Breakdown Of Heme Research Articles

Disruption of hepatic heme biosynthesis after interaction of xenobiotics with cytochrome P‐450

Heme biosynthesis in hepatocytes is controlled by a free heme pool, which regulates delta-aminolevulinic acid synthase. Porphyrinogenic chemicals deplete the regulatory free heme pool by interacting with cytochrome P-450 thereby inhibiting heme biosynthesis and/or causing heme breakdown. Recent developments allow us to predict which groups of chemicals are likely to be porphyrinogenic. One group is exemplified by 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine. Heterocyclic compounds of this type cause mechanism-based inactivation of cytochrome P-450, leading to the formation of N-alkylporphyrins, with ferrochelatase-inhibitory activity resulting in lowering the free heme pool. Allylisopropylacetamide exemplifies a second group. Such compounds containing a terminal olefinic or acetylenic group, cause mechanism-based inactivation of cytochrome P-450. In the process, the heme moiety of cytochrome P-450 is destroyed and the free heme pool is lowered. A third group is exemplified by planar polyhalogenated or polycyclic aromatic hydrocarbons. These compounds induce specific cytochrome P-450 isozymes but are poor substrates. Active oxygen is formed, which interacts with a hepatic substrate to form a uroporphyrinogen decarboxylase inhibitor. Inhibition of this enzyme leads to depletion of the free heme pool.

The effect of hypoxia on hepatic cytochromes and heme turnover in rats in vivo.

We evaluated the effect of hypoxia (7% v/v) on hepatic heme turnover in vivo and microsomal heme protein content in male Sprague-Dawley rats. Hepatic heme protein turnover, measured as 14CO-production during continuous infusion of 5-14C-aminolevulinic acid, a precursor of nonerythrogenic heme, was decreased 60% during hypoxia and returned to control levels promptly after reoxygenation. Hepatic cytochrome P-450 content was decreased in hypoxic and 24-h reoxygenated animals. We conclude that normobaric hypoxia decreases hepatic cytochrome P-450 which could contribute to decreased drug metabolism in hypoxia. This decrease is probably due to heme oxygenase-independent breakdown of hepatic heme.

Regulation of hepatic haem metabolism. Disparate mechanisms of induction of haem oxygenase by drugs and metals.

We studied drug- and metal-mediated increases in activity of haem oxygenase, the rate-controlling enzyme for haem breakdown, in chick-embryo hepatocytes in ovo and in primary culture. Phenobarbitone and phenobarbitone-like drugs (glutethimide, mephenytoin), which are known to increase concentrations of an isoform of cytochrome P-450 in chick-embryo hepatocytes, were found to increase activities of haem oxygenase as well. In contrast, 20-methylcholanthrene, which increases the concentration of a different isoform of cytochrome P-450, had no effect on activity of haem oxygenase. Inhibitors of haem synthesis, 4,6-dioxoheptanoic acid or desferrioxamine, prevented drug-mediated induction of both cytochrome P-450 and haem oxygenase in embryo hepatocytes in ovo or in culture. Addition of haem restored induction of both enzymes. These results are interpreted to indicate that phenobarbitone and its congeners induce haem oxygenase by increasing hepatic haem formation. In contrast, increases in haem oxygenase activity by metals such as cobalt, cadmium and iron were not dependent on increased haem synthesis and were not inhibited by 4,6-dioxoheptanoic acid. We conclude that (1) induction of hepatic haem oxygenase activity by phenobarbitone-type drugs is due to increased haem formation, and (2) induction of haem oxygenase by drugs and metals occurs by different mechanisms.

Hepatic heme synthesis in a new model of experimental hemochromatosis: studies in rats fed finely divided elemental iron.

