Heme degradation by the microsomal heme oxygenase system

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Heme degradation by the microsomal heme oxygenase system Goro Kikuchi and Tadashi Yoshida Heine degradation in the heme oxygenase reaction proceeds essentially as an autocatalytic oxidation o f h e m e which is bound ~o heine oxygenase; in this reaction heme acts as both the substrate and the coenzyme which activates molecular oxygen. Synthesis o f heme oxygenase can be induced by heine itself in a substrate-mediated induction.

The mechanism of conversion of heme and hemoglobin to the bile pigments in vivo remained controversial until as late as 1968 when Schmid's group discovered a heme oxygenase in the microsomal fraction of mammalian tissues such as spleen, liver and kidneyk This heme oxygenase, system catalyses the degradation of heme to yield biliverdin IXct and consumes N A D P H and molecular oxygen. It is now generally accepted that heme oxygenase plays an essential role in the physiological catabolism of heme. Only in the last few years however, has the nature of heme oxygenase been wellcharacterized and the mechanism of heme degradation elucidated. In fact, heme oxygenase was initially thought to involve a cytochrome P-450 as a terminal oxidase because the heme oxygenase reaction had many similarities to those of other hydroxylation reactions catalysed by the microsomal cytochrome P-450 system 2,3. Recently, we purified heme oxygenase to apparent homogeneity from pig spleen microsomes 4,5 and rat liver microsomes 6 and demonstrated that heme oxygenase is independent of any type of cytochrome P-450. Studies with the purified heme oxygenase preparations e,' have revealed that the principal feature of the reaction of heme degradation catalysed by the heme oxygenase system is essentially similar to that of heme decomposition b} the socalled coupled oxidation of myoglobin or hemoglobin and ascorbic acidS."; in the heme oxygenase reaction heme acts as both the substrate and coenzyme, and the heme oxygenase protein provides a suitable site for the rapid autocatalyfic oxidation of the bound heme. The heme oxygenase activity is higher in those tissues normally involved in the breakdown of red cells and hemoglobin; namely, spleen, liver and bone marrow. Heme oxygenase can even be induced in Goro Kikuchi and Tadashi Yoshida are at ,'.he Department o f Biochemistry, Tohoku Universit) School o f Medicine, Sendai 980, Japan.

liver, kidney and macrophages by the administration of heme or hemoglobin 1°,~3, possibly due to a substrate-mediated enzyme induction. This is in contrast to the case of porphyrin biosynthesis in which heme acts to suppress the biosynthetic activity a4. In the present review we will discuss some problems of heme catabolism in animals. Nature of heme oxygenase and the heme oxygenase system The purified heme oxygenase from either pig spleen or rat liver has a minimum mol. wt of 32,000. This is much smaller than the values reported for cytochrome P-450 (45,000 to 65,000). Heme oxygenase is not a hemoprotein by nature, but it binds heme to form a 1 : 1 complex 5. The absorption spectrum of the complex of ferric heme and heme oxygenase is very similar to the absorption spectra of metmyoglobin and methemoglobin, and the complex of ferric heine and heme oxygenase can also bind azide and cyanide. The absorption spectra of the reduced form and the carbon monoxide complex of the reduced form of this complex also resemble those of the corresponding forms of myoglobin and hemoglobin 5,8. Moreover, we could obtain the oxygenated form of the heme-heme oxygenase complex by passing the carbon monoxide complex through a column of Sephadex G-2515. The oxygenated form of the complex showed the absorption maxima at 412, 540 and 575 nm which are very close to those of oxyhemoglobin. The heme binding environment of the heme oxygenase protein may be similar to those of hemoglobin and myoglobin, and heme may bind to heme oxygenase through a coordination linkage with an amino acid residue of the protein. The microsomal heme oxygenase system consists of heine oxygenase and N A D P H cytochrome c reductase (fpT); the latter serves as the electron donor for the heme oxygenase reaction. The oxygenated heme bound to heine oxygenase is

