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Mammalian haem oxygenase — A tale of two enzymes
118 PII: S0307-4412(96)00184-7 MAMMALIAN
HAEM OXYGENASE m A TALE OF TWO ENZYMES
ANN ROWE and JENNIFER D HOUGHTON
D e p a r t m e n t of Biochemistry and M o l e c u l a r Biology University o f L e e d s L e e d s LS2 9JT, U K Introduction
Haem oxygenase is a monooxygenase enzyme, requiring both molecular oxygen and a reducing agent, to act. Originally the role of haem oxygenase in mammals was thought to be solely concerned with the turnover of haemoglobin from senescent red blood cells. Old and damaged blood cells are removed from the circulation and degraded largely by phagocytes in the spleen, liver and bone marrow. Consequently, large amounts of haemoglobin must be degraded every day. Haem arising from erythrocyte catabolism, and from the breakdown of other haemoproteins such as cytochromes, is degraded by haem oxygenase to bile pigments in a reaction which liberates carbon monoxide? The isolation in the mid 1980s of the cDNA for haem oxygenase, and the subsequent elucidation of its sequence enabled a more detailed characterisation of the enzyme, and has revealed sequence homology with a number of haemoproteins. 2 In 1986, it was reported that there are two forms of haem oxygenase which differ in several biochemical properties) These two forms were named haem oxygenase-1 (HO-1) and haem oxygenase-2 (HO-2) and have now been reported to be isozymes arising from different genes? Our understanding of haem oxygenase has rapidly progressed over recent years. Once thought to be just a single enzyme system for the degradation of haem into bile pigments, its two isozymes are now implicated in a diverse array of physiological functions. HO-1, the inducible isozyme is the principal haem degrading form of the enzyme. 1 HO-1 can be induced by a variety of substances including its substrate haem? Recently, HO-1 was reported to be induced by heat shock. 6 Increased transcription of HO-1 m R N A and the consequent rise in levels of HO-1 protein may protect the cell against the effects of elevated temperatures. HO-1 is also a marker of oxidative stress and is found to be induced by a number of oxidative agents such as ultraviolet irradiation, hydrogen peroxide 7 and phagocytosis induced oxidant stress. 8 Free radicals produced by exposure to oxidative agents results in an increase in cellular transcription of HO-1 mRNA, 9 supporting the role of HO-1 as a cellular defence against oxidant damage. In contrast, HO-2 is non-inducible, 3 and is localised predominantly in the brain suggesting its major function is not haem catabolism. 4 The localisation of HO-2 to areas also containing guanyl cyclase led to the suggestion that HO-2 may synthesise carbon monoxide for use as a neurotransmitter. 1° HO-2 synthesised carbon monoxide has been reported to mediate several physiological roles including vasodilation of blood vessels, 11inhibition of platelet aggregation, 12 and may participate in long-term potentiation? 3
Haem catabolism by haem oxygenase-1
Haem oxygenase has traditionally been reported as the enzyme catalysing the rate limiting step in the degradation of haem to form equimolar amounts of the bile pigment biliverdin, carbon monoxide and iron 1 (Fig 1). In reptiles, birds and amphibians, biliverdin is excreted directly. In mammals, however, biliverdin is further converted to bilirubin by biliverdin reductase and conjugated to glucuronate before it is excreted. 14 HO-1 is believed to be the original haem oxygenase enzyme system identified, as it is found predominantly in the spleen and liver consistent with its role in haem degradation, especially that derived from senescent red blood cells. 5 The molecular weight of HO-1 has been disputed due to the confusion regarding the existence of two isoforms of the enzyme, but it is now believed to be approximately 30000 Da in rats 4 and 33000 Da in humans. 15 Reconstituted enzyme systems using purified haem oxygenase 16 and NADPH-cytochrome P-450 reductase 17 have been used to investigate the mechanism of HO-1 catalysis. As a result an outline of the haem degradation pathway has been postulated (Fig 2). In the first step of the reaction ferric (Fe3+) haem binds to an amino acid residue
BIOCHEMICAL EDUCATION 25(3) 1997
119 on the HO-1 enzyme through the fifth co-ordination position of the Fe atom forming a haem-HO-1 complex. 16The ferric iron is subsequently reduced by NADPH-cytochrome P-450 reductase, to the ferrous (Fe z+) state, TM enabling a molecule of oxygen to bind to the complex. 19The resulting ferrous haem-HO-1 complex is then reduced by NADPHcytochrome P-450 reductase to yield an activated oxygen species bound to the haem. It is this activated oxygen species which then initiates haem degradation by hydroxylation of the c~-methene bridge carbon atom, yielding ~-hydroxyprotohaem. The existence of this intermediate has been demonstrated using in vivo radioactivity studies in which ~-hydroxyprotohaem was found to be converted to, and excreted as bilirubin in rats. z° In
Haemoglobin
HAEM
Per°xidases - - m ~ N _ N
Myoglobin
N.~
m - - Catalases ¥
P Cytochromes
~t~ / i N i ~ [ ~ p
Tryptophan Pyrrolase
m 02
NADPH Haem oxygenase
~--q~
CO NADPH-cytochrome P-450 reductase
N A D H ~ Fe
BILIVERDIN-1Xot m
v
H
m
p
p
m
H
m
v
H
NADPH m = Methyl v Vinyl p Propionic acid
Biliverdin reductase
NADP
H
H
H
H
BILIRUBIN-1Xot
Figure 1 Schematic representation of the pathway of haem catabolism in mammals showing the various sources of haem BIOCHEMICAL EDUCATION 25(3) 1997
120 v
In
¥
In
m
In
m
in
v
m
v ~ p m
NADPH NADH a-hydroxyhaem
m
ct-oxyhaem
NAo°; NADH~
v m I n ~ P o
Fe ~,~
v
-
v ~ p m
o
v m
Biliverdin
m
02
mo~~P
J
~
p
~O
688 mm intermediate (unknown structure)
Iron-biliverdin complex
Figure 2 Postulated sequence of intermediates in the degradation of haem to biliverdin the next step of the reaction two further atoms of oxygen are incorporated into this intermediate to yield the linear tetrapyrrole, biliverdin. The methene carbon atom is simultaneously liberated as carbon monoxide21 and the iron atom is released making it available for incorporation into new haemoproteins. Induction of haem oxygenase-1
The use of tin protoporphyrin in the prevention of neonatal hyperbilirubinaemia
HO-1 is inducible by its substrate haem s as well as many other non-haem substances. Metal ions, 22 chemicals such as bromobenzene and trinitrotoluene23 and hormones such as adrenalin and insulin24 are known inducers of HO-1. Various environmental factors such as ultra violet (UV) radiation, 25 heat shock 26 and oxidative stress 27 have also more recently been reported to induce the expression of HO-1. In contrast various compounds are known to inhibit the activity of HO-1. Synthetic metalloporphyrins (eg tin, cobalt, manganese and zinc porphyrins) have been reported to bind more tightly to HO-1 than haem and therefore are competitive inhibitors of the enzyme.28 These competitive inhibitors can have clinically important implications, and tin-protoporphyrin is now used to prevent neonatal hyperbilirubinemia.29
Many new born infants suffer from jaundice. This is a result partly of the rapid degradation of foetal haemoglobin which occurs as it is replaced by adult haemoglobin, and also due to the low levels of bilirubin conjugating enzymes present in the new born liver. As a result of these factors free bilirubin builds up in the blood stream, causing a characteristic yellowing of the skin and of the whites of the eyes. Bilirubin itself is toxic and lipid soluble and is capable of crossing the blood brain barrier. The most serious problem associated with neonatal jaundice is irreversible brain damage as a result of bilirubin accumulation. For this reason neonatal jaundice has always been monitored closely, and a range of therapies have been used to reduce the concentration of circulating bilirubin. Until recently the most common treatment was by UV-light treatment of the skin. This causes photolysis of the bilirubin molecule and produces a water soluble product which is then excreted in the urine. However recent concerns about the links between UV-irradiation and cancer have led a drive to develop alternative therapy strategies. One example of this is in the study of tin protoporphyrin IX as a haem oxygenase inhibitor in new born rats. 29 The effect of this compound is to reduce the rate at which bilirubin is formed, so that the rate of formation does not exceed the rate of the conjugating enzymes, and free bilirubin does not accumulate. This pilot study showed a decrease in the concentration of serum bilirubin within one day following birth, and the effect persisted throughout the 42-day study period. Clearly further studies would be needed to establish the efficacy and safety of this inhibitor in humans.
