Stereochemical mechanism of oxygen transport by haemoglobin

Spectroscopic and chemical evidence speak in favour of the iron-oxygen bond being polar. X-ray analysis shows that the oxygen molecule is inclined at an angle of about 115 degrees to the haem plane. Cooperative binding of oxygen by haemoglobin is due to an equilibrium between two alternative structures, which differ in oxygen affinity by the equivalent of 3-3.5 kcal/mol. I proposed that in the low affinity structure the globin opposes the movement of the iron atom from its five-coordinated pyramidal geometry in the haem of deoxyhaemoglobin to its six-coordinated planar geometry in the haem of oxyhaemoglobin, while in the high affinity structure this restraint is absent. Recent evidence supporting this mechanism is described.     

Water-gated mechanism of proton translocation by cytochrome c oxidase

Cytochrome c oxidase is essential for aerobic life as a membrane-bound energy transducer. O(2) reduction at the haem a(3)-Cu(B) centre consumes electrons transferred via haem a from cytochrome c outside the membrane. Protons are taken up from the inside, both to form water and to be pumped across the membrane (M.K.F. Wikstr?m, Nature 266 (1977) 271; M. Wikstr?m, K. Krab, M. Saraste, Cytochrome Oxidase, A Synthesis, Academic Press, London, 1981 ). The resulting electrochemical proton gradient drives ATP synthesis (P. Mitchell, Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Glynn Research, Bodmin, UK, 1966 ). Here we present a molecular mechanism for proton pumping coupled to oxygen reduction that is based on the unique properties of water in hydrophobic cavities. An array of water molecules conducts protons from a conserved glutamic acid, either to the Delta-propionate of haem a(3) (pumping), or to haem a(3)-Cu(B) (water formation). Switching between these pathways is controlled by the redox-state-dependent electric field between haem a and haem a(3)-Cu(B), which determines the water-dipole orientation, and therefore the proton transfer direction. Proton transfer via the propionate provides a gate to O(2) reduction. This pumping mechanism explains the unique arrangement of the metal cofactors in the structure. It is consistent with the large body of biochemical data, and is shown to be plausible by molecular dynamics simulations.     

Oxygen binding properties of non-mammalian nerve globins

Oxygen-binding globins occur in the nervous systems of both invertebrates and vertebrates. While the function of invertebrate nerve haemoglobins as oxygen stores that extend neural excitability under hypoxia has been convincingly demonstrated, the physiological role of vertebrate neuroglobins is less well understood. Here we provide a detailed analysis of the oxygenation characteristics of nerve haemoglobins from an annelid (Aphrodite aculeata), a nemertean (Cerebratulus lacteus) and a bivalve (Spisula solidissima) and of neuroglobin from zebrafish (Danio rerio). The functional differences have been related to haem coordination: the haem is pentacoordinate (as in human haemoglobin and myoglobin) in A. aculeata and C. lacteus nerve haemoglobins and hexacoordinate in S. solidissima nerve haemoglobin and D. rerio neuroglobin. Whereas pentacoordinate nerve globins lacked Bohr effects at all temperatures investigated and exhibited large enthalpies of oxygenation, the hexacoordinate globins showed reverse Bohr effects (at least at low temperature) and approximately twofold lower oxygenation enthalpies. Only S. solidissima nerve haemoglobin showed apparent cooperativity in oxygen binding, suggesting deoxygenation-linked self-association of the monomeric proteins. These results demonstrate a remarkable diversity in oxygenation characteristics of vertebrate and invertebrate nerve haemoglobins that clearly reflect distinct physiological roles.     

An 18O double-labelling study of haemoglobin catabolism in the rat.

The degradation of haemoglobin to bilirubin in the rat was investigated by 18O labelling of the molecular oxygen consumed by the animal. The oxygen atoms incorporated into bilirubin were derived from two different oxygen molecules. Implications of this finding for the mechanism of haem catabolism in vivo are discussed; both verdohaem and a dioxygen-bridged compound appear to be excluded at intermediates.     

The mechanism of haem catabolism. Bilirubin formation in living rats by [18O]oxygen labelling.