Rats fed chow containing finely divided elemental iron (from carbonyl-iron) develop hepatic iron overload resembling human hereditary hemochromatosis in that deposition of iron is primarily in periportal hepatocytes and with hepatic iron concentrations sufficiently high to be associated in the human disease with hepatic fibrosis or cirrhosis. In recent studies using this model, we reported changes in hepatic hemoproteins and heme oxygenase, the rate-controlling enzyme of heme breakdown. We now report effects of iron-loading on three enzymes of heme synthesis: 5-aminolevulinate synthase; the first and rate-controlling enzyme of the pathway, 5-aminolevulinate dehydrase (or porphobilinogen synthase), and uroporphyrinogen decarboxylase, the activity of which is decreased in porphyria cutanea tarda, a liver disease in which iron is known to play an important but still poorly understood role. Of the three enzymes, only activity of the dehydrase was altered by iron-loading: it was decreased significantly as early as 1 week after starting iron feeding, and with marked iron overload was 30 to 32% of control values. The degree of decrease was inversely related (r = -0.77 to -0.88) to the degree of iron overload and was partially reversed within 1 to 3 days when feeding of the iron-supplemented diet was stopped. The decrease in dehydrase activity was not attributable to lack of reduced glutathione or other disulfide-reducing agents or to zinc deficiency; nor was evidence found for inhibition by iron compounds or other possible inhibitors present in iron-loaded livers.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum pigmentation in Cyclopterus lumpus L

Female lumpsuckers, Cyclopterus lumpus L., have blue‐green blood serum and subcutaneous gelatinous tissue; the male's are red/magenta in colour. The female serum contains a chromoprotein with biliverdin as the prosthetic group; the male serum contains the same biliverdin pigment but this is normally masked by a red pigment, probably phycoerythrin. The female serum also contains the red pigment but at a much lower concentration than in the male. These bile pigments are probably formed by the breakdown of haem in the liver. Lumpsucker gall bladders are relatively small and do not enlarge during starvation. Storage of bile pigments in the serum and subcutaneous jelly may have arisen in response to the problems of regular long‐term starvation. The pigments now appear to have an additional sexual signalling function.

Hepatic heme and drug metabolism in rats with chronic mountain sickness.

Rats chronically exposed to hypobaric conditions develop pulmonary hypertension, right heart failure, hemoglobinemia, and in preliminary studies were recently found to have increased hepatic cytochrome P-450 content and activity of heme oxygenase, the rate-limiting enzyme for heme breakdown. To further delineate effects of chronic hypoxic, hypobaric exposure, on hepatic physiology and biochemistry, we have studied heme and drug metabolism in male Sprague-Dawley rats exposed to hypoxic conditions for 4-5 wk. Hypoxia, produced by exposure of rats to room air under hypobaric conditions (approximately 380 Torr), caused marked polycythemia [hematocrit (Hct) 70% vs. control Hct 43%], plasma hemoglobinemia, depletion of plasma haptoglobin, and decreased hemopexin concentrations. It also led to significant (20-30%) increases in concentrations of total hepatic heme and microsomal cytochrome P-450 and increased activities of heme oxygenase. In contrast, activity of 5-aminolevulinate synthase, the rate-limiting enzyme of hepatic heme synthesis, was significantly decreased in hypoxic rats and was not as inducible as in control normoxic rats. Hypoxia did not alter the rest of the heme synthetic pathway, as shown by a normal rate of conversion of 5-aminolevulinate to heme. Hypoxic exposure had no effect on the concentration of hepatic cytochrome-b5 but decreased activity of NADPH-cytochrome c reductase. Rates of metabolism of aminopyrine, benzphetamine, ethoxyresorufin, and warfarin were similar in hepatic microsomes obtained from hypoxic and normoxic rats. Thus the oxygen-requiring processes of hepatic heme and drug metabolism were well maintained despite chronic profound hypoxia sufficient to cause cardiopulmonary complications.

High-performance liquid chromatographic separation and quantitation of tetrapyrroles from biological materials

We describe a rapid, reverse-phase HPLC procedure for separating and quantifying tetrapyrroles of biological interest. This procedure uses a 5-μm C 18 column and the mobile phase is ammonium phosphate (pH 3.5) with a methanol gradient that is increased from 61 to 100%. Detection is by absorbance at 405 nm or by fluorescence. Porphyrins, heme, and the heme breakdown products, biliverdin and bilirubin, can be separated from a single injection in 25 min. Injections can be made every 40 min. Limits of detection are about 0.1 pmol for porphyrins, 5 pmol for heme, and 10 pmol for biliverdin and bilirubin. We present examples of the use of the system for separating tetrapyrroles formed by primary cultures of chick embryo hepatocytes and homogenates of rat liver.