BIOCHEMICAL EDUCATION 25(3) 1997
121 The rat HO-1 gene is composed of 6830 nucleotides, organised into four introns and five exons. Primer extension analysis and S1 nuclease-mapping, have been used to identify the transcription initiation site and to characterise the 5'-flanking region of the HO-1 gene. 6 Haemin, the Fe 2+ oxidation product of haem, has been shown to strongly induce HO-1 activity and mRNA levels.26The treatment of rat glioma cells with haemin resulted in the accumulation of HO-1 after 1 h, reaching a peak after about 5 h. The maximum activity of HO-1 in haemin treated cells remained constant up to 7 h incubation. These figures were very similar to those obtained in rat glioma cells induced by heat shock the difference being that after heat treatment for 5 h, HO-1 activity decreased suggesting a more rapid turnover of HO-1 at elevated temperatures. 26 The use of cycloheximide and actinomycin D, which inhibit transcription and translation respectively, prevented the increase in HO-1 after haemin treatment. Actinomycin D alone completely inhibited the induction of HO-1 suggesting that haemin induces HO-1 at the transcriptional l e v e l . 26 Later studies using human macrophages confirmed the induction of HO-1 synthesis by its substrate producing a 10-fold increase in HO-1 activity after haemin treatment. 15 To elucidate the effect of metal ions and their porphyrin chelates on the levels of mRNA encoding HO-1, liver HepG2 and Hep3b hepatoma cells were induced by a variety of such agents3°. Five different metal ions were examined: C d 2+, CO2+, Zn 2+, S n 2+ and FeZ+,but only Sn 2+ produced a significant increase in HO-1 mRNA concentrations in both cell lines. The increase in HO-1 mRNA was dependent on the dose and the oxidation state of S n 2 +: thus Sn4+ did not induce the enzyme at all. This may indicate that the lower oxidation state is more stressful to the cell. The porphyrin chelates of Sn 2÷ and Co 2+ had opposing effects on the levels of HO-1 mRNA levels. 3° Sn-protoporphyrin did not induce HO-1 mRNA levels whereas Co-protoporphyrin markedly elevated the levels of HO-I. Sequence analysis of the rat HO-1 gene revealed a motif similar to that found in the metal regulatory element of the metallothionein gene. 6 Metal ions such a s S n 2+ may induce the promoter of the HO-1 gene by activation of a metal responsive transcription factor. 31Consistent with these suggestions is the finding that C d 2 + elevates the levels of HO-1 mRNA and metallothionein mRNA to the same extent, and with the same time course of induction. 3° Similar results have also been obtained with arsenate 32 and antimony.33 Amongst the most potent chemical inducers of HO-1 activity are bromobenzene and trinitrotoluene. Bromobenzene was found to produce an average 15-fold increase in enzyme activity in rats liver after 20 h but produced no increase in HO-1 activity in the kidney or spleen. 34 Induction of HO-1 by bromobenzene was blocked by cycloheximide but not by actinomycin D, suggesting that bromobenzene stimulates de novo synthesis at the level of translation. Trinitrotoluene administration was similarly found to induce HO-1 activity in rats after inter peritoneal injection. 23 Administration of insulin and adrenalin has been shown to induce haem oxygenase activity in the rat liver.24 The administration of these two hormones caused only moderate induction of haem oxygenase, comparable to induction by haemin. This suggests that hormone induction of haem oxygenase is mediated by haem, resulting from an increase of haem flowing into the liver from an unknown source. Human chorionic gonadotrophin hormone is also reported to induce HO-1 activity in rat testis. 35 The mechanism of this induction has not yet been elucidated. The effect of tissue injury on the activity of HO-1 is such that skin microsomal fractions are able to form bilirubin from haem when incubated with N A D P H . 36 The requirement for a cofactor, localised induction by haem and metals and the kinetics of the skin enzyme were found to be very similar to HO-1 in the liver. Furthermore skin fibroblasts, were shown to contain haem 37 and haem oxygenase activity inducible by metals. 38 It has therefore been concluded that the increase in skin haem oxygenase activity is a response to the cellular components (such as haemoglobin and myoglobin) released as a result of tissue damage and that skin HO-1 activity is regulated in the same way as that in other tissues.
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122 Bruising
Heat shock and oxidative stress
Reactive oxygen species
When skin is bruised, it exhibits a characteristic colour change varying from deep blue, through green to yellow before the pigment is finally cleared from the skin. We now know that the deep blue pigment is a haemoglobin derivative, the green pigment is biliverdin, and the yellow pigment is bilirubin. This change in colour suggests that skin is capable of oxidatively degrading haem to bilirubin, and that haem oxygenase is present in skin. Prompted by this observation, further studies have shown that haem oxygenase is indeed expressed in skin tissue, and may have an important role to play not only following stress by injury, but also in heat stress and in response to UV-irradiation.
Heat shock proteins (HSPs) are produced by many animal, plant and bacteria cells on exposure to elevated but non-lethal temperatures, and partially protect the cell against damage. The increase in the concentration of these proteins arises due to the activation of a small number of specific genes which remain non-expressed or only expressed at low levels when the cells are living at physiological temperatures. HO-1 has been implicated as a heat shock protein. The cloning and sequencing of the haem oxygenase gene from the rat revealed a variety of D N A regulatory sequences located in the 5'-flanking region. 6 Amongst these regulatory elements was a potential heat shock element (HSE), situated 273 base pairs upstream from the transcription start site. Consequently, a series of experiments were carried out by Shibahara and co-workers to investigate the effect of heat shock on HO-1. 26 Rat glioma cells were incubated at 42°C and the rate of synthesis of HO-1 was assayed by measuring the incorporation of [3H]-leucine. HO-1 activity began to increase after an incubation period of 1 h and reached a maximum after approximately 5 h. There was no significant increase in cells treated at 37°C over this period. R N A prepared from heat-treated cells was also examined and a similar result was obtained. Incubation of cells at 42°C resulted in an increase of m R N A after approximately 1 h and the maximum was obtained after a 3-h incubation period. There was no increase in HO-1 m R N A from cells incubated at 37°C, confirming rat HO-1 as a heat shock protein. In order to deduce the nature of the HO-1 heat shock response, Shibahara et al, carried out further incubation experiments using cycloheximide and actinomycin D. Actinomycin D inhibited the increase in HO-1 activity in heat treated cells completely which implies that heat shock induces HO-1 at the transcriptional level. Cycloheximide however only partially prevented the m R N A increase, which suggests that protein synthesis is necessary to achieve the full induction of mRNA. Recently HO-1 has been found to be induced by heat in human Hep3b c e l l s . 39 Oxidative stress results from a disturbance in the balance between the production of reactive oxygen species and a cells antioxidant defence mechanisms, a potentially harmful situation for the cell. Glutathione is involved in that it acts as a sulphydryl buffer keeping the cysteine residues of red cell proteins in the reduced state, thereby protecting the cells from oxidative damage. Many mammalian cells respond to oxidative stress by rapidly enhancing transcription of a number of genes, human HO-1 is believed to be one such gene. 4° The induction of HO-1 was shown to be affected by the redox state of the cell because lowering cellular glutathione leads to a significant increase in HO-1 mRNA. 41 When the level of glutathione is reduced the anti-oxidant defence system is also lowered, subsequent increases in the level of HO-1 in the cell therefore prevent oxidative damage. Hydrogen peroxide is generated by a reaction between two highly reactive species. A potentially damaging superoxide anion (02-) is formed by the one electron reduction of a molecule of oxygen. Protonation of the superoxide anion yields hydroperoxy radical (HO2) which, in turn, can react with a second superoxide anion to form hydrogen peroxide (H202). All these species can cause oxidative damage to a cell unless they are detoxified by enzymes such as catalase, which degrades hydrogen peroxide to oxygen and water. If hydrogen peroxide is allowed to accumulate inside a cell it can result in cell death, formation of D N A strand breaks, induction of sister chromatid crossovers42 and the induction of gene mutation? 3 Proteins which reduce oxygen, such as cytochrome oxidase, do not usually release superoxide anions so that cellular damage is avoided.
BIOCHEMICAL EDUCATION 25(3) 1997
123 However, a small amount of these species are formed during the oxidation of the ferrohaem (Fe 2+) group of haemoglobin to ferrihaem (Fe 3+) by oxygen. The treatment of human fibroblast cells with either hydrogen peroxide or ultraviolet A irradiation induces both HO-1 m R N A and protein in the cell: This increase in HO-1 activity has two effects. Firstly there is a reduction in the cellular pool of haem and haemoproteins by increasing their degradation. Secondly, the induction of HO-1 activity enables a cell to synthesise biliverdin and bilirubin (in the presence of biliverdin reductase). Unconjugated bilirubin is a potent antioxidant, scavenging singlet oxygen and reacting with superoxide anion and peroxyl radicals. 44 In scavenging two hydroperoxy radicals, bilirubin is oxidised to biliverdin which is then rapidly reconverted to bilirubin. For these reasons it is believed that the induction of HO-1 may be a general response to oxidative stress providing the cell with a defence mechanism against oxidative damage. Hydrogen peroxide is thought to produce its biological effects through a Fenton reaction (Fig 3) with naturally occurring iron complexes to yield the highly reactive hydroxyl radical. The Fenton reaction involves hydrogen peroxide and iron (II) salts reacting with a variety of organic molecules and causing a series of radical reactions. The evidence for this reaction mechanism comes from studies on mammalian cells using the membrane permeable iron chelator, o-phenanthroline. The presence of this iron chelator was shown to lower the H202 induction of both HO-1 m R N A and protein by between 50 and 80%. 45o-Phenanthroline prevents the Fenton reaction by rendering the iron unavailable for use in catalysis and thereby prevents generation of the hydroxyl radical. Glucose oxidase is a generator of hydrogen peroxide and consequently it is an oxidative stress agent. During recent experiments, glucose oxidase, the sulphydrylreactive heavy metal cadmium and the electrophilic agent DEM, have also been shown to induce HO-1 after 6-12 h exposure. 46 This further confirms the role of HO-1 in protection against oxidative stress. Both macrophages and neutrophils are phagocytic white blood cells which ingest and destroy foreign particles such as micro-organisms, cell debris and damaged erythrocytes. During phagocytosis, a significant change in oxygen utilisation occurs and superoxide ions (O2-.) and hydrogen peroxide (H202) are produced. These reduced oxygen species along with singlet oxygen (102) help to destroy the ingested micro-organism but may also cause oxidative damage to the host cells, possibly through the Fenton reaction. The The Fenton Reaction
Formation of the hydroxyl radical:
Fe2+ + H202
~ Fe3+ + OH" + OH-
Traces of Fe3+ can then react further with hydrogen peroxide:
Fe3+ + H202
.~ Fe2+ + 0 2 - + 2H+
Even more reactions are possible:
OH'+H20 2 ~
H 2 0 + H + + O 2-
0 2- + F e 3+ ~
Fe2 + + O 2
OH" + Fe2+ .