1. The pathway of haem breakdown in living rats was studied by using 18O in the oxygen that the animals consumed. By cannulation of the common bile duct and collection of bile, labelled bilirubin was isolated and its mass spectrum determined. One set of results was obtained for a rat to which haemoglobin had been intravenously administered and another set obtained for a rat that was not given exogenous haem. Isomerization of bilirubin IXalpha to the XIIIalpha and IIIalpha isomers did not occur to any significant extent. The 18O-labelling pattern obtained in the bilirubin was consistent with a Two-Molecule Mechanism, whereby the terminal lactam oxygen atoms of bilirubin are derived from different oxygen molecules. The consequences of this mechanism are discussed in terms of the possible intermediates of the catabolic pathway. 2. 18O-labelled bilirubin appeared in the bile in less than 10 min after exposure of the animals to labelled oxygen. This result suggests that all of the chemical transformations involving production of biliverdin, reduction to bilirubin and conjugation of the bilirubin are fast processes. 3. The quantitative recovery of label obtained in the experiments suggests that there is little or no exchange of newly synthesized bilirubin with existing bilirubin pools in the animal.     

Plasma clearance of glycoproteins with terminal mannose and N-acetylglucosamine by liver non-parenchymal cells. Studies with beta-glucuronidase, N-acetyl-beta-D-glucosaminidase, ribonuclease B and agalacto-orosomucoid.

The mechanism of bile-pigment formation from haem breakdown was studied by using 18O labelling of the molecular oxygen required for macrocyclic ring cleavage. For haem degradation by the spleen microsomal haem oxygenase system, mass spectrometry of the product bilirubin revealed that cleavage occurred by the Two-Molecule Mechanism, i.e. the terminal lactam oxygen atoms in bilirubin were derived from two different oxygen molecules. Similarly, degradation of myoglobin by coupled oxidation with ascorbate and oxygen proceeded via the Two-Molecule Mechanism. Cobalt and manganese complexes of protoporphyrin IX were not degraded by either the haem oxygenase system or the coupled oxidation system. This result suggests that the iron atom possesses unique properties in facilitating porphyrin breakdown.     

Structure and mechanism in the bacterial dihaem cytochrome c peroxidases

The bacterial cytochrome c peroxidases contain an electron-transferring haem c (E) and a peroxidatic haem c (P). Many are isolated in an inactive oxidised state. Reduction of the E haem promotes Ca(2+)-dependent spin state and coordination changes at the P haem rendering it accessible to ligand. Recent crystallographic work on the oxidised and mixed valence enzymes has suggested a mechanism by which an electron entering the E haem remotely triggers this activation of the P haem. Binding of hydrogen peroxide at the activated P haem leads to an intermediate catalytic form containing two oxidising equivalents, one of which is a ferryl oxene. This form of the enzyme is then reduced by two single electron transfers to the E haem delivered by small redox proteins such as cytochromes or cupredoxins. The binding of these small redox proteins is dominated by global electrostatic forces but the interfaces of the electron transfer complexes that are formed are largely hydrophobic and relatively non-specific. These features allow very high electron transfer rates in the steady state.     

Water-gated mechanism of proton translocation by cytochrome c oxidase

Cytochrome c oxidase is essential for aerobic life as a membrane-bound energy transducer. O₂ reduction at the haem a₃-Cu_B centre consumes electrons transferred via haem a from cytochrome c outside the membrane. Protons are taken up from the inside, both to form water and to be pumped across the membrane (M.K.F. Wikström, Nature 266 (1977) 271 [1]; M. Wikström, K. Krab, M. Saraste, Cytochrome Oxidase, A Synthesis, Academic Press, London, 1981 [2]). The resulting electrochemical proton gradient drives ATP synthesis (P. Mitchell, Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Glynn Research, Bodmin, UK, 1966 [3]). Here we present a molecular mechanism for proton pumping coupled to oxygen reduction that is based on the unique properties of water in hydrophobic cavities. An array of water molecules conducts protons from a conserved glutamic acid, either to the Δ-propionate of haem a₃ (pumping), or to haem a₃-Cu_B (water formation). Switching between these pathways is controlled by the redox-state-dependent electric field between haem a and haem a₃-Cu_B, which determines the water-dipole orientation, and therefore the proton transfer direction. Proton transfer via the propionate provides a gate to O₂ reduction. This pumping mechanism explains the unique arrangement of the metal cofactors in the structure. It is consistent with the large body of biochemical data, and is shown to be plausible by molecular dynamics simulations.     

Stereospecific haem cleavage. A model for the formation of bile-pigment isomers in vivo and in vitro.

