Non-enzymic nature of the pyridine haemochrome-cleaving activity of mammalian tissue extracts (`haem α-methyl oxygenase'')

1. The pyridine haemochrome-cleaving activity of extracts from mammalian liver and other tissues is shown conclusively to be entirely non-enzymic in nature and attributable to coupled oxidation with ascorbate. 2. Reduced glutathione probably contributes to the activity indirectly by continuously regenerating the ascorbate to the reduced form. 3. The cleavage shows no specificity for the alpha-methine bridge of pyridine haemochrome. 4. Results are presented suggesting some probable reasons for the erroneous characterization of the activity as an alpha-methine-specific haem-cleaving enzyme (;haem alpha-methenyl oxygenase') by Nakajima and co-workers (e.g. Nakajima, Takemura, Nakajima & Yamaoka, 1963; Nakajima & Gray, 1967).     

Reactivities of hydroxylamine and sodium bisulphite with carbonyl-containing haems with the prosthetic groups of the erythrocyte green haemoproteins

The reactivities of alkaline NH(2)OH and neutral NaHSO(3) with carbonyl and olefinic groups conjugated with the tetrapyrrole nucleus of haems were studied. The reactions were carried out with 2-3nmol of haem a, spirographis haem, isospirographis haem, 2,4-diacetyldeuterohaem and protohaem. Vinyl side chains were found to be insensitive to the chemical action of both alkaline NH(2)OH and neutral NaHSO(3). The formyl-containing haems reacted rapidly with both reagents at room temperature, as evidenced by sizable hypsochromic shifts of the reduced pyridine haemochrome spectrum. In less alkaline solution, the reactions of these formyl-containing haems with NH(2)OH were much slower. 2,4-Diacetyldeuterohaem reacted with alkaline NH(2)OH, but not with neutral NaHSO(3). These rapid, simple and straightforward tests are readily usable in differentiating among formyl, acetyl and other electron-withdrawing side chains conjugated with the tetrapyrrole ring of haems. We applied these observations to an investigation of the two unique prosthetic groups of the bovine erythrocyte green haemoproteins. The prosthetic groups of these two proteins were isolated and spectrally characterized. Under the conditions used, the haems did not react with either NH(2)OH or NaHSO(3), but were altered by dithionite, suggesting that the previous interpretation that a formyl group was present [Hultquist, Dean & Reed (1976) J. Biol. Chem.251, 3927-3932] may have been premature. These studies also provide evidence that the alpha-hydroxyfarnesylethyl side chain of haem a affects the alpha-band maximum, but not the beta- or Soret bands of the reduced pyridine haemochrome spectrum of haem a.     

Mesohaem substitution reveals how haem electronic properties can influence the kinetic and catalytic parameters of neuronal NO synthase

NOSs (NO synthases, EC 1.14.13.39) are haem-thiolate enzymes that catalyse a two-step oxidation of L-arginine to generate NO. The structural and electronic features that regulate their NO synthesis activity are incompletely understood. To investigate how haem electronics govern the catalytic properties of NOS, we utilized a bacterial haem transporter protein to overexpress a mesohaem-containing nNOS (neuronal NOS) and characterized the enzyme using a variety of techniques. Mesohaem-nNOS catalysed NO synthesis and retained a coupled NADPH consumption much like the wild-type enzyme. However, mesohaem-nNOS had a decreased rate of Fe(III) haem reduction and had increased rates for haem-dioxy transformation, Fe(III) haem-NO dissociation and Fe(II) haem-NO reaction with O2. These changes are largely related to the 48 mV decrease in haem midpoint potential that we measured for the bound mesohaem cofactor. Mesohaem nNOS displayed a significantly lower Vmax and KmO2 value for its NO synthesis activity compared with wild-type nNOS. Computer simulation showed that these altered catalytic behaviours of mesohaem-nNOS are consistent with the changes in the kinetic parameters. Taken together, the results of the present study reveal that several key kinetic parameters are sensitive to changes in haem electronics in nNOS, and show how these changes combine to alter its catalytic behaviour.     