Management of children with hyperbilirubinemia and green teeth

Discoloration of the teeth is a well-known complication of tetracycline administration in the child with developing teethJ ,2 Reports of liquid iron preparations causing incorporation of a black iron stain into dental structures and pigment deposition in the teeth of patients with disorders of porphyrin metabolism have also been described? ,4 However, the presence of green pigmented teeth in a child is a rare finding? -7 Previously, green staining of the teeth occurred in a small minority of newborn infants who survived severe jaundice from erythroblastosis fetalis. Advances in the diagnosis and management of infants with extrahepatic biliary atresia and neonatal hepatitis have allowed a population of children with cholestatic liver disease to survive beyond infancy. These children are often plagued by long-standing cholestasis and lifelong jaundice. We report two cases of children with green pigmented primary teeth. The presumed cause of the tooth staining was deposition of bilirubin and heme breakdown products in the tooth structure. CASE REPORTS

Hepatic heme metabolism and cytochrome P450 in cirrhotic rat liver

Cytochrome P450 levels are often low in the cirrhotic liver but the reason for this has not been established. Because changes in heme metabolism may reduce hepatic levels of cytochrome P450, the relationship of heme turnover to cytochrome P450 levels has been examined in rats with cirrhosis. Cirrhosis was produced by repeated carbon tetrachloride inhalation. In animals with cirrhosis, hepatic microsomal cytochrome P450 content was significantly Jess (32%) than in controls. Heme synthesis was assessed by measuring the activity of mitochondrial δ-amino-levulinic acid synthetase and also by determining the incorporation (within 30 min) of radiolabeled δ-aminolevulinic acid into the microsomal heme fraction. Both these parameters were normal in rats with CCl4-induced cirrhosis. In addition, the activity of microsomal heme oxygenase, the rate-limiting enzyme in catabolism of heme to bilirubin, was not altered. Cytochrome P450 heme degradation was then determined directly by injecting radiolabeled δ-aminolevulinic acid and measuring radioactivity in CO-binding particles (microsomes incubated with protease to remove cytochrome b5) prepared at various times thereafter. By this method, the degradation rate of cytochrome P450 heme did not differ between rats with cirrhosis and controls. Finally, the availability of hepatic heme for formation of hemoproteins was deemed to be satisfactory in cirrhotic liver because tryptophan pyrrolase saturation was comparable with controls, and also because heme administered in vivo did not enhance hepatic clearance of the cytochrome P450 substrate antipyrine. The failure to find defective heme biosynthesis or accelerated heme breakdown and the evidence that heme is available in amounts that do not restrict hemoprotein formation indicate that aberrant heme metabolism is not the cause of low cytochrome P450 levels in this rat model of cirrhosis.

Heme metabolism in liver and spleen of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU)-treated rats

The effect of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), an anticancer alkylating agent of the nitrosourea group, on liver and spleen enzymes involved in the control of heme metabolism was studied. A single oral dose of 50 or 100 mg/kg CCNU caused a time-dependent loss in weight of both spleen and liver. Seven days after CCNU treatment (lOO mg/kg) the weights were at 45 and 65% of controls respectively. The activity of δ-aminolevulinic acid synthetase (ALA-S), the rate-limiting enzyme in heme biosynthesis, declined in spleen and liver to 11 and 24% of control values, respectively, 7 days after CCNU treatment. Heme oxygenase activity, the rate-limiting enzyme of heme breakdown, was moderately increased in liver and spleen following CCNU administration. In liver, heme oxygenase activity was 142% of control values at 24 hr, and in spleen the activity was 180% of controls at 1 week. Pretreatment of the animals with phenobarbital (PB) (40 mg/kg/day, i.p.) for 4 days caused a reversal in the decline of liver weight with no effect on the decline in spleen weight following CCNU treatment. Similarly, PB pretreatment reversed the decline in hepatic ALA-S activity after CCNU administration but had no effect on the decline in splenic ALA-S activity. This study indicates that CCNU causes significant decreases in the activity of enzymes of heme biosynthesis in spleen and liver. The CCNU hemotoxicity in the liver was reversed by PB pretreatment whereas the splenic hemotoxicity was unchanged.

Control of heme and cytochrome P-450 metabolism by inorganic metals, organometals and synthetic metalloporphyrins.