~ Fe3+ + HO-
Figure 3 The Fenton reaction, showing the formation of hydroxyl radicals (adapted from ref 58) B I O C H E M I C A L E D U C A T I O N 25(3) 1997
124 stress response of human macrophages during phagocytosis of Staphylococcus aureus was investigated 8. In the presence of iron de n o v o synthesis of HO-1 was induced. Other oxidative agents induced the same response in mouse macrophages. 47 Approximately 3% of the body's circulating haemoglobin is oxidised to methemoglobin daily. Neutrophils are a site of this oxidation reaction which results in the generation of a superoxide (02-) ion and potential oxidant stress. Exposure to methemoglobin, however, sensitises the host cell to oxidant damage via the initiation of HO-1 transcription and the cells become resistant to the action of oxidants. Phagocytosis mediated oxidant damage to vascular endothelium is likely to be involved in various vasculopathies including atherosclerosis and pulmonary leak syndromes such as adult respiratory distress syndrome. Vascular cells are frequently exposed to high oxidant concentrations derived from phagocytes. HO-1 appears to provide a cellular defence against this and is increased 50-fold in response to phagocytic induced oxidant stress. 48 UV radiation
UV-A radiation (from 320 to 380 nm) is a component of sunlight and is known to be carcinogenic to animals. The elucidation of the mechanism of cell damage by UV-A could have important clinical consequences in the prevention of human skin cancer. UV-A has been reported to increase HO-1 levels in the cell 7'6'45. The increase in the expression of HO-1 results from an enhanced transcription rate of the HO-1 gene as suggested by the elevated levels of HO-1 mRNA. 9 UV-A treatment of cells is known to lead to the formation of hydrogen peroxide (H202) and superoxide anion ( 0 2 ) . U V A induction of HO-1 was concluded to be mediated by a hydroxyl radical (OH') generated by the iron catalysed Fenton reaction. However, iron is also involved in reactions other than those which generate a hydroxyl radical, such reactions include decomposition of lipid hydroperoxides and auto oxidation of a number of biomolecules. Therefore, iron catalysed reactions could also lead to the generation of other active oxygen species such as the lipid peroxyl radical and singlet oxygen. In order to examine the involvement of both singlet oxygen and hydroxyl radicals in the UV-A induction of HO-1, two techniques were used? 9 Firstly, human skin fibroblasts were irradiated in the presence of deuterium oxide (D20), in which singlet oxygen has a longer half life. In D20 treated cells the wavelength-dependent increase of HO-1 is enhanced, suggesting that singlet oxygen generated by UV-A and near-visible light is the primary effector in the induction of the HO-1 gene. Secondly dimethyl sulphoxide (DMSO) and mannitol, which are effective scavengers of O H were used prior to irradiation of the cells. These were found to have no significant effect on the wavelength-dependent accumulation of HO-1. In conclusion, singlet oxygen constitutes an important intermediate in the signal transduction events which lead to the induction of HO-1 by UV-A and near-visible light. The mechanism of transcriptional activation of HO-1 by UV-A radiation was examined using nuclear extracts from untreated and UV-A treated human skin fibroblasts. 25 These were then assayed for their ability to bind to a 147 base pair fragment of the promoter of the HO-1 gene. A combination of gel retardation assays and DNase 1 footprinting analyses revealed a region within the promoter of the HO-1 gene that binds a specific protein complex present only in extracts from UV-A treated cells. This protein binds to the promoter of HO-1 and initiates transcription in response to oxidative stress.
Haem catabolism by haem oxygenase-2
The existence of a second isoform of haem oxygenase, HO-2, was first reported in 1986 as a non-inducible form of the enzyme? HO-2 is expressed constitutively throughout the body with a high concentration in the brain. 4 Purification of HO-2 from rat testes enabled a full characterisation of the enzyme 5° and HO-2 was found to differ from HO-1 in a number of ways including regulation, activation, immunochemical properties and molecular weight. HO-2 is the only detectable haem oxygenase species in adult rat brain, 51 although HO-1 may be induced by heat shock in some glial cells and certain neurones 52. The discrete localisation of HO-2 m R N A in areas of the brain containing guanyl cyclase, suggests that HO-2 may have a function in the synthesis of carbon monoxide for use as a neurotransmitter, l° Carbon monoxide (CO) is formed endogenously during haem catabolism by the action of haem oxygenase (Fig 4), the only known reaction which forms CO in the body.
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125 Haem Haemoxygenase > CO + Biliverdin+ Fe
membrane v diffusion ~ / ~ a n y (~ l cyclase
{/
oTP
ooMP
Figure 4 Carbon monoxide may enter cells by diffusion and mediate its physiological effects by stimulating guanyl cyclase. Guanyl cyclase then catalyses the formation of cGMP which functions as a second messenger CO has a number of physiological effects including the vasodilation of blood vessels 11 and the inhibition of platelet aggregation?2 Nitric oxide (NO), a recently discovered gaseous vasodilator mediates its effect by activating guanyl cyclase and consequently increasing cyclic guanosine 3,5-monophosphate (cGMP) levels. 53 CO is also able to activate guanyl cyclase by binding to its iron atom 12and it was therefore postulated that CO may play a physiological role similar to that of It has been demonstrated that zinc protoporphyrin, which inhibits haem oxygenase activity, depletes cGMP in primary cultures of rat olfactory neurones. 1° CO has also been demonstrated to inhibit the contractility of vascular smooth muscle by causing a time dependent increase in cGMP levels.11 In mammals the hippocampus, a part of the cerebral cortex in the brain, plays a special role in learning. Short bursts of repetitive firing of synaptic vesicles at the presynaptic cells cause long-term potentiation (LTP). Zinc protoporphyrin was shown to block the induction of LTP in hippocampal slices from adult male guinea pigs, 13 and in mice and rats, 55 providing evidence that CO is involved in LTP in mammals. Zinc protoporphyrin is also synthesised endogenously, formed when zinc replaces iron for insertion into protoporphyrin under iron deficient conditions and in lead poisoning.56 Endogenous zinc protoporphyrin may, by inhibiting haem oxygenase, modulate synthesis of CO from haem and therefore modulate LTP, possibly explaining the neurological problems associated with lead poisoning. 57 CO has also been shown to inhibit platelet aggregation. This inhibition can be overcome by illumination with light of the wavelength 400 nm, suggesting that CO may exert its inhibitory action by binding to a reduced haem-containing protein such as guanyl cyclase. In the presence of CO the intracellular levels of cGMP were increased dramatically. Studies with sodium nitroprusside, which activates guanyl cyclase, showed that platelet aggregation was sensitive to even the smallest increases in cGMP concentrations. This combination of experimental results has lead to the suggestion that CO probably inhibits platelet aggregation through a signal transducing pathway involving cGMP. 12
NO.54
Comparison of haem oxygenases-1 and -2
The two isoforms of haem oxygenase were first identified in 1986 in rat liver microsomes. 3 Initially, it was impossible to infer whether the two forms were isozymes, representing products of different genes or isoforms, having the same gene but representing products of post-transcriptional modification. Sequencing characterisation and subsequent comparison of the two haem oxygenase species has provided conclusive evidence that HO-1 and HO-2 are products of different genes. 4 Detailed studies of the two forms of the enzyme has revealed a number of biochemical differences between them. One of the first differences reported between the HO-1 and HO-2 was their different regulatory mechanisms. The activity of HO-1 can be increased up to 1000-fold in response to various agents including metals, = chemicals 34 and haematin. In contrast, the activity of HO-2 remained almost constant despite exposure to known inducers of H O - I ? Kinetic
BIOCHEMICAL EDUCATION 25(3) 1997
126 studies of the two isoforms have revealed a 3-fold difference in apparent Km values between the two isozymes. 3 Kinetic properties of haem oxygenases I and II
Although haem oxygenases I and II carry out the same chemical reaction. Their kinetic properties are different. The maximum velocity (gm) and the Michaelis constant (Vm~) values of the two isoenzymes arise as a result of very small changes in the three dimensional shape of the two isoenzymes, which is itself a result of their differing amino acid sequences. The overall effect of these differences is to produce a pair of enzymes whose maximum rates and substrate affinities are closely matched to their physiological roles. Thus haem oxygenase I, the main function of which is to catalyse the breakdown of haemoproteins, has a rather low Km and a high value for Vmax. This means that the enzyme is active even at low substrate concentrations, and its activity increases as the concentration of haem increases within the physiological range. Conversely haem oxygenase II, which functions to catalyse the production of pharmacological quantities of carbon monoxide, has a higher Km and a significantly lower value for Vinos.This means that the enzyme has low activity at low substrate concentrations, and even its maximum activity is limited to less than 10% of that of haem oxygenase I. The Km and V,,o~values of the two haem oxygenase isoenzymes are shown below (from Maines et al 1986 ref 3):
Isoenzyme HO-1 HO-2
Km
Vm~
0.24#m 0.67pm
3.4 pmol/mg/h 0.24 pmol/mg/h
For HO-1 the Km value was 0.24 #m whereas the value for HO-2 was 0.67 pm. The lower Km value for the inducible form of the enzyme, HO-2, is thought to have an important biological role especially under conditions when the level of endogenous haem substrate (eg cytochrome P-450) are low. Alternatively, the difference in Kmvalues could arise due to a different substrate specificity, for example HO-1 may exhibit a preference for a particular isomer of haem (eg haem a, b or c which have differently substituted porphyrin rings). Different types of haem are found in the various haemoproteins and therefore the differences in K,, could signify preferences of the two isozymes of haem oxygenase towards various haemoproteins. In addition to their regulatory properties, the tissue distribution of the two haem oxygenase isozymes also differs. It was the initial resistance of the testis and brain haem oxygenase to respond to inducers such as haem that led to the discovery of the non-inducible HO-2 which predominates in these organs? HO-1 is the predominant species in the spleen and after exposure of a tissue to inducing agents. The primary structures of HO-1 and HO-2 differ significantly. 4 Specifically, the two isozymes vary in their amino acid compositions, containing dissimilar amounts of asparagine, glycine, aspartate, histidine, isoleucine, leucine and cysteine residues. The most striking difference being that HO-2 contains three cysteine residues which are absent in HO-1. The molecular weights of the proteins differ by 6000 Da on sodium dodecyl sulphate electrophoresis gels. HO-1 has an apparent M, of 30000 Da while that of HO-2 is higher at 36000 Da? ° Discussion
The variety of roles assigned to haem oxygenase can, in part, be explained by the existence of two isozymes of the enzyme which have different regulatory mechanisms, sequences and properties. The principle role of HO-1 seems to be catabolism of haem from red blood cells and haemoproteins, but the evidence implicating HO-1 as a heat shock protein is increasing, and strongly suggests that induction occurs at the transcriptional level via a heat shock element. The evidence supporting induction of HO-1 by oxidative stress is also mounting. Further investigation into the nature of the induction of HO-1 is needed, in particular in characterising the cis-acting element which confers the inducibility to haemin.