Oxidative stress promotes degradation of the Irr protein to regulate haem biosynthesis in Bradyrhizobium japonicum

The haem proteins catalase and peroxidase are stress response proteins that detoxify reactive oxygen species. In the bacterium Bradyrhizobium japonicum, expression of the gene encoding the haem biosynthesis enzyme delta-aminolevulinic acid dehydratase (ALAD) is normally repressed by the Irr protein in iron-limited cells. Irr degrades in the presence of iron, which requires haem binding to the protein. Here, we found that ALAD levels were elevated in iron-limited cells of a catalase-deficient mutant, which corresponded with aberrantly low levels of Irr. Irr was undetectable in wild-type cells within 90 min after exposure to exogenous H2O2, but not in a haem-deficient mutant strain. In addition, Irr did not degrade in response to iron in the absence of O2. The findings indicate that reactive oxygen species promote Irr turnover mediated by haem, and are involved in iron-dependent degradation. We demonstrated Irr oxidation in vitro, which required haem, O2 and a reductant. A truncated Irr mutant unable to bind ferrous haem does not degrade in vivo, and was not oxidized in vitro. We suggest that Irr oxidation is a signal for its degradation, and that cells sense and respond to oxidative stress through Irr to regulate haem biosynthesis.     

Crystal structure of protoporphyrinogen IX oxidase: a key enzyme in haem and chlorophyll biosynthesis

Protoporphyrinogen IX oxidase (PPO), the last common enzyme of haem and chlorophyll biosynthesis, catalyses the oxidation of protoporphyrinogen IX to protoporphyrin IX. The membrane-embedded flavoprotein is the target of a large class of herbicides. In humans, a defect in PPO is responsible for the dominantly inherited disease variegate porphyria. Here we present the crystal structure of mitochondrial PPO from tobacco complexed with a phenyl-pyrazol inhibitor. PPO forms a loosely associated dimer and folds into an FAD-binding domain of the p-hydroxybenzoate-hydrolase fold and a substrate-binding domain that enclose a narrow active site cavity beneath the FAD and an alpha-helical membrane-binding domain. The active site architecture suggests a specific substrate-binding mode compatible with the unusual six-electron oxidation. The membrane-binding domains can be docked onto the dimeric structure of human ferrochelatase, the next enzyme in haem biosynthesis, embedded in the opposite side of the membrane. This modelled transmembrane complex provides a structural explanation for the uncoupling of haem biosynthesis observed in variegate porphyria patients and in plants after inhibiting PPO.     

Tryptophan pyrrolase in haem regulation. Experiments with administered haematin and the relationship between the haem saturation of tryptophan pyrrolase and the activity of 5-aminolaevulinate synthase in rat liver.

1. Administration of haematin to rats decreases 5-aminolaevulinate synthase activity in whole liver homogenates. 2. An inverse relationship between this decrease and the increase in saturation of apo-(tryptophan pyrrolase) with haem is observed during the initial phase of treatment with haematin. 3. Significant changes in both functions are caused by a 1 mg/kg dose of haematin, whereas the maximum effects are achieved by the 5 mg/kg dose. 4. Prevention by allopurinol of the conjugation of exogenously administered haematin with apo-(tryptophan pyrrolase) renders this haem available for further repression of 5 aminolaevulinate synthase. 5. The various aspects of the relationship between synthase activity and the haem saturation of tryptophan pyrrolase are discussed.     

Oxidation and haem loss kinetics of poly(ethylene glycol)-conjugated haemoglobin (MP4): dissociation between in vitro and in vivo oxidation rates

Haemoglobin-based oxygen carriers can undergo oxidation of ferrous haemoglobin into a non-functional ferric form with enhanced rates of haem loss. A recently developed human haemoglobin conjugated to maleimide-activated poly(ethylene glycol), termed MP4, has unique physicochemical properties (increased molecular radius, high oxygen affinity and low cooperativity) and lacks the typical hypertensive response observed with most cell-free haemoglobin solutions. The rate of in vitro MP4 autoxidation is higher compared with the rate for unmodified SFHb (stroma-free haemoglobin), both at room temperature (20-22 degrees C) and at 37 degrees C (P<0.001). This appears to be attributable to residual catalase activity in SFHb but not MP4. In contrast, MP4 and SFHb showed the same susceptibility to oxidation by reactive oxygen species generated by a xanthine-xanthine oxidase system. Once fully oxidized to methaemoglobin, the rate of in vitro haem loss was five times higher in MP4 compared with SFHb in the fast phase, which we assign to the beta subunits, whereas the slow phase (i.e. haem loss from alpha chains) showed similar rates for the two haemoglobins. Formation of MP4 methaemoglobin in vivo following transfusion in rats and humans was slower than predicted by its first-order in vitro autoxidation rate, and there was no appreciable accumulation of MP4 methaemoglobin in plasma before disappearing from the circulation. These results show that MP4 oxidation and haem loss characteristics observed in vitro provide information regarding the effect of poly(ethylene glycol) conjugation on the stability of the haemoglobin molecule, but do not correspond to the oxidation behaviour of MP4 in vivo.     