The heme-cytochrome P-450 complexes represent sensitive metabolic systems for examining the biological impact of metals on important cellular functions. Many metals, both in the inorganic form and bound to organic moieties, potently induce heme oxygenase, the rate limiting enzyme of heme degradation. The resulting increase in the rate of heme breakdown is reflected in a marked depression of cellular cytochrome P-450 content and impairment of the oxidative metabolism of natural and foreign chemicals dependent on this hemeprotein. Organometal complexes do not mimic in all their aspects the actions of the inorganic elements which they contain. For example, organotins, in contrast to inorganic tin, produce a prolonged induction response of heme oxygenase in the liver but not in the kidney. Co-protoporphyrin is a much more potent inducer of heme oxygenase in liver than is inorganic cobalt; and Sn-protoporphyrin inhibits heme oxygenase activity nearly completely, whereas inorganic tin is a powerful inducer of the renal enzyme. Contrasting effects on heme metabolism exist as well within the metalloporphyrin species as demonstrated by the effects in vivo of Co-protoporphyrin and Sn-protoporphyrin on heme oxygenase activity; the former induces the enzyme whereas the latter potently inhibits it. In vitro, however, both compounds competitively inhibit heme oxidation activity. These differences, among others which characterize metal actions in vivo and in vitro attest to the importance of pharmacokinetic, adaptive and other host factors in defining the responses of the heme-cytochrome P-450 systems to the impact of metals in the whole animal.

A novel method for continuous monitoring of bilirubin production in unstressed rats.

We describe a device for continuous infusion and monitoring of exhaled 14CO as a test of hepatic bilirubin production in rats. A Silastic catheter, implanted into a jugular vein under light ether anesthesia, was protected with a spring shield and a cannula swivel. The animals were kept in a modified Bollman cage. delta-[5-14C]aminolevulinic acid, a heme precursor yielding 14CO upon breakdown of heme to bilirubin, was infused at a constant rate. Exhaled 14CO was oxidized to 14CO2 and collected in ethanolamine. The efficiency of the system averaged 97.8%. In untreated animals 14CO production reached a plateau within 12 h; thereafter, it increased by 2.8% per day. The responsiveness of the system was tested by fasting the animals, which stimulated hepatic bilirubin production. Fasting increased 14CO production by 32.8 +/- 8% (mean +/- SD, P less than 0.005) after 72 h. This was associated with an increase in hepatic heme oxygenase activity (+48%, P less than 0.05) and a decrease in microsomal cytochrome P-450 content (-45%, P less than 0.05). Thus, our approach permits continuous monitoring of hepatic bilirubin production without subjecting the animals to the stress of handling, restraint, or anesthesia. The method can easily be applied to other breath tests involving formation of 14CO2.

Glutathione and hepatic mixed-function oxidase activity.

It is apparent that hepatic GSH may function in drug metabolism not only as a substrate for conjugation but also in regulation of cytochrome P-450 activity. The remarkable aspect of the latter activity is its specificity. Loss of hepatic GSH depresses N-demethylation of DAB while ring hydroxylation is unaffected. On the other hand, the effect is to some degree nonspecific in that control as well as PB- or MC-induced N-demethylation is inhibited. Thus the response may not simply be specific to one isozyme of cytochrome P-450 but may be associated with one aspect of the enzymic activity of several cytochrome P-450 isozymes (i.e., N-demethylation). We have postulated that sensitivity of this activity to lipid peroxidation underlies the relationship to GSH since the tripeptide serves as a major protection against hepatic lipid peroxidation and its consequences. It is as yet not clear as to how or why this particular aspect of P-450 activity is more sensitive to lipid peroxidation than are other activities such as ring hydroxylation. Ongoing investigations include attempts to identify the cytochrome P-450 isozyme(s) which inhibit this response to GSH depletion. GSH-lipid peroxidation relationships have already been reported with isolated hepatocytes, and there may be a possible connection between this and the relative instability of cytochrome P-450 in cultured hepatocytes. Another factor which may be involved is heme oxygenase activity, which is markedly induced in the liver after GSH depletion, after cobalt administration (which also depresses cytochrome P-450 activity), and during incubation of isolated hepatocytes. This enzyme catalyzes the rate-limiting step in heme breakdown and may contribute to the loss of cytochrome P-450 activity associated with GSH depletion.