A new approach is suggested for an explanation of sterospecific haem degradation to biliverdin and bilirubin. A model is proposed in which an oxygen molecule, bound to the haem iron atom, attacks a methene-bridge carbon atom in an intramolecular reaction. Specificity of macrocyclic ring cleavage is explained on the basis of the different accessibilities of the bound oxygen molecule to the four methene bridges. The consequences of these ideas are assessed in relation to coupled oxidation in model systems and to haem catabolism.     

Hyperbaric oxygen induces apoptosis via a mitochondrial mechanism

During therapeutic hyperbaric oxygenation lymphocytes are exposed to high partial pressures of oxygen. This study aimed to analyze the mechanism of apoptosis induction by hyperbaric oxygen. For intervals of 0.5–4 h Jurkat-T-cells were exposed to ambient air or oxygen atmospheres at 1–3 absolute atmospheres. Apoptosis was analyzed by phosphatidylserine externalization, caspase-3 activation and DNA-fragmentation using flow cytometry. Apoptosis was already induced after 30 min of hyperbaric oxygenation (HBO, P < 0.05). The death receptor Fas was downregulated. Inhibition of caspase-9 but not caspase-8 blocked apoptosis induction by HBO. Hyperbaric oxygen caused a loss of mitochondrial membrane potential and caspase-9 induction. The mitochondrial pro-survival protein Bcl-2 was upregulated, and antagonizing Bcl-2 function potentiated apoptosis induction by HBO. In conclusion, a single exposure to hyperbaric oxygenation induces lymphocyte apoptosis by a mitochondrial and not a Fas-related mechanism. Regulation of Fas and Bcl-2 may be regarded as protective measures of the cell in response to hyperbaric oxygen.     

The NtrY/X two-component system of Brucella spp. acts as a redox sensor and regulates the expression of nitrogen respiration enzymes

Brucella spp. are facultative intracellular bacteria pathogenic for many mammalian species including humans, causing a disease called brucellosis. Learning how Brucella adapts to its intracellular niche is crucial for understanding its pathogenesis mechanism, allowing for the development of new and more effective vaccines and treatments against brucellosis. Brucella pathogenesis resides mostly in its ability to adapt to the harsh environmental conditions encountered during host infection such as the oxygen depletion. The mechanism by which Brucella senses the oxygen tension and triggers its environmental adaptation is unknown. In this work we show that the Brucella abortus NtrY/NtrX two-component system is involved in oxygen sensing through a haem group contained in a Per-ARNT-SIM (PAS) domain of the NtrY histidine kinase. The NtrY haem iron can be reduced to the ferrous form and is rapidly oxidized to the ferric form in presence of oxygen. Importantly, we show that the oxidation state of the haem iron modulates the autokinase activity, being the anoxygenic reduced ferrous form the signalling state of NtrY. Also, we show that ntrY gene expression increases under low oxygen tension and that NtrY transfers its signal to its cognate response regulator NtrX, regulating in this way the expression of nitrogen respiration enzymes. Based on these findings, we postulate that NtrY acts as a redox sensor in Brucella spp.     

Oxygenation mechanism in the oxidation of xenobiotic aldehyde to carboxylic acid by mouse hepatic microsomes

11-Oxo-delta 8-tetrahydrocannabinol was oxidized to delta 8-tetrahydrocannabinol-11-oic acid by mouse hepatic microsomes. The oxygenation mechanism in the reaction was confirmed by the incorporation of oxygen-18 from molecular oxygen into delta 8-tetrahydrocannabinol-11-oic acid. The oxygenation of aldehyde to carboxylic acid represents a novel mechanism in biological oxidation of aldehyde to carboxylic acid.     

Influence of anions and protons on the Adair coefficients of haemoglobins A and Cowtown (His HC3(146) beta----Leu)

We have measured the contribution of the alkaline Bohr effect of the C-terminal histidine residues of the beta-chains of haemoglobin A by comparing haemoglobin A with haemoglobin Cowtown in which those histidine residues are replaced by leucine. Oxygenation of a stripped 2.5 mM (haem) solution of haemoglobin A yielded 0.19 H+/haem, while oxygenation of a similar solution of haemoglobin Cowtown produced no change of pH. Oxygen equilibria measured at 60 microM-haem in 0.1 M-Hepes buffer gave an alkaline Bohr effect of -0.21 H+/haem for haemoglobin A and only -0.01 H+/haem for haemoglobin Cowtown, even though its Hill's coefficient was greater than 2 throughout the pH range studied. These results prove that the chloride-independent part of the alkaline Bohr effect is due to the C-terminal histidine residues of the beta-chains. Oxygen equilibria measured in 0.095 M-bis-Tris buffers with minimal chloride or with 0.1 M-chloride showed the contribution of those histidine residues to the alkaline Bohr effect to be about 0.2 H+/haem, independent of chloride concentration. Determination of the individual Adair coefficients in the three different buffers indicated that pH and chloride tend to have their greatest effects at the second or third steps of oxygenation when the change of quaternary structure is most likely to occur; between pH 7 and 9, the fourth Adair coefficient is only very slightly affected by pH and not significantly by chloride.     