Helix movements and the reconstruction of the haem pocket during the evolution of the cytochrome c family

Analysis of cytochromes c (tuna), c2 (Rhodospirillum rubrum), c550 (Paracoccus denitrificans) and c551 (Pseudomonas aeruginosa) shows that they contain 48 residues identifiable as homologous from superposition of the structures. The other 34 to 64 residues are in loops that vary greatly in sequence, length and conformation, or in alpha-helices that are found in only some of the structures. Of the 48 homologous residues, 17 are in three segments which pack onto the haem faces. In all four structures, these segments have the same conformations, and the same locations relative to the haem. The other 31 residues are in three alpha-helices which are in contact with each other. These form the back and one side of the haem pocket. In cytochrome c551 the positions of the three alpha-helices have shifted and rotated, in comparison with cytochromes c and c2, by up to 5 A and 25 degrees relative to the haem. These shifts, facilitated by mutations at the helix-helix interfaces, are related to the reconstruction of the propionic acid side of the haem pocket described by Almassy & Dickerson (1978). Together these effects produce alternative structures for the haem pocket. This mechanism of adaptation to mutation contrasts with that observed in the globins. In the globins, mutations also produce changes in helix interfaces and shifts of packed helices, but in the globins these shifts are coupled to conserve the structure of the haem pocket.     

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.     

Terminal steps of haem biosynthesis

The terminal three steps in haem biosynthesis are the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX, followed by the six-electron oxidation of protoporphyrinogen to protoporphyrin IX, and finally the insertion of ferrous iron to form haem. Interestingly, Nature has evolved distinct enzymic machinery to deal with the antepenultimate (coproporphyrinogen oxidase) and penultimate (protoporphyrinogen oxidase) steps for aerobic compared with anaerobic organisms. The terminal step is catalysed by the enzyme ferrochelatase. This enzyme is clearly conserved with regard to a small set of essential catalytic residues, but varies significantly with regard to size, subunit composition, cellular location and the presence or absence of a [2Fe-2S] cluster. Coproporphyrinogen oxidase and protoporphyrinogen oxidase are reviewed with regard to their enzymic and physical characteristics. Ferrochelatase, which is the best characterized of these three enzymes, will be described with particular emphasis paid to what has been learned from the crystal structure of the Bacillus subtilis and human enzymes.     

Redox, haem and CO in enzymatic catalysis and regulation

The present paper describes general principles of redox catalysis and redox regulation in two diverse systems. The first is microbial metabolism of CO by the Wood-Ljungdahl pathway, which involves the conversion of CO or H2/CO2 into acetyl-CoA, which then serves as a source of ATP and cell carbon. The focus is on two enzymes that make and utilize CO, CODH (carbon monoxide dehydrogenase) and ACS (acetyl-CoA synthase). In this pathway, CODH converts CO2 into CO and ACS generates acetyl-CoA in a reaction involving Ni·CO, methyl-Ni and acetyl-Ni as catalytic intermediates. A 70 ? (1 ?=0.1?nm) channel guides CO, generated at the active site of CODH, to a CO 'cage' near the ACS active site to sequester this reactive species and assure its rapid availability to participate in a kinetically coupled reaction with an unstable Ni(I) state that was recently trapped by photolytic, rapid kinetic and spectroscopic studies. The present paper also describes studies of two haem-regulated systems that involve a principle of metabolic regulation interlinking redox, haem and CO. Recent studies with HO2 (haem oxygenase-2), a K+ ion channel (the BK channel) and a nuclear receptor (Rev-Erb) demonstrate that this mode of regulation involves a thiol-disulfide redox switch that regulates haem binding and that gas signalling molecules (CO and NO) modulate the effect of haem.     

Oxidation of glycine by Phaseolus leghaemoglobin with associated catabolic reactions at the haem.

Leghaemoglobin from the root nodules of kidney bean (Phaseolus vulgaris) reacts in alkaline glycine solutions as a glycine oxidase in a reaction that may also be regarded as a coupled oxidation. Leghaemoglobin is reduced to the ferrous form by glycinate, the oxygen complex is formed, and finally the haem is attacked to yield a green reaction product. Glycine is simultaneously oxidized to glyoxylate, and hydrogen peroxide is generated. The initial velocity of the formation of the green product is proportional to the concentrations of leghaemoglobin and glycine, and the optimum pH for the reaction is 10.2. The green product is not formed if carbon monoxide, azide of imidazole is bound to the haem, whereas oxidation of glycine to glyoxylate is not inhibited by azide and not essentially by carbon monoxide. Haem breakdown is activated by digestion of leghaemoglobin by carboxypeptidase, and partly inhibited by catalase and superoxide dismutase.     