readily auto-oxidizable at pH 6.0 although it is quite stable at neutral pH ~5. There was no indication of heme degradation during auto-oxidation of the oxygenated heme, but when the oxygenated complex was incubated with the fpT system, the oxygenated heme was converted readily and quantitatively to biliverdin, indicating that a reducing equivalent is indispensable for the onset of heme degradation from the oxygenated form of the complex 15. With microsomes, however, the heme oxygenase reaction is also supported to some extent by N A D H . In this case N A D H serves as the electron donor by way of fpT. We have observed that the NADH-supported heme degradation in the microsomal system, as well as the NADPH-supported heme degradation, was completely inhibited by the addition of anti-fpT IgG but not at all by the antibody prepared against N A D H - c y t o c h r o m e b5 reductase (fpD) TM. Moreover, we have observed in the reconstituted heme oxygenase system that no biliverdin was formed when the oxygenated heme-heme oxygenase complex was incubated with the fpD-cytochrome b~-NADH system, while the ferric heme bound to heme oxygenase could easily be reduced by the fpD-cytochrome b s - N A D H system, eventually giving rise to the formation of the oxygenated form of the complex ~7. It is interesting that the fpD-cytochrome bs system was unable to donate a significant number of electrons to the oxygenated form of the complex to initiate the heme degradation, while the fpD-cytochrome bs system could readily reduce the ferric form of the complex. Mechanism of the heme oxygenase-dependent heme degradation The possible reaction sequence of heme degradation in the heme oxygenase reaction is shown in Fig. 1. The possible chemical sequence of heme degradation is depicted in Fig. 2. First, protoheme binds to the heme oxygenase protein to form a ferric heme-heme oxygenase complex. The ferric heme bound to heine oxygenase is then reduced to ferrous heine by the fpT system followed by binding with molecular oxygen to form the oxygenated h e m e heine oxygenase complex. For the onset of the heine degradation, the second electron is indispensable, and the first step of heme oxidation is assumed to be hydroxylation at the a-methene carbon of protoheme. In the reconstituted heine oxygenase reaction, the apparent Km for protoheme islow at about 1/~M 7. The heme oxygenase reaction is not inhibited by various scavengers of singlet 0 Elsevier/North-Holland Biomedical Press

1980

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oxygen, superoxide anion and hydroxy radicaF. The molecular oxygen which is bound to heme on heme oxygenase is probably activated in the presence of the fpT system and is directly utilized to oxidize the heme moiety. The activated oxygen appears to attack specifically the a-methene of heme since only the a-type of biliverdin is obtained as the final product of heme degradation in the heme oxygenase reaction. The heme bound to heme oxygenase also decomposes when the complex is incubated with ascorbic acid 7,1~, suggesting that the mechanism of heme degradation in the heme oxygenase reaction is essentially similar to that in the coupled oxidation. In

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the reaction system with ascorbic acid, however, the reaction rate is far lower than the rate in the reaction system with fpT, and the final oxidation product of the heme bound to heme oxygenase is not biliverdin but a biliverdin-iron complex from which biliverdin can readily be liberated by the addition of desferrioxamine, a ferric iron chelator 7. The release of iron and biliverdin from the biliverdin-iron complex is also facilitated by the addition of the fpT system 7. For release of iron from the biliverdin-iron chelate, the iron may have to be reduced to the ferrous state by the fpT system. Ascorbic acid can hardly reduce the biliverdin-iron chelate, probably because the oxidation-reduction potential of