BIOCHEMICAL EDUCATION 25(3) 1997
127 The principal role of HO-2 seems to be the production of carbon monoxide for use as a n e u r o t r a n s m i t t e r . H O - 2 p r o d u c e s c a r b o n m o n o x i d e i n t h e s a m e w a y as H O - 1 b y t h e degradation of haem but, due to the localisation of HO-2 to areas of the brain containing guanyl cyclase, the carbon monoxide can activate this second messenger system producing cGMP and thereby mediate numerous physiological processes. Future work may elucidate further the role of carbon monoxide in the activation of guanyl cyclase and its s u b s e q u e n t c e l l u l a r t a r g e t s .
References
1. Tenhunen R., Marver H. S. and Schmid R., The enzymatic conversion of heme to bilirubin by microsomal haem oxygenase. Proc. NatlAcad. Sci. (USA), 1968, 61, 748-755. 2. Shibahara S., Muller R. M., Taguchi H. and Yoshida T., Cloning and expression of cDNA for rat heme oxygenase. Proc. Natl Acad. Sci. (USA), 1985, 82, 7865-7869. 3. Maines M. D., Trakshel M. G. and Kutty K. R., Characterisation of two constitutive forms of rat liver microsomal heme oxygenase: only one molecular species of the enzyme is inducible. J. Biol. Chem., 1986, 261, 411-419. 4. Cruse I. and Maines M. D., Evidence suggesting that the two forms of heme oxygenase are products of different genes. J. BioL Chem., 1988, 263, 3348-3353. 5. Tenhunen R., Marver H. S. and Schmid R., The enzymatic catabolism of haemoglobin: stimulation of microsomal heme oxygenase by haemin. J. Lab. Clin. Med., 1970, 75, 410-421. 6. Muller R. M., Taguchi H. and Shibahara S., 1987) Nucleotide sequence and organisation of the rat heme oxygenase gene. J. Biol. Chem., 1987, 262, 6795-6802. 7. Keyse S. M. and Tyrrell R. M., Both near ultraviolet radiation and the oxidising agent hydrogen peroxide induce a 32KDa stress protein in human skin fibroblasts. J. BioL Chem., 1987, 262, 14821-14825. 8. Kantengwa S. and Polla B. S., Phagocytosis of Staphylococcus aureus induces a selective stress response in human monocytes-macrophages (Mphi). Infect. Immun., 1993, 61, 1281-1287. 9. Keyse S. M. and Tyrrell R. M., Induction of the haem oxygenase gene in human skin fibroblasts by hydrogen peroxide and UVA (365 nm) radiation: evidence for the involvement of the hydroxy radical. Carcinogenesis, 1990, 11, 787-791. 10. Verma A., Hirsch D. J., Glatt C. E., Ronnett G. V. and Synder S. H., Carbon monoxide: a putative neural messenger. Science, 1993, 259, 381-384. 11. Ramos K. S., Lin H. and McGrath J. J., Modulation of cyclic guanosine monophosphate levels in cultured aortic smooth muscle cells by carbon monoxide. Biochem. Pharmacol., 1989, 38, 1368-1370. 12. Brune B. and Ullrich V., Inhibition of platelet aggregation by carbon monoxide is mediated by the activation of guanylate cyclase. Molec. Pharm., 1987, 32, 497-504. 13. Zhuo M., Small S. A., Kandel E. R. and Hawkins R. D., Nitric oxide and carbon monoxide produce activitydependent long-term synaptic enhancement in hippocampus. Science, 1993, 260, 1946-1950. 14. Singleton J. W. and Laster L., Biliverdin reductase in guinea pig liver. J. Biol. Chem., 1965, 240, 4780-4789. 15. Yoshida T., Biro P., Cohen T., Muller R. M. and Shibahara S., Human heme oxygenase cDNA and induction of its mRNA by hemin. Eur. J. Biochem., 1988, 171, 457-461. 16. Yoshida T. and Kikuchi G., Purification and properties of heme oxygenase from pig spleen microsomes. J. Biochem., 1978, 253, 4224-4229. 17. Yasukochi Y., Peterson J. A. and Masters B. S. S., NADPH-cytochrome c P-450a reductase: Spectrophotometric stopped flow kinetic studies on the formation of reduced flavoprotein intermediates. J. Biol. Chem., 1979, 254, 7097-7104. 18. Schacter B. A., Nelson E. B., Marver H. S. and Masters B. S. S., Immunochemical evidence for an assosiation of heme oxygenase with the microsomal electron transport system. J. Biol. Chem., 1972, 247, 3601-3607. 19. Yoshida T., Noguchi M. and Kikuchi G., Oxygenated form and heme.heme oxygenase complex and requirement for a second electron to initiate heme degradation from the oxygenated complex. J. Biol. Chem., 1980, 255, 4418-4420. 20. Kondo T., Nicholson D. C., Jackson A. H. and Kenner G. W., Isotopic studies of the conversion of oxophlorins and their ferrihaems into bile pigments in the rat. Biochem. J., 1971, 121, 601-607. 21. Tenhunen R., Ross M., Marver H. S. and Schmid R., Microsomal heme oxygenase: characterisation of the enzyme. J. BioL Chem., 1969, 244, 6388-6394. 22. Maines M. D. and Kappas A., Metals as regulators of heme metabolism: physiological and toxicological implications. Science, 1977, 198, 1215-1221. 23. Tenhunen R., Zitting A, Nickels and Savolainen H., Trinitrotoluene-induced effects on rat haem metabolism. Exp. Mol. Pathol., 1984, 40, 362-366. 24. Bakken A. A., Thaler M. M. and Schmid R., Metabolic regulation of heme catabolism and biliverdin production. J. Clin. Invest., 1972, 51, 530-536. 25. Nascimento A. L., Luscher P. and Tyrrell R. M., Ultraviolet A (320-380 nm) radiation causes an alteration in the binding of a specific protein-protein complex to a short region of the promoter of the human heme oxygenase gene. Nucl. Acid. Res., 1993, 21, 1103-1109. 26. Shibahara S., Muller R. M. and Taguchi H., Transcriptional control of rat heine oxygenase by heat shock. J. Biol. Chem., 1987, 262, 12889-12892. 27. Tyrrell R. M. and Basu-Modak S., Transient enhancement of heme oxygenase-1 mRNA accumulation: a marker of oxidative stress in eukaryotic cells. Methods Enzymol., 1994, 234, 224-235. 28. Yoshinga .T, Sassa S. and Kappas A., Purification and properties of bovine spleen heme oxygenase: amino acid composition and sites of action of inhibitors of heme oxidation. J. BioL Chem., 1982, 257, 7778-7785. 29. Drummond G. S. and Kappas A., Prevention of neonatal hyperbilirubinemia by tin porphyrin-IX, a potent competitive inhibitor of heme oxidation. Proc. NatlAcad. Sci. (USA), 1981, 78, 6466-6470. 30. Mitani K., Fujita H., Fukuda Y., Kappas A. and Sassa S., The role of inorganic metals and metalloporphyrins in the induction of haem oxygenase and heat shock protein 70 in human hepatoma cells. Biochem. J., 1993, 290, 819-825.