Effect of anaerobiosis and glucose on the content of haem and its precursors in intact yeast cells

The content of haem and its precursors was determined in yeast cells grown under various conditions. The cells grown aerobically on 2% galactose contain about three times more haem (about 300 nmoles/g dry wt.) than the cells grown on 10% glucose. A trace amount of haem was found in anoxia irrespective of the carbon source used. The "efficiency" of the first enzyme of the haem biosynthetic pathway--delta-aminolevulinic acid (ALA) synthetase--expressed as the sum of all intermediates of the pathway in the cells grown on galactose, is similar in anaerobic and aerobic cells. The "efficiency" of the second enzyme--ALA dehydratase--is lower about three times both in anoxia and under conditions of glucose repression. In anoxia, not haem but delta-aminolevulinic acid is the main biosynthetic product. The role of glucose repression and of the feedback mechanisms in regulation of haem synthesis in yeast is discussed. A method for haem determination in the intact yeast cells, based on the formation of pyridine haemochrome, is described.     

Kinetics of haem binding to human albumin and haemopexin: nonspecific interactions of haem with proteins

The kinetics of haem binding to human serum albumin and haemopexin were studied by means of the stopped flow technique. The reaction could be divided into three kinetically clearly distinguished steps: (1) extremely fast reaction of haem with nonspecific binding sites on the surface of the apoprotein molecule; this type of haem binding site seems to exist in proteins in general; (2) by meaas of equilibrium with its monomer, haem is transferred to the specific binding site; this second order reaction takes about 1–2 s, the reaction rate constant amounts to ≈10⁶ l mol^?1 s^?1 both for albumin and haemopexin: (3) conformational changes of haemoprotein molecule, accompanied by changes of absorption spectra in the Soret region; this series of slow monomolecular reactions takes about 20 min. These results are discussed in connection with the mechanism of haem transport from blood to liver cells.     

Phenylhydrazine-mediated induction of haem oxygenase activity in rat liver and kidney and development of hyperbilirubinaemia. Inhibition by zinc-protoporphyrin.

Phenylhydrazine was found to be a potent inducer of microsomal haem oxygenase activity in rat liver and kidney, but not in spleen. The phenylhydrazine-mediated increase in haem oxygenase activity was time-dependent. Maximum activity was attained 12h after treatment in the liver, and 24h after treatment in the kidney. The increases in the activity of haem oxygenase in the liver and the kidney could be inhibited by cycloheximide. Furthermore, the increases could not be elicited by the treatment of microsomal preparations in vitro with phenylhydrazine. In consonance with the increased haem oxygenase activity, a marked increase (16-fold) was observed in the serum total bilirubin concentration in phenylhydrazine-treated rats. The mechanism of haem degradation promoted by phenylhydrazine in vivo appears to differ from that in vitro; only in the former case is bilirubin formed as the end-product of haem degradation. When rats were given zinc-protoporphyrin (40 mumol/kg) 12h before and after phenylhydrazine treatment, the phenylhydrazine-mediated increases in haem oxygenase activity in the liver and the kidney were effectively blocked. Treatment of rats in vivo with the metalloporphyrin also inhibited the activity of splenic haem oxygenase, and promoted a major decrease in the serum bilirubin levels. In phenylhydrazine-treated animals, the microsomal content of cytochrome P-450 was significantly decreased in the absence of a decrease in the microsomal haem concentration. The decrease in cytochrome P-450 content was accompanied by an increased absorption in the 420nm region of the reduced CO-difference spectrum, suggesting the conversion of the cytochrome to an inactive form. The marked depletion of cellular glutathione levels suggests that this conversion may be related to the action of active intermediates and free radicals formed in the course of the interaction of phenylhydrazine with the haem moiety of cytochrome P-450.     

