NO stress and are discussed in relation to the energy metabolism of E. coli in natural O(2)-depleted environments. (+info)
Direct binding of hydroxylamine to the heme iron of Arthromyces ramosus peroxidase. Substrate analogue that inhibits compound I formation in a competetive manner.
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The interaction of hydroxylamine (HA) with Arthromyces ramosus peroxidase (ARP) was investigated by kinetic, spectroscopic, and x-ray crystallographic techniques. HA inhibited the reaction of native ARP with H(2)O(2) in a competitive manner. Electron absorption and resonance Raman spectroscopic studies indicated that pentacoordinate high spin species of native ARP are converted to hexacoordinate low spin species upon the addition of HA, strongly suggesting the occurrence of a direct interaction of HA with ARP heme iron. Kinetic analysis exhibited that the apparent dissociation constant is 6.2 mm at pH 7.0 and that only one HA molecule likely binds to the vicinity of the heme. pH dependence of HA binding suggested that the nitrogen atom of HA could be involved in the interaction with the heme iron. X-ray crystallographic analysis of ARP in complex with HA at 2.0 A resolution revealed that the electron density ascribed to HA is located in the distal pocket between the heme iron and the distal His(56). HA seems to directly interact with the heme iron but is too far away to interact with Arg(52). In HA, it is likely that the nitrogen atom is coordinated to the heme iron and that hydroxyl group is hydrogen bonded to the distal His(56). (+info)
Nitric-oxide dioxygenase activity and function of flavohemoglobins. sensitivity to nitric oxide and carbon monoxide inhibition.
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Widely distributed flavohemoglobins (flavoHbs) function as NO dioxygenases and confer upon cells a resistance to NO toxicity. FlavoHbs from Saccharomyces cerevisiae, Alcaligenes eutrophus, and Escherichia coli share similar spectra, O(2), NO, and CO binding kinetics, and steady-state NO dioxygenation kinetics. Turnover numbers (V(max)) for S. cerevisiae, A. eutrophus, and E. coli flavoHbs are 112, 290, and 365 NO heme(-1) s(-1), respectively, at 37 degrees C with 200 microm O(2). The K(M) values for NO are low and range from 0.1 to 0.25 microm. V(max)/K(M)(NO) ratios of 900-2900 microm(-1) s(-1) indicate an extremely efficient dioxygenation mechanism. Approximate K(M) values for O(2) range from 60 to 90 microm. NO inhibits the dioxygenases at NO:O(2) ratios of > or =1:100 and makes true K(M)(O(2)) values difficult to determine. High and roughly equal second order rate constants for O(2) and NO association with the reduced flavoHbs (17-50 microm(-1) s(-1)) and small NO dissociation rate constants suggest that NO inhibits the dioxygenase reaction by forming inactive flavoHbNO complexes. Carbon monoxide also binds reduced flavoHbs with high affinity and competitively inhibits NO dioxygenases with respect to O(2) (K(I)(CO) = approximately 1 microm). These results suggest that flavoHbs and related hemoglobins evolved as NO detoxifying components of nitrogen metabolism capable of discriminating O(2) from inhibitory NO and CO. (+info)
Expression of prokaryotic and eukaryotic cytochromes c in Escherichia coli.
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C-type cytochromes from various sources show substantial structural conservation. For the covalent attachment of heme groups to apocytochromes, however, three different enzyme systems have been described so far. We have examined the ability of the heme ligation systems of Escherichia coli and of Saccharomyces cerevisiae to process cytochromes from S. cerevisiae, Paracoccus denitrificans, and Synechocystis sp. PCC 6803. E. coli's maturation system with at least eight different proteins accepted all these cytochromes for heme ligation. The single subunit heme lyase from S. cerevisiae mitochondria, on the other hand, failed to attach heme groups to cytochromes of prokaryotic origin. (+info)
Dissociation and recombination between ligands and heme in a CO-sensing transcriptional activator CooA. A flash photolysis study.
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CooA from Rhodospirillum rubrum is a transcriptional activator in which a heme prosthetic group acts as a CO sensor and regulates the activity of the protein. In this study, the electronic relaxation of the heme, and the concurrent recombination between ligands and the heme at approximately 280 K were examined in an effort to understand the environment around the heme and the dynamics of the ligands. Upon photoexcitation of the reduced CooA at 400 nm, electronic relaxation of the heme occurred with time constants of 0.8 and 1.7 ps. The ligand rebinding was substantially completed with a time constant of 6.5 ps, followed by a slow relaxation process with a time constant of 173 ps. In the case of CO-bound CooA, relaxation of the excited heme occurred with two time constants, 1.1 and 2.4 ps, which were largely similar to those with reduced CooA. The subsequent CO recombination process was remarkably fast compared with that of other CO-bound heme proteins. It was well described as a biphasic geminate recombination process with time constants of 78 ps (60%) and 386 ps (30%). About 10% of the excited heme remained unligated at 1.9 ns. The dynamics of rebinding of CO thus will help us to understand how the physiologically relevant diatomic molecule approaches the heme binding site in CooA with picosecond resolution. (+info)
Crystal structure of a nonsymbiotic plant hemoglobin.
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BACKGROUND: Nonsymbiotic hemoglobins (nsHbs) form a new class of plant proteins that is distinct genetically and structurally from leghemoglobins. They are found ubiquitously in plants and are expressed in low concentrations in a variety of tissues including roots and leaves. Their function involves a biochemical response to growth under limited O(2) conditions. RESULTS: The first X-ray crystal structure of a member of this class of proteins, riceHb1, has been determined to 2.4 A resolution using a combination of phasing techniques. The active site of ferric riceHb1 differs significantly from those of traditional hemoglobins and myoglobins. The proximal and distal histidine sidechains coordinate directly to the heme iron, forming a hemichrome with spectral properties similar to those of cytochrome b(5). The crystal structure also shows that riceHb1 is a dimer with a novel interface formed by close contacts between the G helix and the region between the B and C helices of the partner subunit. CONCLUSIONS: The bis-histidyl heme coordination found in riceHb1 is unusual for a protein that binds O(2) reversibly. However, the distal His73 is rapidly displaced by ferrous ligands, and the overall O(2) affinity is ultra-high (K(D) approximately 1 nM). Our crystallographic model suggests that ligand binding occurs by an upward and outward movement of the E helix, concomitant dissociation of the distal histidine, possible repacking of the CD corner and folding of the D helix. Although the functional relevance of quaternary structure in nsHbs is unclear, the role of two conserved residues in stabilizing the dimer interface has been identified. (+info)
Haem-polypeptide interactions during cytochrome c maturation.
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Cytochrome c maturation involves the translocation of a polypeptide, the apocytochrome, and its cofactor, haem, through a membrane, before the two molecules are ligated covalently. This review article focuses on the current knowledge on the journey of haem during this process, which is known best in the Gram-negative bacterium Escherichia coli. As haem always occurs bound to protein, its passage across the cytoplasmic membrane and incorporation into the apocytochrome appears to be mediated by a set of proteinaceous maturation factors, the Ccm (cytochrome c maturation) proteins. At least three of them, CcmC, CcmE and CcmF, are thought to interact directly with haem. CcmE binds haem covalently, thus representing an intermediate of the haem trafficking pathway. CcmC is required for binding of haem to CcmE, and CcmF for releasing it from CcmE and transferring it onto the apocytochrome. The mechanism by which haem crosses the cytoplasmic membrane is currently unknown. (+info)