B I O C H E M I C A L E D U C A T I O N 25(3) 1997
128 31. Alam J., Shibahara S. and Smith A., Transcriptional activation of the heme oxygenase gene by hemin and cadmium in mouse hepatoma cells. J. Biol. Chem., 1989, 264, 6371-6375. 32. Sardana M. K., Drummond G. S., Sassa S. and Kappas A., The potent heine oxygenase inducing action of arsenic and paraciticidal arsenicals. Pharmacol., 1981, 23, 247-253. 33. Drummond G. S. and Kappas A., Potent heme-degrading action of antimony and antimony-containing parasiticidal agents. J. Exp. Med., 1981, 153, 245-256. 34. Guzelian P. S. and Elshourbagy N. A., Induction of hepatic heme oxygenase activity by bromobenzene. Arch. Biochem. Biophys., 1979, 196, 178-185. 35. Kutty R. K. and Maines M. D., Selective induction of heme oxygenase-1 isozyme in rat testis by human chorionic gonadotrophin. Arch. Biochem. Biophys., 1989, 268, 100-107. 36. Maines M. D. and Cohn J., Bile pigment formation by skin heme oxygenase: studies on the response of the enzyme to heme compounds and tissue injury. J. Exp. Med., 1977, 145, 1054-1059. 37. Gemsa D., Woo C. H., Fudenberg H. H. and Schmid R., Erythrocyte catabolism by macrophages in vitro. The effect of hydrocortisone on erythrophagocytosis and on the induction of heme oxygenase. J. Clin. Invest, 1973, 52, 812-822. 38. Tenhunen R., Ross M., Marver H. S. and Schmid R., Reduced nicotinamide adenine-dinucleotide phosphatedependent biliverdin reductase: partial purification and characterisation. Biochem., 1970, 20, 298-303. 39. Mitani K., Fujita H., Sassa S. and Kappas A., Activation of heme oxygenase and heat shock protein 70 genes by stress in human hepatoma cells. Biochem. Biophys. Res. Commun., 1990, 166, 1429-1434. 40. Applegate L. A., Luscher P. and Tyrrell R. M., Induction of heme oxygenase: a general response to oxidant stress in cultured mammalian cells. Cancer Res., 1991, 51, 974-978. 41. Lautier D., Luscher P. and Tyrrell R. M., Endogenous glutathionone levels modulate both constitutive and UVA radiation/hydrogen peroxide inducible expression of the human heme oxygen gene. Carcinogenesis (London), 1992, 13, 227-232. 42. Larramendy R., Mello Filho A. C., Marlins E. A. L. and Meneghini R., Iron mediated induction of sister chromatid exchanges by hydrogen peroxide and superoxide anion. Mutat. Res., 1987, 187, 57-63. 43. Nassi-Calo L., Mello Filho A. C. and Meneghini R., O-phenanthroline protects mammalian cells from hydrogen peroxide-induced gene mutation and morphological transformation. Carcinogenesis, 1987, 10, 1055-1057. 44. Stocker R., Yamantoto Y., McDonagh A. F., Glazer A. N. and Ames B. N., Bilirubin is an antioxidant of possible physiological importance. Science, 1987, 235, 1043-1046. 45. Keyse S. M. and Tyrrell R. M., Haem oxygenase is the major 32 KDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide and sodium arsenite. Proc. Natl Acad. Sci., 1989, 86, 99-103. 46. Siow R. C., Ishii T., Sato H., Taketani S., Lecke D. S., Sweiry J. H., Pearson J. D., Bannai S. and Mann G. E., Induction of the antioxidant stress proteins heme oxygenase-1 and MSP23 by stress agents and oxidised LDL in cultured vascular smooth muscle cells. FEBS Lett., 1995, 368, 239-242. 47. Sato H., Ishii T., Sugita Y., Tateishi N. and Bannal S., Induction of a 28KDa stress protein by oxidative and sulphydryl reactive agents in mouse peritoneal macrophages. Biochim. Biophys. Acta, 1993, 1148, 127-132. 48. Balla J., Jacobs H. S., Balla G., Nath K. and Vercellotti G. M., Endothelial cell heme oxygenase and ferritin induction by heme proteins: A possible mechanism limiting shock damage. Trans. Assoc. Am. Phys., 1992, 105, 1-6. 49. Basu-Modak S. and Tyrrell R. M., Singlet oxygen: a primary effector in the ultraviolet A/near visible light induction of the human heme oxygenase gene. Cancer Res., 1993, 53, 4504-4510. 50. Trakshel M. G., Kutty K. R. and Maines M. D., Purification and characterisation of the major constitutive form of testicular heme oxygenase: the non-inducible isoform. J. Biol. Chem., 1986, 261, 11 131-11 137. 51. Trakshel M. G., Kutty K. R. and Maines M. D., Resolution of rat brain haem oxygenase activity. Absence of a detectable amount of the inducible form (HO-1). Arch. Biochem. Biophys., 1988, 260, 732-739. 52. Ewing J. F. and Maines M. D., Rapid induction of heme oxygenase-1 mRNA and protein by hyperthermia in rat brain: Heme oxygenase-2 is not a heat shock protein. Proc. NatlAcad. Sci. (USA), 1991, 88, 5364-5368. 53. Moncada S., Palmer R. M. and Higgs E. A., Nitric oxide: physiology, pathophysiology and pharmacology (Review). Pharmacol. Rev., 1995, 43, 109-142. 54. Marks G. S., Brien J. F., Nakatsu K. and Mclaughlin B. E., Does carbon monoxide have a physiological function (Review). Trends Pharmacol. Sci., 1991, 12, 185-188. 55. Stevens C. F. and Wang Y., Reversal of long term potentiation by inhibitors of heine oxygenase. Nature (London), 1993, 36, 149-417. 56. Marks G. S., Nakatsu K. and Brien J. F., Does endogenous zinc protoporphyrin modulate carbon monoxide formation of heme? Implications for long term potentiation, memory and cognitive function. Can. J. Physiol. Pharmacol., 1993, 71, 753-754. 57. Goyer, R. A., Toxic effects of metals. In Casarett and Doull, Toxicology. The Basic Science of Poisons, 3rd edn, Chap. 19, eds C. D. Klassen, M. O. Amdur and J. Doull. Mcmillan, New York, 1986, pp 582-535. 58. HaUiwell, B. and Gutteragc, J. M. C. Free Radicals in Biology and Medicine. Clarendon Press, Oxford, 1989.
BIOCHEMICAL EDUCATION 25(3) 1997
HAEM OXYGENASE m A TALE OF TWO ENZYMES
ANN ROWE and JENNIFER D HOUGHTON
D e p a r t m e n t of Biochemistry and M o l e c u l a r Biology University o f L e e d s L e e d s LS2 9JT, U K Introduction
Haem oxygenase is a monooxygenase enzyme, requiring both molecular oxygen and a reducing agent, to act. Originally the role of haem oxygenase in mammals was thought to be solely concerned with the turnover of haemoglobin from senescent red blood cells. Old and damaged blood cells are removed from the circulation and degraded largely by phagocytes in the spleen, liver and bone marrow. Consequently, large amounts of haemoglobin must be degraded every day. Haem arising from erythrocyte catabolism, and from the breakdown of other haemoproteins such as cytochromes, is degraded by haem oxygenase to bile pigments in a reaction which liberates carbon monoxide? The isolation in the mid 1980s of the cDNA for haem oxygenase, and the subsequent elucidation of its sequence enabled a more detailed characterisation of the enzyme, and has revealed sequence homology with a number of haemoproteins. 2 In 1986, it was reported that there are two forms of haem oxygenase which differ in several biochemical properties) These two forms were named haem oxygenase-1 (HO-1) and haem oxygenase-2 (HO-2) and have now been reported to be isozymes arising from different genes? Our understanding of haem oxygenase has rapidly progressed over recent years. Once thought to be just a single enzyme system for the degradation of haem into bile pigments, its two isozymes are now implicated in a diverse array of physiological functions. HO-1, the inducible isozyme is the principal haem degrading form of the enzyme. 1 HO-1 can be induced by a variety of substances including its substrate haem? Recently, HO-1 was reported to be induced by heat shock. 6 Increased transcription of HO-1 m R N A and the consequent rise in levels of HO-1 protein may protect the cell against the effects of elevated temperatures. HO-1 is also a marker of oxidative stress and is found to be induced by a number of oxidative agents such as ultraviolet irradiation, hydrogen peroxide 7 and phagocytosis induced oxidant stress. 8 Free radicals produced by exposure to oxidative agents results in an increase in cellular transcription of HO-1 mRNA, 9 supporting the role of HO-1 as a cellular defence against oxidant damage. In contrast, HO-2 is non-inducible, 3 and is localised predominantly in the brain suggesting its major function is not haem catabolism. 4 The localisation of HO-2 to areas also containing guanyl cyclase led to the suggestion that HO-2 may synthesise carbon monoxide for use as a neurotransmitter. 1° HO-2 synthesised carbon monoxide has been reported to mediate several physiological roles including vasodilation of blood vessels, 11inhibition of platelet aggregation, 12 and may participate in long-term potentiation? 3
Haem catabolism by haem oxygenase-1
Haem oxygenase has traditionally been reported as the enzyme catalysing the rate limiting step in the degradation of haem to form equimolar amounts of the bile pigment biliverdin, carbon monoxide and iron 1 (Fig 1). In reptiles, birds and amphibians, biliverdin is excreted directly. In mammals, however, biliverdin is further converted to bilirubin by biliverdin reductase and conjugated to glucuronate before it is excreted. 14 HO-1 is believed to be the original haem oxygenase enzyme system identified, as it is found predominantly in the spleen and liver consistent with its role in haem degradation, especially that derived from senescent red blood cells. 5 The molecular weight of HO-1 has been disputed due to the confusion regarding the existence of two isoforms of the enzyme, but it is now believed to be approximately 30000 Da in rats 4 and 33000 Da in humans. 15 Reconstituted enzyme systems using purified haem oxygenase 16 and NADPH-cytochrome P-450 reductase 17 have been used to investigate the mechanism of HO-1 catalysis. As a result an outline of the haem degradation pathway has been postulated (Fig 2). In the first step of the reaction ferric (Fe3+) haem binds to an amino acid residue