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ascorbic acid is very high (E0', + 0.080 V). It should be noted that the whole process of heme degradation is catalysed by a single enzyme, heme oxygenase, provided that appropriate reducing equivalents are supplied. The specific feature of the heme oxygenase reaction may be the outcome of the specific structure of the heme oxygenase protein which provides a suitable environment for autocatalytic oxidative degradation of the heme moiety. In this respect the catabolism of heme is quite unique as compared with that of other biological substances. The most difficult unsolved problem in the heme oxygenase reaction is the chemical sequence from hydroxyheme to iron-biliverdin complex which involves liberation of carbon monoxide. According to Brown and King ls,19, two molecules of oxygen are probably consumed in a sequential manner during the course of opening the ring of hydroxyheme. Recently we have found a new intermediate which occurs between hydroxyheme and the biliverdin-iron complex *°. The intermediate stays in the ferrous state in the heme oxygenase reaction with either the fpT system or ascorbic acid and shows an absorption maximum at 688 nm. This intermediate has an extremely high affinity for carbon monoxide and the carbon monoxide complex shows a sharp absorption maximum at 638 nm. Therefore, when the reaction is performed with a gas mixture containing air and carbon monoxide, the reaction stops almost completely at the level of this 638 nm compound. This 638 nm compound is easily converted to biliverdin-iron complex when carbon monoxide is eliminated, but only when the intermediate is kept in a ferrous state. The chemical nature of this newly found intermediate remains to be elucidated. It is also unclear whether carbon monoxide is liberated before or after the formation of this intermediate. At any rate, it appears to be derived from hydroxyheme by consuming a molecular oxygen and is converted to the biliverdin-iron complex consuming another molecular oxygen. It seems quite conceivable that every step of heme degradation up to the formation of biliverdiniron chelate requires molecular oxygen which is activated by binding with the ferrous state of the heme iron. Induction of heine oxygenase

By the combined use of a tracer method with [3H]leucine and an immunochemical method with a rabbit antibody (IgG) prepared against a purified pig spleen heme oxygenase we have demonstrated that the

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hemin-induced increase of the heme oxygenase activity in cultured pig alveolar macrophages is actually due to increased synthesis of the heme oxygenase protein TM. Cell-free synthesis in a reticulocyte lysate system with polysomes isolated from hemin-treated macrophages has shown that free polysomes are the major sites of synthesis of heme oxygenase~L The polysome-directed, cell-free synthesis of heme oxygenase also revealed that the functional m R N A for heme oxygenase was actually increased in the hemin-induced cells, indicating that hemin stimulated synthesis of the m R N A specific for heme oxygenase in alveolar macrophages TM. Increased heme concentration in the cell, on the other hand, brings about the repression of synthesis of 8-aminolevulinate synthase, the rate-limiting enzy~e in the heme synthetic pathway ~4. Increased heme also prevents the transfer of lhe newly synthesized 8-aminolevulinate synthase from the cytosol into the mitochondria which is the site where 8-aminolevulinate

synthase functions, and this would also contribute to some extent to reduce the rate of heme synthesis*L Apparently the heme metabolism in the cell is regulated principally by the concentration of heme itself, and this mechanism would represent a beautiful example of self-control in the metabolic regulation functioning at the cell level.

References 1 Tenhunen, R., Marver, H. S. and Schmid, R. (1968) Proc. Natl. Acad. Sci. U.S.A. 61,748-755

2 Tenhunen, R., Marver, H. S. and Schmid, R. (1969)J. Biol. Chem. 244, 6388-6394 3 Tenhunen, R., Marver, H. S., Pimstone, N. R., Trager, W. F., Cooper, D. Y. and Schmid, R. (1972) Biochemistry 11, 1716-1720 4 Yoshida,T. and Kikuchi,G. (1977) J. Biochem. 81,265-268 5 Yoshida,T. and Kikuchi,G. (1978)J. Biol. Chem. 253, 4224-4229 6 Yoshida,T. and Kikuchi,G. (1979)J. Biol. Chem. 254, 4487-4491 7 Yoshida,T. and Kikuchi,G. (1978)J. Biol. Chem. 253, 4230--4236 8 Lemberg, R. (1956) Rev. Pure Appl. Chem. 6, 1-23

Immunoglobulin RNA processing Randolph Wall Recent studies indicate that, in addition to generating messenger R N A , R N A processing plays an important role in controlling the expression o f immunoglobulin genes in the development o f the immune response.

The immense diversity of antibody specificities and the complex developmental switches in the expression of antibody genes have long intrigued molecular biologists. Molecular cloning has now provided many insights into the structure, multiplicity and origins of diversity in immunoglobulin genes. Parallel studies on immunoglobulin RNA have complemented gene cloning approaches in defining the structure of active immunoglobulin genes (i.e. transcription units) and in resolving the events in messenger RNA (mRNA) processing in eukaryotic cells. This brief review will concentrate on recent studies which indicate that an early developmental transition in the expression of genes for immunoglobulin heavy chains is regulated through R N A l:,rocessing. These studies provide striking s~apport for the long standing proposal thai: different types of post-transcriptional processing of RNA may be used to control the expression of eukaryotic genes. Randolph Wall is at the Molecular Biology Institute and Department o f Microbiology and Immunology, UCLA School o f Medicine, Los Angeles. CA 90024, U.S.A.

