Anoxic function for the Escherichia coli flavohaemoglobin (Hmp): reversible binding of nitric oxide and reduction to nitrous oxide.
The flavohaemoglobin Hmp of Escherichia coli is inducible by nitric oxide (NO) and provides protection both aerobically and anaerobically from inhibition of growth by NO and agents that cause nitrosative stress. Here we report rapid kinetic studies of NO binding to Fe(III) Hmp with a second order rate constant of 7.5 x 10(5) M(-1) s(-1) to generate a nitrosyl adduct that was stable anoxically but decayed in the presence of air to reform the Fe(III) protein. NO displaced CO bound to dithionite-reduced Hmp but, remarkably, CO recombined after only 2 s at room temperature indicative of NO reduction and dissociation from the haem. Addition of NO to anoxic NADH-reduced Hmp also generated a nitrosyl species which persisted while NADH was oxidised. These results are consistent with direct demonstration by membrane-inlet mass spectrometry of NO consumption and nitrous oxide production during anoxic incubation of NADH-reduced Hmp. The results demonstrate a new mechanism by which Hmp may eliminate NO under anoxic growth conditions. (+info)
Five-coordinate iron-porphyrin as a model for the active site of hemoproteins. Characterization and coordination properties.
Preparation of iron(III)-deuteroporphyrin 6(7)-methyl ester, 7(6)-(histidine methyl ester) by coupling histidine methyl ester to deuterohemin has been performed using the mixed carboxylic/carbonic-acid-anhydride method. This compound, which is very soluble in various organic solvents, can be considered as a prosthetic group model for the active site of five-coordinate hemoproteins. In the oxidized state a basic, a neutral or an acid form can be isolated. The basic and acid forms are monomeric at all concentrations. The neutral form is found dimeric in concentrated solutions while it is monomeric at low concentration. The coordination state of iron in these various species is investigated. The neutral form reacts with ligands, such as nitrogenous organic bases, leading to six-coordinate well-known hemichromes which exhibit low-spin electron spin resonance (ESR) spectra. The reaction of anionic ligands, such as F-, CN-, NO-2 and N-3, with the neutral form model leads to unsymmetrical six-coordinate complexes whose optical and ESR spectra are similar to those of synthetic deuteromyoglobin. In benzene, toluene or dichloromethane solutions iron (II)-deuteroporphyrin 6(7)-methyl ester, 7(6)-histidine methyl ester), obtained from ferric forms by heterogeneous reduction with aqueous dithionite, exhibits an optical spectrum characteristic of a five-coordinate high-spin ferrous complex. At low temperature important spectral modifications are observed indicating a dimeric association. At room temperature it binds one nitrogenous base molecule leading to the well-known hemochrome. It reacts also with carbon monoxide with a very high affinity constant (4.5 X 10(8) M-1), comparable to that of the isolated human hemoglobin alpha and beta chains, but much higher than the values reported for other various models, suggesting that the side-chain length bearing the fifth ligand may have an important influence upon the reactivity of the sixth position of the iron ion. At low temperature in toluene or dichloromethane, this compound reversibly binds oxygen without oxidation of the iron ion while oxidation occurs at room temperature. The significance of these results is discussed in relation with the local environment, the electronic nature of the base and the immobilization of the heme group in hemoproteins. (+info)
An anomaly in the resonance Raman spectra of cytochrome P-450cam in the ferrous high-spin state.
Resonance Raman spectra of cytochrome P-450cam (P-450cam) and its enzymatically inactive form (P-420) in various oxidation and spin states were measured for the first time. The Raman spectrum of reduced P-450cam was unusual in the sense that the "oxidation-state marker" appeared at an unexpectedly lower frequency (1346 cm-1) in comparison with those of other reduced hemoproteins (approximately 1355-approximately 1365 cm-1), whereas that of oxidized P-450cam was located at a normal frequency. This anomaly in the Raman spectrum of reduced P-450cam can be explained by assuming electron delocalization from the fifth ligand, presumably a thiolate anion, to the antibonding pi orbital of the porphyrin ring. The corresponding Raman line of reduced P-420 appeared at a normal frequency (1360 cm-1), suggesting a status change or replacement of the fifth ligand upon conversion from P-450cam to P-420. The Raman spectrum of reduced P-450cam-metyrapone complex was very similar to that of ferrous cytochrome b5. (+info)
Structure and function of a cysBJIH gene cluster in the purple sulphur bacterium Thiocapsa roseopersicina.
A gene cluster containing homologues of the genes cysB, cysJI and cysH was found in the genome of the sulphur-oxidizing purple bacterium Thiocapsa roseopersicina. The nucleotide sequence indicated four open reading frames encoding homologues of 3'-phosphoadenylylsulphate (PAPS) reductase (CysH), sulphite reductase flavoprotein (CysJ) and haem protein (CysI) subunits, and a transcriptional regulator (CysB). Genes cysJIH are separated by a short cis-active intergenic region from cysB which is transcribed divergently. cysB encodes a polypeptide of 35.9 kDa consisting of 323 amino acid residues with 40% identity to the CysB regulator from enterobacteria. cysH encodes a protein with 239 amino acid residues and a calculated mass of 27.7 kDa; cysJ encodes a protein with 522 amino acid residues and a mass of 57.8 kDa; and cysI encodes a protein with 559 amino acid residues and a mass of 62.3 kDa. The cysJIH gene products have been expressed and used for complementation of cys mutants from Escherichia coli Biochemical analysis. The gene product CysH is a thioredoxin-dependent PAPS reductase (EC 188.8.131.52). It was repressed under photoautotrophic growth using hydrogen sulphide as electron donor and derepressed under conditions of sulphate deficiency. Products of the cysJI genes were identified as the two subunits of NADPH-sulphite reductase (EC 184.108.40.206). cysJ encoded the flavoprotein, with > or = 39% identity to the protein from E. coli, and cysI encoded the haem protein, with > or = 53% identity. A cysI clone was used to complement the corresponding mutant from E. coli and to express enzymically active methylviologen-sulphite reductase. (+info)
Oxygen sensing in yeast: evidence for the involvement of the respiratory chain in regulating the transcription of a subset of hypoxic genes.