Peroxidase-catalyzed S-oxygenation: mechanism of oxygen transfer for lactoperoxidase

The mechanism of organosulfur oxygenation by peroxidases [lactoperoxidase (LPX), chloroperoxidase, thyroid peroxidase, and horseradish peroxidase] and hydrogen peroxide was investigated by use of para-substituted thiobenzamides and thioanisoles. The rate constants for thiobenzamide oxygenation by LPX/H2O2 were found to correlate with calculated vertical ionization potentials, suggesting rate-limiting single-electron transfer between LPX compound I and the organosulfur substrate. The incorporation of oxygen from 18O-labeled hydrogen peroxide, water, and molecular oxygen into sulfoxides during peroxidase-catalyzed S-oxygenation reactions was determined by LC- and GC-MS. All peroxidases tested catalyzed essentially quantitative oxygen transfer from 18O-labeled hydrogen peroxide into thiobenzamide S-oxide, suggesting that oxygen rebound from the oxoferryl heme is tightly coupled with the initial electron transfer in the active site. Experiments using H2(18)O2, 18O2, and H2(18)O showed that LPX catalyzed approximately 85, 22, and 0% 18O-incorporation into thioanisole sulfoxide oxygen, respectively. These results are consistent with a active site controlled mechanism in which the protein radical form of LPX compound I is an intermediate in LPX-mediated sulfoxidation reactions.     

Formation of cobalt protoporphyrin in the liver of rats. A mechanism for the inhibition of liver haem biosynthesis by inorganic cobalt.

1. Treatment of rats with small doses of CoCl2 decreases liver 5-aminolaevulinate synthase (EC 2.3.1.37) activity and impairs incorporation of 5-amino[14C]laevulinate into liver haem. Salts of other metals (cadmium, nickel, manganese and zinc) are all relatively inactive. 2. The dose-response curves obtained for both these effects closely mirror the accumulation in the liver of a compound that is labelled by 5-amino[14C]laevulinate and is unextractable by acetone/HCl. 3. Incorporation of 5-amino[14C]laevulinate into unextractable compound is also obtained in vitro by incubating liver homogenates with label in the presence of cobalt:isotope-dilution experiments show that the radioactivity passes through pools of porphobilinogen and protoporphyrin, but not of haem. 4. The unextractable compound is not covalently bound to protein and possesses the same extraction and spectral properties as authentic cobalt protoporphyrin. 5. It is concluded (a) that cobalt protoporphyrin is readily formed not only in vitro, but also in vivo, and (b) that its formation accounts for the impaired incorporation of 5-aminolaevulinate into haem and may also be responsible for the action of cobalt on 5-aminolaevulinate synthase.     

The purification and some equilibrium properties of the nitrite reductase of the bacterium Wolinella succinogenes.

The bacterium Wolinella succinogenes produces a nitrite reductase enzyme that can be purified to homogeneity in high yield by a combination of detergent extraction, hydroxyapatite chromatography and Mr fractionation. Nitrite reductase activity is found to be present in both a high- and a low-Mr fraction. The high-Mr fraction has been shown to consist of the low-Mr nitrite reductase enzyme associated with a hydrophobic 'binding protein'. The amino acid composition for both proteins is reported. The nitrite reductase enzyme shows spectral characteristics indicative of the presence of c-type haem groups. Measurements at 610 nm indicate the presence of some high-spin haem groups at neutral pH. This haem subgroup undergoes a pH-linked high-spin - low-spin transition at alkaline pH. Approximately two of the six haem groups present within the enzyme bind CO with low affinity (KD = 0.4 mM). The enzyme also shows a range of redox activities with various inorganic reagents. The enzyme has been shown to exhibit dithionite reductase, oxygen reductase and CO2 reductase activities.     

Evidence that in chick embryos destruction of hepatic microsomal cytochrome P-450 haem is a general mechanism of induction of delta-aminolaevulinate synthase by porphyria-causing drugs.