BIOCHEMICAL EDUCATION 25(3) 1997
119 on the HO-1 enzyme through the fifth co-ordination position of the Fe atom forming a haem-HO-1 complex. 16The ferric iron is subsequently reduced by NADPH-cytochrome P-450 reductase, to the ferrous (Fe z+) state, TM enabling a molecule of oxygen to bind to the complex. 19The resulting ferrous haem-HO-1 complex is then reduced by NADPHcytochrome P-450 reductase to yield an activated oxygen species bound to the haem. It is this activated oxygen species which then initiates haem degradation by hydroxylation of the c~-methene bridge carbon atom, yielding ~-hydroxyprotohaem. The existence of this intermediate has been demonstrated using in vivo radioactivity studies in which ~-hydroxyprotohaem was found to be converted to, and excreted as bilirubin in rats. z° In
Haemoglobin
HAEM
Per°xidases - - m ~ N _ N
Myoglobin
N.~
m - - Catalases ¥
P Cytochromes
~t~ / i N i ~ [ ~ p
Tryptophan Pyrrolase
m 02
NADPH Haem oxygenase
~--q~
CO NADPH-cytochrome P-450 reductase
N A D H ~ Fe
BILIVERDIN-1Xot m
v
H
m
p
p
m
H
m
v
H
NADPH m = Methyl v Vinyl p Propionic acid
Biliverdin reductase
NADP
H
H
H
H
BILIRUBIN-1Xot
Figure 1 Schematic representation of the pathway of haem catabolism in mammals showing the various sources of haem BIOCHEMICAL EDUCATION 25(3) 1997
120 v
In
¥
In
m
In
m
in
v
m
v ~ p m
NADPH NADH a-hydroxyhaem
m
ct-oxyhaem
NAo°; NADH~
v m I n ~ P o
Fe ~,~
v
-
v ~ p m
o
v m
Biliverdin
m
02
mo~~P
J
~
p
~O
688 mm intermediate (unknown structure)
Iron-biliverdin complex
Figure 2 Postulated sequence of intermediates in the degradation of haem to biliverdin the next step of the reaction two further atoms of oxygen are incorporated into this intermediate to yield the linear tetrapyrrole, biliverdin. The methene carbon atom is simultaneously liberated as carbon monoxide21 and the iron atom is released making it available for incorporation into new haemoproteins. Induction of haem oxygenase-1
The use of tin protoporphyrin in the prevention of neonatal hyperbilirubinaemia
HO-1 is inducible by its substrate haem s as well as many other non-haem substances. Metal ions, 22 chemicals such as bromobenzene and trinitrotoluene23 and hormones such as adrenalin and insulin24 are known inducers of HO-1. Various environmental factors such as ultra violet (UV) radiation, 25 heat shock 26 and oxidative stress 27 have also more recently been reported to induce the expression of HO-1. In contrast various compounds are known to inhibit the activity of HO-1. Synthetic metalloporphyrins (eg tin, cobalt, manganese and zinc porphyrins) have been reported to bind more tightly to HO-1 than haem and therefore are competitive inhibitors of the enzyme.28 These competitive inhibitors can have clinically important implications, and tin-protoporphyrin is now used to prevent neonatal hyperbilirubinemia.29
Many new born infants suffer from jaundice. This is a result partly of the rapid degradation of foetal haemoglobin which occurs as it is replaced by adult haemoglobin, and also due to the low levels of bilirubin conjugating enzymes present in the new born liver. As a result of these factors free bilirubin builds up in the blood stream, causing a characteristic yellowing of the skin and of the whites of the eyes. Bilirubin itself is toxic and lipid soluble and is capable of crossing the blood brain barrier. The most serious problem associated with neonatal jaundice is irreversible brain damage as a result of bilirubin accumulation. For this reason neonatal jaundice has always been monitored closely, and a range of therapies have been used to reduce the concentration of circulating bilirubin. Until recently the most common treatment was by UV-light treatment of the skin. This causes photolysis of the bilirubin molecule and produces a water soluble product which is then excreted in the urine. However recent concerns about the links between UV-irradiation and cancer have led a drive to develop alternative therapy strategies. One example of this is in the study of tin protoporphyrin IX as a haem oxygenase inhibitor in new born rats. 29 The effect of this compound is to reduce the rate at which bilirubin is formed, so that the rate of formation does not exceed the rate of the conjugating enzymes, and free bilirubin does not accumulate. This pilot study showed a decrease in the concentration of serum bilirubin within one day following birth, and the effect persisted throughout the 42-day study period. Clearly further studies would be needed to establish the efficacy and safety of this inhibitor in humans.
BIOCHEMICAL EDUCATION 25(3) 1997
121 The rat HO-1 gene is composed of 6830 nucleotides, organised into four introns and five exons. Primer extension analysis and S1 nuclease-mapping, have been used to identify the transcription initiation site and to characterise the 5'-flanking region of the HO-1 gene. 6 Haemin, the Fe 2+ oxidation product of haem, has been shown to strongly induce HO-1 activity and mRNA levels.26The treatment of rat glioma cells with haemin resulted in the accumulation of HO-1 after 1 h, reaching a peak after about 5 h. The maximum activity of HO-1 in haemin treated cells remained constant up to 7 h incubation. These figures were very similar to those obtained in rat glioma cells induced by heat shock the difference being that after heat treatment for 5 h, HO-1 activity decreased suggesting a more rapid turnover of HO-1 at elevated temperatures. 26 The use of cycloheximide and actinomycin D, which inhibit transcription and translation respectively, prevented the increase in HO-1 after haemin treatment. Actinomycin D alone completely inhibited the induction of HO-1 suggesting that haemin induces HO-1 at the transcriptional l e v e l . 26 Later studies using human macrophages confirmed the induction of HO-1 synthesis by its substrate producing a 10-fold increase in HO-1 activity after haemin treatment. 15 To elucidate the effect of metal ions and their porphyrin chelates on the levels of mRNA encoding HO-1, liver HepG2 and Hep3b hepatoma cells were induced by a variety of such agents3°. Five different metal ions were examined: C d 2+, CO2+, Zn 2+, S n 2+ and FeZ+,but only Sn 2+ produced a significant increase in HO-1 mRNA concentrations in both cell lines. The increase in HO-1 mRNA was dependent on the dose and the oxidation state of S n 2 +: thus Sn4+ did not induce the enzyme at all. This may indicate that the lower oxidation state is more stressful to the cell. The porphyrin chelates of Sn 2÷ and Co 2+ had opposing effects on the levels of HO-1 mRNA levels. 3° Sn-protoporphyrin did not induce HO-1 mRNA levels whereas Co-protoporphyrin markedly elevated the levels of HO-I. Sequence analysis of the rat HO-1 gene revealed a motif similar to that found in the metal regulatory element of the metallothionein gene. 6 Metal ions such a s S n 2+ may induce the promoter of the HO-1 gene by activation of a metal responsive transcription factor. 31Consistent with these suggestions is the finding that C d 2 + elevates the levels of HO-1 mRNA and metallothionein mRNA to the same extent, and with the same time course of induction. 3° Similar results have also been obtained with arsenate 32 and antimony.33 Amongst the most potent chemical inducers of HO-1 activity are bromobenzene and trinitrotoluene. Bromobenzene was found to produce an average 15-fold increase in enzyme activity in rats liver after 20 h but produced no increase in HO-1 activity in the kidney or spleen. 34 Induction of HO-1 by bromobenzene was blocked by cycloheximide but not by actinomycin D, suggesting that bromobenzene stimulates de novo synthesis at the level of translation. Trinitrotoluene administration was similarly found to induce HO-1 activity in rats after inter peritoneal injection. 23 Administration of insulin and adrenalin has been shown to induce haem oxygenase activity in the rat liver.24 The administration of these two hormones caused only moderate induction of haem oxygenase, comparable to induction by haemin. This suggests that hormone induction of haem oxygenase is mediated by haem, resulting from an increase of haem flowing into the liver from an unknown source. Human chorionic gonadotrophin hormone is also reported to induce HO-1 activity in rat testis. 35 The mechanism of this induction has not yet been elucidated. The effect of tissue injury on the activity of HO-1 is such that skin microsomal fractions are able to form bilirubin from haem when incubated with N A D P H . 36 The requirement for a cofactor, localised induction by haem and metals and the kinetics of the skin enzyme were found to be very similar to HO-1 in the liver. Furthermore skin fibroblasts, were shown to contain haem 37 and haem oxygenase activity inducible by metals. 38 It has therefore been concluded that the increase in skin haem oxygenase activity is a response to the cellular components (such as haemoglobin and myoglobin) released as a result of tissue damage and that skin HO-1 activity is regulated in the same way as that in other tissues.