Immunoglobulin mRNA processing in fully differentiated cells The immunoglobulin protein molecule consists of two identical light chains and two identical heavy chains. Immunoglobulin light chains contain two functional domains: the variable region (VL) at the NH2-terminus, which is involved in antibody specificity, and the constant region (C0 at the COOH-terminus. Heavy chains have a variable region (VH) which also contributes to antibody specificity, along with either three or four constant region domains (CH1, CH2, etc.) homologous to the constant region in the light chains. Coding sequences for variable and constant regions a~e, widely separated in DNA from germline and somatic cells but are rearranged into closer proximity in immunoglobulin-producing cells 1. However, even in the rearranged active immunoglobulin gene, sequences coding for variable and constant regions (called exons) are separated by non-coding intervening sequences (called introns). The separated variable and constant regions are transcribed directly into large nuclear RNA molecules and then spliced together

9 0 ' C a r r a , P. (1975) in Porphyrins and Metalloporphyrins (Smith, K. M., ed.), pp. 123-153, Elsevier, Amsterdam 10 Pimstone, N. R., Engel, P., Tenhunen, R., Seitz, P. T., Marver, H. S. and Schmid, R. (1971)J. Clin. Invest. 50, 2042-2050 11 Gemsa, D., Woo, C. H., Fundenberg, H. H. and Schmid, R. (1973)J. Clin. Invest. 52, 812822 12 Shibahara, S., Yoshida, T. and Kikuchi, G. (1978) Arch. Biochem. Biophys. 188,243-250 13 Sibahara, S., Yoshida, T. and Kikuchi, G. (1979) Arch. Biochem. Biophys. 197,607-617 14 Granick, S. and Sassa, S. (1971) in Metabolic Pathways (Vogel, H. J., ed.), Vol. 5, pp. 77-141, Academic Press, New York 15 Yoshida, T., Noguchi, M. and Kikuchi, G. (1980) J. Biol. Chem. 255, 4418--4420 16 Noguchi, M., Yoshida, T. and Kikuchi, G. (1979) FEBS Lett. 98, 281-284 17 Yoshida, T., Noguchi, M. and Kikuchi, G. (1980) FEBS Lett. 115,278--280 18 Brown, S. B. and King, R. F. G. J. (1975) Biochem. J. 150, 565-567 19 Brown, S. B. and King, R. F. G. J. (1976) Biochem. Soc. Trans. 4, 197-201 20 Yoshida, T., Noguchi, M. and Kikuchi, G. (1980) J. Biochem. 88, 557-563 21 Yamauchi, K., Hayashi, N. and Kikuchi, G. (1980)J. Biol. Chem. 255, 1746-1751

in a complex series of R N A processing events. Considerable evidence now indicates that these large nuclear R N A molecules are the precursors to immunoglobulin mRNA; this evidence includes formal proof from pulse-chase experiments in which immunoglobulin m R N A sequences, exclusively in large nuclear RNA molecules, were quantitatively processed into cytoplasmic m R N A after all further transcription was abolished 2-4. The posttranscriptional processing events which generate immunoglobulin m R N A from large RNA precursors apparently occur in a specific order. One of the earliest events after transcription is the addition of poly(A) at the 3' end of the primary transcript 2-4. Then the RNA is spliced to remove introns and join the separated exons2-L The fully processed immunoglobulin m R N A molecules appear to be immediatedly transported to the cytoplasm. Myeloma cells were used in these studies on the processing of immunoglobulin RNA. These tumor cells correspond to fully differentiated antibody-secreting cells, where a substantial fraction (up to 20%) of the total cell protein synthesized is immunoglobulin. The transcriptional and post-transcriptional processes which generate immunoglobulin m R N A in these fully differentiated cells are extremely efficients,4. Reasonable estimates for the transcriptional activity of immunoglobulin genes in myeloma cells approximate to © Elsevier/North-HollandBiomedicalPress 1980