A variety of prophyrinogenic compounds were tested for their effect in ovo on chick-embryo liver microsomal cytochrome P-450 haem concentration and mitochondrial delta-aminolaevulinate synthase activity. With all drugs tested, there was a 30--50% decrease in cytochrome P-450 haem concentration within 1 h of treatment, and this was closely followed by an increase in delta-aminolaevulinate synthase activity. The relationship was independent of the extent of enzyme induction and is consistent with the proposal that drug-mediated destruction of cytochrome P-450 haem is the primary mechanism of induction of delta-aminolaevulinate synthase. After induction, synthesis of delta-aminolaevulinate synthase could be maintained by inhibiting further haem synthesis. These studies suggest that induction of porphyria is a combination of two distinct processes: (a) induction of delta-aminolaevulinate synthase synthesis by destruction of cytochrome P-450 haem and consequent depletion of cellular free haem; (b) maintenance of continued delta-aminolaevulinate synthase synthesis by preventing replenishment of cellular haem either by inhibiting haem synthesis and/or by promoting continuous removal of newly synthesized haem.     

The mechanism of the oxidation of d-amphetamine by rabbit liver oxygenase. Oxygen-18 studies

The oxidation of d-amphetamine by rabbit liver microsomes has been studied using oxygen-18 as the source of oxygen. Incorporation of heavy oxygen into the two major metabolites phenylacetone oxime and phenylacetone, was 93–95% and 25–31% respectively. These data are consistant with a mechanism in which the initial step is the hydroxylation of the substrate at the carbon atom α to the amino group. The carbinol amine which is formed by this reaction then serves as the key intermediate from which ketone and oxime are formed. Thus, oxime can form from carbinol amine in two step, (1) dehydration of carbinol amine and (2) oxygenation of the resulting imine. Phenylacetone can form by two pathways (1) loss of a molecule of ammonia from carbinol amine (incorporation of oxygen from molecular oxygen) and (2) hydrolysis of oxime (incorporation of oxygen from water). In the case of d-amphetamine the hydrolytic route appears to be the more important as suggested by Hucker, et al. (4, 5).     

Electron-transport pathway of the NADH-dependent haem oxygenase system of rat liver microsomal fraction induced by cobalt chloride.

The hepatic microsomal haem oxygenase activity of rats treated with CoCl2 was studied kinetically by measuring biliverdin, the immediate product of the reaction. Biliverdin was extracted with diethyl ether/ethanol mixture, and was determined by the difference between A690 and A800. The apparent Km value for NADPH (at 50 microM-haematin) was about 0.2 microM when an NADPH-generating system was used, whereas that for NADH was about 630 microM. Essentially the same Vmax. values were obtained for both the NADH- and NADPH-dependent haem oxygenase reactions. No synergism was observed with NADH and NADPH. The NADH-dependent reaction was competitively inhibited by NADP+, with a Ki of about 10 microM. The inhibitoin of the NADH-dependent reaction by the antibody against rat liver microsomal NADPH-cytochrome c reductase was essentially complete, with a pattern similar to that of the NADPH-dependent reaction. The immunochemical experiment and the comparison of the kinetic values with the reported data on isolated NADH-cytochrome b5 reductase and NADPH--cytochrome c reductase indicated the involvement of the latter enzyme in NADH-dependent haem oxygenation by microsomal fraction in situ.     

The IsdG‐family of haem oxygenases degrades haem to a novel chromophore

Enzymatic haem catabolism by haem oxygenases is conserved from bacteria to humans and proceeds through a common mechanism leading to the formation of iron, carbon monoxide and biliverdin. The first members of a novel class of haem oxygenases were recently identified in Staphylococcus aureus (IsdG and IsdI) and were termed the IsdG‐family of haem oxygenases. Enzymes of the IsdG‐family form tertiary structures distinct from those of the canonical haem oxygenase family, suggesting that IsdG‐family members degrade haem via a unique reaction mechanism. Herein we report that the IsdG‐family of haem oxygenases degrade haem to the oxo‐bilirubin chromophore staphylobilin. We also present the crystal structure of haem‐bound IsdI in which haem ruffling and constrained binding of oxygen is consistent with cleavage of the porphyrin ring at the β‐ or δ‐meso carbons. Combined, these data establish that the IsdG‐family of haem oxygenases degrades haem to a novel chromophore distinct from biliverdin.