BIOCHEMICAL EDUCATION 25(3) 1997
122 Bruising
Heat shock and oxidative stress
Reactive oxygen species
When skin is bruised, it exhibits a characteristic colour change varying from deep blue, through green to yellow before the pigment is finally cleared from the skin. We now know that the deep blue pigment is a haemoglobin derivative, the green pigment is biliverdin, and the yellow pigment is bilirubin. This change in colour suggests that skin is capable of oxidatively degrading haem to bilirubin, and that haem oxygenase is present in skin. Prompted by this observation, further studies have shown that haem oxygenase is indeed expressed in skin tissue, and may have an important role to play not only following stress by injury, but also in heat stress and in response to UV-irradiation.
Heat shock proteins (HSPs) are produced by many animal, plant and bacteria cells on exposure to elevated but non-lethal temperatures, and partially protect the cell against damage. The increase in the concentration of these proteins arises due to the activation of a small number of specific genes which remain non-expressed or only expressed at low levels when the cells are living at physiological temperatures. HO-1 has been implicated as a heat shock protein. The cloning and sequencing of the haem oxygenase gene from the rat revealed a variety of D N A regulatory sequences located in the 5'-flanking region. 6 Amongst these regulatory elements was a potential heat shock element (HSE), situated 273 base pairs upstream from the transcription start site. Consequently, a series of experiments were carried out by Shibahara and co-workers to investigate the effect of heat shock on HO-1. 26 Rat glioma cells were incubated at 42°C and the rate of synthesis of HO-1 was assayed by measuring the incorporation of [3H]-leucine. HO-1 activity began to increase after an incubation period of 1 h and reached a maximum after approximately 5 h. There was no significant increase in cells treated at 37°C over this period. R N A prepared from heat-treated cells was also examined and a similar result was obtained. Incubation of cells at 42°C resulted in an increase of m R N A after approximately 1 h and the maximum was obtained after a 3-h incubation period. There was no increase in HO-1 m R N A from cells incubated at 37°C, confirming rat HO-1 as a heat shock protein. In order to deduce the nature of the HO-1 heat shock response, Shibahara et al, carried out further incubation experiments using cycloheximide and actinomycin D. Actinomycin D inhibited the increase in HO-1 activity in heat treated cells completely which implies that heat shock induces HO-1 at the transcriptional level. Cycloheximide however only partially prevented the m R N A increase, which suggests that protein synthesis is necessary to achieve the full induction of mRNA. Recently HO-1 has been found to be induced by heat in human Hep3b c e l l s . 39 Oxidative stress results from a disturbance in the balance between the production of reactive oxygen species and a cells antioxidant defence mechanisms, a potentially harmful situation for the cell. Glutathione is involved in that it acts as a sulphydryl buffer keeping the cysteine residues of red cell proteins in the reduced state, thereby protecting the cells from oxidative damage. Many mammalian cells respond to oxidative stress by rapidly enhancing transcription of a number of genes, human HO-1 is believed to be one such gene. 4° The induction of HO-1 was shown to be affected by the redox state of the cell because lowering cellular glutathione leads to a significant increase in HO-1 mRNA. 41 When the level of glutathione is reduced the anti-oxidant defence system is also lowered, subsequent increases in the level of HO-1 in the cell therefore prevent oxidative damage. Hydrogen peroxide is generated by a reaction between two highly reactive species. A potentially damaging superoxide anion (02-) is formed by the one electron reduction of a molecule of oxygen. Protonation of the superoxide anion yields hydroperoxy radical (HO2) which, in turn, can react with a second superoxide anion to form hydrogen peroxide (H202). All these species can cause oxidative damage to a cell unless they are detoxified by enzymes such as catalase, which degrades hydrogen peroxide to oxygen and water. If hydrogen peroxide is allowed to accumulate inside a cell it can result in cell death, formation of D N A strand breaks, induction of sister chromatid crossovers42 and the induction of gene mutation? 3 Proteins which reduce oxygen, such as cytochrome oxidase, do not usually release superoxide anions so that cellular damage is avoided.
BIOCHEMICAL EDUCATION 25(3) 1997
123 However, a small amount of these species are formed during the oxidation of the ferrohaem (Fe 2+) group of haemoglobin to ferrihaem (Fe 3+) by oxygen. The treatment of human fibroblast cells with either hydrogen peroxide or ultraviolet A irradiation induces both HO-1 m R N A and protein in the cell: This increase in HO-1 activity has two effects. Firstly there is a reduction in the cellular pool of haem and haemoproteins by increasing their degradation. Secondly, the induction of HO-1 activity enables a cell to synthesise biliverdin and bilirubin (in the presence of biliverdin reductase). Unconjugated bilirubin is a potent antioxidant, scavenging singlet oxygen and reacting with superoxide anion and peroxyl radicals. 44 In scavenging two hydroperoxy radicals, bilirubin is oxidised to biliverdin which is then rapidly reconverted to bilirubin. For these reasons it is believed that the induction of HO-1 may be a general response to oxidative stress providing the cell with a defence mechanism against oxidative damage. Hydrogen peroxide is thought to produce its biological effects through a Fenton reaction (Fig 3) with naturally occurring iron complexes to yield the highly reactive hydroxyl radical. The Fenton reaction involves hydrogen peroxide and iron (II) salts reacting with a variety of organic molecules and causing a series of radical reactions. The evidence for this reaction mechanism comes from studies on mammalian cells using the membrane permeable iron chelator, o-phenanthroline. The presence of this iron chelator was shown to lower the H202 induction of both HO-1 m R N A and protein by between 50 and 80%. 45o-Phenanthroline prevents the Fenton reaction by rendering the iron unavailable for use in catalysis and thereby prevents generation of the hydroxyl radical. Glucose oxidase is a generator of hydrogen peroxide and consequently it is an oxidative stress agent. During recent experiments, glucose oxidase, the sulphydrylreactive heavy metal cadmium and the electrophilic agent DEM, have also been shown to induce HO-1 after 6-12 h exposure. 46 This further confirms the role of HO-1 in protection against oxidative stress. Both macrophages and neutrophils are phagocytic white blood cells which ingest and destroy foreign particles such as micro-organisms, cell debris and damaged erythrocytes. During phagocytosis, a significant change in oxygen utilisation occurs and superoxide ions (O2-.) and hydrogen peroxide (H202) are produced. These reduced oxygen species along with singlet oxygen (102) help to destroy the ingested micro-organism but may also cause oxidative damage to the host cells, possibly through the Fenton reaction. The The Fenton Reaction
Formation of the hydroxyl radical:
Fe2+ + H202
~ Fe3+ + OH" + OH-
Traces of Fe3+ can then react further with hydrogen peroxide:
Fe3+ + H202
.~ Fe2+ + 0 2 - + 2H+
Even more reactions are possible:
OH'+H20 2 ~
H 2 0 + H + + O 2-
0 2- + F e 3+ ~
Fe2 + + O 2
OH" + Fe2+ .
~ Fe3+ + HO-
Figure 3 The Fenton reaction, showing the formation of hydroxyl radicals (adapted from ref 58) B I O C H E M I C A L E D U C A T I O N 25(3) 1997
124 stress response of human macrophages during phagocytosis of Staphylococcus aureus was investigated 8. In the presence of iron de n o v o synthesis of HO-1 was induced. Other oxidative agents induced the same response in mouse macrophages. 47 Approximately 3% of the body's circulating haemoglobin is oxidised to methemoglobin daily. Neutrophils are a site of this oxidation reaction which results in the generation of a superoxide (02-) ion and potential oxidant stress. Exposure to methemoglobin, however, sensitises the host cell to oxidant damage via the initiation of HO-1 transcription and the cells become resistant to the action of oxidants. Phagocytosis mediated oxidant damage to vascular endothelium is likely to be involved in various vasculopathies including atherosclerosis and pulmonary leak syndromes such as adult respiratory distress syndrome. Vascular cells are frequently exposed to high oxidant concentrations derived from phagocytes. HO-1 appears to provide a cellular defence against this and is increased 50-fold in response to phagocytic induced oxidant stress. 48 UV radiation
UV-A radiation (from 320 to 380 nm) is a component of sunlight and is known to be carcinogenic to animals. The elucidation of the mechanism of cell damage by UV-A could have important clinical consequences in the prevention of human skin cancer. UV-A has been reported to increase HO-1 levels in the cell 7'6'45. The increase in the expression of HO-1 results from an enhanced transcription rate of the HO-1 gene as suggested by the elevated levels of HO-1 mRNA. 9 UV-A treatment of cells is known to lead to the formation of hydrogen peroxide (H202) and superoxide anion ( 0 2 ) . U V A induction of HO-1 was concluded to be mediated by a hydroxyl radical (OH') generated by the iron catalysed Fenton reaction. However, iron is also involved in reactions other than those which generate a hydroxyl radical, such reactions include decomposition of lipid hydroperoxides and auto oxidation of a number of biomolecules. Therefore, iron catalysed reactions could also lead to the generation of other active oxygen species such as the lipid peroxyl radical and singlet oxygen. In order to examine the involvement of both singlet oxygen and hydroxyl radicals in the UV-A induction of HO-1, two techniques were used? 9 Firstly, human skin fibroblasts were irradiated in the presence of deuterium oxide (D20), in which singlet oxygen has a longer half life. In D20 treated cells the wavelength-dependent increase of HO-1 is enhanced, suggesting that singlet oxygen generated by UV-A and near-visible light is the primary effector in the induction of the HO-1 gene. Secondly dimethyl sulphoxide (DMSO) and mannitol, which are effective scavengers of O H were used prior to irradiation of the cells. These were found to have no significant effect on the wavelength-dependent accumulation of HO-1. In conclusion, singlet oxygen constitutes an important intermediate in the signal transduction events which lead to the induction of HO-1 by UV-A and near-visible light. The mechanism of transcriptional activation of HO-1 by UV-A radiation was examined using nuclear extracts from untreated and UV-A treated human skin fibroblasts. 25 These were then assayed for their ability to bind to a 147 base pair fragment of the promoter of the HO-1 gene. A combination of gel retardation assays and DNase 1 footprinting analyses revealed a region within the promoter of the HO-1 gene that binds a specific protein complex present only in extracts from UV-A treated cells. This protein binds to the promoter of HO-1 and initiates transcription in response to oxidative stress.