Absence of effects of lead feedings and growth-retardation on mitochondrial and microsomal cytochromes in the developing brain

Heme-containing enzymes were measured by absorption spectroscopy in mitochondria and microsomes isolated from the brains of PbCO 3-fed rat pups. Brain mitochondrial cytochromes also were measured in lead-fed adult rats and in growth-retarded pups. In normal animals, brain mitochondrial and microsomal cytochrome levels increased about twofold from 1 week of age to adulthood. In pups fed lead from birth, the resulting encephalopathy occurred most frequently in the first 2 weeks. Cerebellar mitochondrial cytochromes were decreased but only after 4 weeks of feedings. In pups fed lead from 2 weeks of age, the encephalopathy occurred after 2 weeks of feedings. There were no decreases in cerebral or cerebellar mitochondrial cytochromes. In both groups of pups, brain microsomal cytochromes tended to be higher in lead-fed than in control animals. Lead-feedings did not alter adult brain mitochondrial cytochrome levels. Brain mitochondrial levels also were unchanged in growth-retarded compared to normal rat pups. We conclude that the cytochrome concentrations of mitochondria and microsomes from both the immature and adult rat brain are relatively unaffected by factors such as inorganic lead exposure, known to influence pathways of heme breakdown and synthesis in other tissues. Second, changes in brain mitochondrial cytochrome contents are not associated with the development of lead encephalopathy in the rat pup.

Orientation of oxygen in oxyhaemoproteins and its implications for haem catabolism

Haem is degraded to bile pigments in the catabolism of haemoproteins in mammals and in the formation of photosynthetic pigments in algae. The first stage of this reaction involves oxygen attack at one of the four methene-bridge carbon atoms, which is ultimately eliminated as CO(ref. 1). The four bridges are not sterically equivalent (Fig. 1) and the bilirubin in mammalian bile and algal bile pigments consists almost exclusively of the alpha-isomers. Little is known about the structures of the ring-cleaving enzymes responsible, although microsomal haem oxygenase, which catalyses the breakdown of haem to biliverdin in mammals, has very similar spectroscopic properties to myoglobin. The degradation process has been simulated in vitro by a 'coupled oxidation' method in which the proportions of the four possible isomeric products depend on the nature of the globin moiety to which the haem is bound. We report here the use of an interactive computer display system to explore the relative accessibilities of the four methene bridges to a haem-bound oxygen molecule in myoglobin and in the alpha and beta chains of haemoglobin. Our calculated interaction energies agree well with the proportions of the four isomers that are observed experimentally.

Degradation of cytochrome P-450 haem by carbon tetrachloride and 2-allyl-2-isopropylacetamide in rat liver in vivo and in vitro. Involvement of non-carbon monoxide-forming mechanisms.

Degradation of intrinsic hepatic [(14)C]haem was analysed as (14)CO formation in living rats and in hepatic microsomal fractions prepared from these animals 16h after pulse-labelling with 5-amino[5-(14)C]laevulinic acid, a precursor that labels bridge carbons of haem in non-erythroid tissues. NADPH-catalysed peroxidation of microsomal lipids in vitro (measured as malondialdehyde) was accompanied by loss of cytochrome P-450 and microsome-associated [(14)C]haem (largely cytochrome P-450 haem), but little (14)CO formation. No additional (14)CO was formed when carbon tetrachloride and 2-allyl-2-isopropylacetamide were added to stimulate lipid peroxidation and increase loss of cytochrome P-450 [(14)C]haem. Because the latter effect persisted despite inhibition of lipid peroxidation with MnCl(2) or phenyl-t-butylnitrone(a spin-trapping agent for free radicals), it was concluded that carbon tetrachloride, as reported for 2-allyl-2-isopropylacetamide, may promote loss of cytochrome P-450 haem through a non-CO-forming mechanism independent of lipid peroxidation. By comparison with breakdown of intrinsic haem, catabolism of [(14)C]methaemalbumin by microsomal haem oxygenase in vitro produced equimolar quantities of (14)CO and bilirubin, although these catabolites reflected only 18% of the degraded [(14)C]haem. This value was increased to 100% by addition of MnCl(2), which suggests that lipid peroxidation may be involved in degradation of exogenous haem to products other than CO. Phenyl-t-butylnitrone completely blocked haem oxygenase activity, which suggests that hydroxy free radicals may represent a species of active oxygen used by this enzyme system. After administration of carbon tetrachloride or 2-allyl-2-isopropylacetamide to labelled rats, hepatic [(14)C]haem was decreased and haem oxygenase activity was unchanged; however, (14)CO excretion was either unchanged (carbon tetrachloride) or decreased (2-allyl-2-isopropylacetamide). These changes were unaffected by cycloheximide pretreatment. From the lack of parallel losses of cytochrome P-450 [(14)C]haem and (14)CO excretion, one may infer that an important fraction of hepatic [(14)C]haem in normal rats is degraded by endogenous pathways not involving CO. We conclude that carbon tetrachloride and 2-allyl-2-isopropylacetamide accelerate catabolism of cytochrome P-450 haem through mechanisms that do not yield CO as an end product, and that are insensitive to cycloheximide and independent of haem oxygenase activity.