Role of haem in the synthesis and assembly of cytochrome P-450

By using 3-amino-1,2,4-triazole, an inhibitor of haem synthesis, and 2-allyl-2-isopropylacetamide, a drug that degrades the haem moiety of cytochrome P-450, the involvement of haem in cytochrome P-450 synthesis and assembly was investigated. Phenobarbital was used to stimulate apo-(cytochrome P-450) synthesis. Degradation of preformed cytochrome P-450 haem does not result in a concomitant release of the apoprotein from the endoplasmic reticulum. The availability of haem for cytochrome P-450 synthesis in the normal animal is not rate-limiting. Prolonged inhibition of haem synthesis in vivo decreases the rate of apo-(cytochrome P-450) synthesis, although this effect is not discernible under conditions of short-term inhibition of haem synthesis. Under the former conditions exogenous haemin is able to counteract the decrease in the rate of apoprotein synthesis. In animals receiving successive injections of phenobarbital plus 3-amino-1,2,4-triazole, compared with those receiving phenobarbital only, the holo-(cytochrome P-450) content measured spectrally shows a greater decrease than could be accounted for by the decrease in the content of the total apoprotein. In addition to less haem being available under these conditions, the free apoprotein appears to have undergone some modification, such that its haem-binding capacity is considerably decreased. This particular effect could be due to a direct interaction of 3-amino-1,2,4-triazole or its metabolites with cytochrome P-450 rather than a consequence of haem deficiency. Apo-(cytochrome P-450) is capable of binding to the endoplasmic reticulum in a form and at a site, which can be reconstituted with haemin to yield the functional protein.     

Bile pigment--protein interactions. Coupled oxidation of cytochrome c

A new methodology is described for the chemical modification of the heme prosthetic group of horse heart cytochrome c. The selective modification of the heme moiety of cytochrome c is facilitated by utilizing coupling oxidation conditions. Comparison of the absorption spectra of this chemically modified cytochrome c species in two different solvents (aqueous pyridine and carbon monoxide saturated 6 M guanidinium chloride) with those of two model compounds [bis(pyridine)(2,3,7,8,12,13,17,18-octaethyl-5-oxaporphyrinato)iron(II) tetrafluoroborate salt and (pyridine)carbonyl-(2,3,7,8,12,13,17,18-octaethyl-5-oxaporphyrinato)iron(II) tetrafluoroborate salt] shows that coupled oxidation of cytochrome c affords a new protein with a covalently bound iron(II) oxaporphyrin prosthetic group. Amino acid analysis of this protein-bound iron(II) oxaporphyrin species reveals that only limited modification of the primary structure of the apoprotein occurs during coupled oxidation of cytochrome c. This protein-bound iron(II) oxaporphyrin species is also interconvertible to a protein-bound bilatriene species under hydrolytic conditions. The synthetic utility of the coupled oxidation of cytochrome c for the preparation of chromoproteins which possess covalently bound iron(II) oxaporphyrin and bilatriene prosthetic groups is considered.     

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.     

The mechanism of haem degradation in vitro. Kinetic evidence for the formation of a haem-oxygen complex.

The rate of haem degradation in aqueous pyridine shows a saturation dependence on O2 concentration. CO competitively inhibits the reaction. This is evidence for formation of an iron-O2 complex and is consistent with an intramolecular pathway for haem degradation.     

Oxidation of myosin by haem proteins generates myosin radicals and protein cross-links

Previous studies have reported that myosin can be modified by oxidative stress and particularly by activated haem proteins. These reactions have been implicated in changes in the properties of this protein in food samples (changes in meat tenderness and palatability), in human physiology (alteration of myocyte function and force generation) and in disease (e.g. cardiomyopathy, chronic heart failure). The oxidant species, mechanisms of reaction and consequences of these reactions are incompletely characterized. In the present study, the nature of the transient species generated on myosin as a result of the reaction with activated haem proteins (horseradish peroxidase/H2O2) and met-myoglobin/H2O2) has been investigated by EPR spectroscopy and amino-acid consumption, product formation has been characterized by HPLC, and changes in protein integrity have been determined by SDS/PAGE. Multiple radical species have been detected by EPR in both the presence and the absence of spin traps. Evidence has been obtained for the presence of thiyl, tyrosyl and other unidentified radical species on myosin as a result of damage-transfer from oxidized myoglobin or horseradish peroxidase. The generation of thiyl and tyrosyl radicals is consistent with the observed consumption of cysteine and tyrosine residues, the detection of di-tyrosine by HPLC and the detection of both reducible (disulfide bond) and non-reducible cross-links between myosin molecules by SDS/PAGE. The time course of radical formation on myosin, product generation and cross-link induction are consistent with these processes being interlinked. These changes are consistent with the altered function and properties of myosin in muscle tissue exposed to oxidative stress arising from disease or from food processing.