Haem catabolism by haem oxygenase-2
The existence of a second isoform of haem oxygenase, HO-2, was first reported in 1986 as a non-inducible form of the enzyme? HO-2 is expressed constitutively throughout the body with a high concentration in the brain. 4 Purification of HO-2 from rat testes enabled a full characterisation of the enzyme 5° and HO-2 was found to differ from HO-1 in a number of ways including regulation, activation, immunochemical properties and molecular weight. HO-2 is the only detectable haem oxygenase species in adult rat brain, 51 although HO-1 may be induced by heat shock in some glial cells and certain neurones 52. The discrete localisation of HO-2 m R N A in areas of the brain containing guanyl cyclase, suggests that HO-2 may have a function in the synthesis of carbon monoxide for use as a neurotransmitter, l° Carbon monoxide (CO) is formed endogenously during haem catabolism by the action of haem oxygenase (Fig 4), the only known reaction which forms CO in the body.
BIOCHEMICAL EDUCATION 25(3) 1997
125 Haem Haemoxygenase > CO + Biliverdin+ Fe
membrane v diffusion ~ / ~ a n y (~ l cyclase
{/
oTP
ooMP
Figure 4 Carbon monoxide may enter cells by diffusion and mediate its physiological effects by stimulating guanyl cyclase. Guanyl cyclase then catalyses the formation of cGMP which functions as a second messenger CO has a number of physiological effects including the vasodilation of blood vessels 11 and the inhibition of platelet aggregation?2 Nitric oxide (NO), a recently discovered gaseous vasodilator mediates its effect by activating guanyl cyclase and consequently increasing cyclic guanosine 3,5-monophosphate (cGMP) levels. 53 CO is also able to activate guanyl cyclase by binding to its iron atom 12and it was therefore postulated that CO may play a physiological role similar to that of It has been demonstrated that zinc protoporphyrin, which inhibits haem oxygenase activity, depletes cGMP in primary cultures of rat olfactory neurones. 1° CO has also been demonstrated to inhibit the contractility of vascular smooth muscle by causing a time dependent increase in cGMP levels.11 In mammals the hippocampus, a part of the cerebral cortex in the brain, plays a special role in learning. Short bursts of repetitive firing of synaptic vesicles at the presynaptic cells cause long-term potentiation (LTP). Zinc protoporphyrin was shown to block the induction of LTP in hippocampal slices from adult male guinea pigs, 13 and in mice and rats, 55 providing evidence that CO is involved in LTP in mammals. Zinc protoporphyrin is also synthesised endogenously, formed when zinc replaces iron for insertion into protoporphyrin under iron deficient conditions and in lead poisoning.56 Endogenous zinc protoporphyrin may, by inhibiting haem oxygenase, modulate synthesis of CO from haem and therefore modulate LTP, possibly explaining the neurological problems associated with lead poisoning. 57 CO has also been shown to inhibit platelet aggregation. This inhibition can be overcome by illumination with light of the wavelength 400 nm, suggesting that CO may exert its inhibitory action by binding to a reduced haem-containing protein such as guanyl cyclase. In the presence of CO the intracellular levels of cGMP were increased dramatically. Studies with sodium nitroprusside, which activates guanyl cyclase, showed that platelet aggregation was sensitive to even the smallest increases in cGMP concentrations. This combination of experimental results has lead to the suggestion that CO probably inhibits platelet aggregation through a signal transducing pathway involving cGMP. 12
NO.54
Comparison of haem oxygenases-1 and -2
The two isoforms of haem oxygenase were first identified in 1986 in rat liver microsomes. 3 Initially, it was impossible to infer whether the two forms were isozymes, representing products of different genes or isoforms, having the same gene but representing products of post-transcriptional modification. Sequencing characterisation and subsequent comparison of the two haem oxygenase species has provided conclusive evidence that HO-1 and HO-2 are products of different genes. 4 Detailed studies of the two forms of the enzyme has revealed a number of biochemical differences between them. One of the first differences reported between the HO-1 and HO-2 was their different regulatory mechanisms. The activity of HO-1 can be increased up to 1000-fold in response to various agents including metals, = chemicals 34 and haematin. In contrast, the activity of HO-2 remained almost constant despite exposure to known inducers of H O - I ? Kinetic
BIOCHEMICAL EDUCATION 25(3) 1997
126 studies of the two isoforms have revealed a 3-fold difference in apparent Km values between the two isozymes. 3 Kinetic properties of haem oxygenases I and II
Although haem oxygenases I and II carry out the same chemical reaction. Their kinetic properties are different. The maximum velocity (gm) and the Michaelis constant (Vm~) values of the two isoenzymes arise as a result of very small changes in the three dimensional shape of the two isoenzymes, which is itself a result of their differing amino acid sequences. The overall effect of these differences is to produce a pair of enzymes whose maximum rates and substrate affinities are closely matched to their physiological roles. Thus haem oxygenase I, the main function of which is to catalyse the breakdown of haemoproteins, has a rather low Km and a high value for Vmax. This means that the enzyme is active even at low substrate concentrations, and its activity increases as the concentration of haem increases within the physiological range. Conversely haem oxygenase II, which functions to catalyse the production of pharmacological quantities of carbon monoxide, has a higher Km and a significantly lower value for Vinos.This means that the enzyme has low activity at low substrate concentrations, and even its maximum activity is limited to less than 10% of that of haem oxygenase I. The Km and V,,o~values of the two haem oxygenase isoenzymes are shown below (from Maines et al 1986 ref 3):
Isoenzyme HO-1 HO-2
Km
Vm~
0.24#m 0.67pm
3.4 pmol/mg/h 0.24 pmol/mg/h
For HO-1 the Km value was 0.24 #m whereas the value for HO-2 was 0.67 pm. The lower Km value for the inducible form of the enzyme, HO-2, is thought to have an important biological role especially under conditions when the level of endogenous haem substrate (eg cytochrome P-450) are low. Alternatively, the difference in Kmvalues could arise due to a different substrate specificity, for example HO-1 may exhibit a preference for a particular isomer of haem (eg haem a, b or c which have differently substituted porphyrin rings). Different types of haem are found in the various haemoproteins and therefore the differences in K,, could signify preferences of the two isozymes of haem oxygenase towards various haemoproteins. In addition to their regulatory properties, the tissue distribution of the two haem oxygenase isozymes also differs. It was the initial resistance of the testis and brain haem oxygenase to respond to inducers such as haem that led to the discovery of the non-inducible HO-2 which predominates in these organs? HO-1 is the predominant species in the spleen and after exposure of a tissue to inducing agents. The primary structures of HO-1 and HO-2 differ significantly. 4 Specifically, the two isozymes vary in their amino acid compositions, containing dissimilar amounts of asparagine, glycine, aspartate, histidine, isoleucine, leucine and cysteine residues. The most striking difference being that HO-2 contains three cysteine residues which are absent in HO-1. The molecular weights of the proteins differ by 6000 Da on sodium dodecyl sulphate electrophoresis gels. HO-1 has an apparent M, of 30000 Da while that of HO-2 is higher at 36000 Da? ° Discussion
The variety of roles assigned to haem oxygenase can, in part, be explained by the existence of two isozymes of the enzyme which have different regulatory mechanisms, sequences and properties. The principle role of HO-1 seems to be catabolism of haem from red blood cells and haemoproteins, but the evidence implicating HO-1 as a heat shock protein is increasing, and strongly suggests that induction occurs at the transcriptional level via a heat shock element. The evidence supporting induction of HO-1 by oxidative stress is also mounting. Further investigation into the nature of the induction of HO-1 is needed, in particular in characterising the cis-acting element which confers the inducibility to haemin.
BIOCHEMICAL EDUCATION 25(3) 1997
127 The principal role of HO-2 seems to be the production of carbon monoxide for use as a n e u r o t r a n s m i t t e r . H O - 2 p r o d u c e s c a r b o n m o n o x i d e i n t h e s a m e w a y as H O - 1 b y t h e degradation of haem but, due to the localisation of HO-2 to areas of the brain containing guanyl cyclase, the carbon monoxide can activate this second messenger system producing cGMP and thereby mediate numerous physiological processes. Future work may elucidate further the role of carbon monoxide in the activation of guanyl cyclase and its s u b s e q u e n t c e l l u l a r t a r g e t s .
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