Inhibition by cycloheximide of degradation of cytochrome P-450 in primary cultures of adult rat liver parenchymal cells and in vivo.

Degradation of cytochrome P-450 was studied in adult rat liver parenchymal cells in primary monolayer culture. In cells incubated in standard culture medium, the amount of cytochrome P-450 decreased at an accelerated rate relative to either the rate of degradation of total protein in the cells or the turnover of cytochrome P-450 in vivo. This change was succeeded by a spontaneous increase in the activity of haem oxygenase, an enzyme system that converts haem into bilirubin in vitro, measured in extracts from the cultured cells. This finding suggests that the rate of cytochrome P-450 breakdown may be controlled by factor(s) other than the activity of haem oxygenase. The decline in cytochrome P-450 and the subsequent increase in haem oxygenase activity was prevented by incubation of hepatocytes in medium containing an inhibitor of protein synthesis such as cycloheximide, puromycin, actinomycin D, or azaserine. The effect of cycloheximide appeared to be due to decreased breakdown of microsomal (14)C-labelled haem. By contrast, cycloheximide was without effect on the degradation of total protein, measured either in homogenates or in microsomal fractions prepared from the cultured cells. These results suggest that the conditions of cell culture stimulate selective degradation of cytochrome P-450 by a process that is inhibited by cycloheximide and hence may require protein synthesis. The findings in culture were verified in parallel studies of cytochrome P-450 degradation in vivo. After administration of bromobenzene, the degradation of the haem moiety of cytochrome P-450 was accelerated in vivo in a manner resembling that observed in cultured hepatocytes. Administration of cycloheximide to either bromobenzene-treated rats or to untreated rats decreased the degradation of the haem moiety of cytochrome P-450. However, the drug failed to affect degradation of haem not associated with cytochrome P-450, suggesting that cycloheximide is not a general inhibitor of haem oxidation in the liver. These findings confirm that the catabolism of hepatic cytochrome P-450 haem is controlled by similar cycloheximide-sensitive processes in the basal steady state in vivo, as stimulated by bromobenzene in vivo, or in hepatocytes under the conditions of cell culture. We conclude that the rate-limiting step in this process appears to require protein synthesis and precedes cleavage of the haem ring.

Comparative aspects of bile pigment formation and excretion

Breakdown of haem which is of key importance in most organisms, involves oxidative CO-evolving cleavage of the macrocyclic ring with formation of biliverdin-IX. In two major pathways established so far formation of biliverdin-IXα is followed by (a) biliary secretion or (b) reduction to bilirubin-IXα, formation of more hydrophilic derivatives (usually glycosidic conjugates) and biliary secretion. The scattered comparative information available indicates marked species variation with regard to the methin-bridge carbon atom removed from haem and the metabolic site of cleavage, the nature of bilirubin conjugates and the developmental sequence of maturation of enzyme activities and transport proteins involved in the chain of events leading from breakdown of haem to the excretion of the final end products.

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.

The mechanism of haem catabolism. A study of haem breakdown in spleen microsomal fraction and in a model system by 18O labelling and metal substitution.

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.