Oxygen availability affects the transcription of a number of genes in nearly all organisms. Although the molecular mechanisms for sensing oxygen are not precisely known, heme is thought to play a pivotal role. Here, we address the possibility that oxygen sensing in yeast, as in mammals, involves a redox-sensitive hemoprotein. We have found that carbon monoxide (CO) completely blocks the anoxia-induced expression of two hypoxic genes, OLE1 and CYC7, partially blocks the induction of a third gene, COX5b, and has no effect on the expression of other hypoxic or aerobic genes. In addition, transition metals (Co and Ni) induce the expression of OLE1 and CYC7 in a concentration-dependent manner under aerobic conditions. These findings suggest that the redox state of an oxygen-binding hemoprotein is involved in controlling the expression of at least two hypoxic yeast genes. By using mutants deficient in each of the two major yeast CO-binding hemoproteins (cytochrome c oxidase and flavohemoglobin), respiratory inhibitors, and cob1 and rho0 mutants, we have found that the respiratory chain is involved in the anoxic induction of these two genes and that cytochrome c oxidase is likely the hemoprotein "sensor." Our findings also indicate that there are at least two classes of hypoxic genes in yeast (CO sensitive and CO insensitive) and imply that multiple pathways/mechanisms are involved in modulating the expression of hypoxic yeast genes. (+info)
Non-enzymatic nitric oxide synthesis in biological systems.
Nitric oxide (NO) is an important regulator of a variety of biological functions, and also has a role in the pathogenesis of cellular injury. It had been generally accepted that NO is solely generated in biological tissues by specific nitric oxide synthases (NOS) which metabolize arginine to citrulline with the formation of NO. However, NO can also be generated in tissues by either direct disproportionation or reduction of nitrite to NO under the acidic and highly reduced conditions which occur in disease states, such as ischemia. This NO formation is not blocked by NOS inhibitors and with long periods of ischemia progressing to necrosis, this mechanism of NO formation predominates. In postischemic tissues, NOS-independent NO generation has been observed to result in cellular injury with a loss of organ function. The kinetics and magnitude of nitrite disproportionation have been recently characterized and the corresponding rate law of NO formation derived. It was observed that the generation and accumulation of NO from typical nitrite concentrations found in biological tissues increases 100-fold when the pH falls from 7.4 to 5.5. It was also observed that ischemic cardiac tissue contains reducing equivalents which reduce nitrite to NO, further increasing the rate of NO formation more than 40-fold. Under these conditions, the magnitude of enzyme-independent NO generation exceeds that which can be generated by tissue concentrations of NOS. The existence of this enzyme-independent mechanism of NO formation has important implications in our understanding of the pathogenesis and treatment of tissue injury. (+info)
Nitric oxide and iron proteins.
Nitric oxide interactions with iron are the most important biological reactions in which NO participates. Reversible binding to ferrous haem iron is responsible for the observed activation of guanylate cyclase and inhibition of cytochrome oxidase. Unlike carbon monoxide or oxygen, NO can also bind reversibly to ferric iron. The latter reaction is responsible for the inhibition of catalase by NO. NO reacts with the oxygen adduct of ferrous haem proteins (e.g. oxyhaemoglobin) to generate nitrate and ferric haem; this reaction is responsible for the majority of NO metabolism in the vasculature. NO can also interact with iron-sulphur enzymes (e.g. aconitase, NADH dehydrogenase). This review describes the underlying kinetics, thermodynamics, mechanisms and biological role of the interactions of NO with iron species (protein and non-protein bound). The possible significance of iron reactions with reactive NO metabolites, in particular peroxynitrite and nitroxyl anion, is also discussed. (+info)
Phospholipid bound to the flavohemoprotein from Alcaligenes eutrophus.
The structurally characterized flavohemoprotein from Alcaligenes eutrophus (FHP) contains a phospholipid-binding site with 1-16 : 0-2-cyclo-17 : 0-diacyl-glycerophospho-ethanolamine and 1-16 : 0-2-cyclo-17 : 0-diacyl-glycerophospho-glycerol as the major occupying compounds. The structure of the phospholipid is characterized by its compact form, due to the -sc/beta/-sc conformation of the glycerol and the nonlinear arrangement of the sn-1- and sn-2-fatty acid chains. The phospholipid-binding site is located adjacent to the heme molecule at the bottom of a large cavity. The fatty acid chains form a large number of van der Waal's contacts with nonpolar side chains, whereas the glycerophosphate moiety, which points towards the entrance of the channel, is linked to the protein matrix by polar interactions. The thermodynamically stable globin module of FHP, obtained after cleaving off the oxidoreductase module, also contains the phospholipid and can therefore be considered as a phospholipid-binding protein. Single amino acid exchanges designed to decrease the lipid-binding site revealed both the possibility of blocking incorporation of the phospholipid and its capability to evade steric barriers. Conformational changes in the phospholipid can also be induced by binding heme-ligating compounds. Phospholipid binding is not a general feature of flavohemoproteins, because the Escherichia coli and the yeast protein exhibit less and no lipid affinity, respectively. (+info)