Tadashi Yoshida

Mechanism of heme degradation by heme oxygenase

Heme oxygenase catalyzes the three step-wise oxidation of hemin to alpha-biliverdin, via alpha-meso-hydroxyhemin, verdoheme, and ferric iron-biliverdin complex. This enzyme is a simple protein which does not have any prosthetic groups. However, heme and its two metabolites, alpha-meso-hydroxyhemin and verdoheme, combine with the enzyme and activate oxygen during the heme oxygenase reaction. In the conversion of hemin to alpha-meso-hydroxyhemin, the active species of oxygen is Fe-OOH, which self-hydroxylates heme to form alpha-meso-hydroxyhemin. This step determines the alpha-specificity of the reaction. For the formation of verdoheme and liberation of CO from alpha-meso-hydroxyhemin, oxygen and one reducing equivalent are both required. However, the ferrous iron of the alpha-meso-hydroxyheme is not involved in the oxygen activation and unactivated oxygen is reacted on the activated heme edge of the porphyrin ring. For the conversion of verdoheme to the ferric iron-biliverdin complex, both oxygen and reducing agents are necessary, although the precise mechanism has not been clear. The reduction of iron is required for the release of iron from the ferric iron-biliverdin complex to complete total heme oxygenase reaction.

Crystal structure of rat biliverdin reductase.

Biliverdin reductase (BVR) is a soluble cytoplasmic enzyme that catalyzes the conversion of biliverdin to bilirubin using NADH or NADPH as electron donor. Bilirubin is a significant biological antioxidant, but it is also neurotoxic and the cause of kernicterus. In this study, we have determined the crystal structure of rat BVR at 1.4 Å resolution. The structure contains two domains: an N-terminal domain characteristic of a dinucleotide binding fold (Rossmann fold) and a C-terminal domain that is predominantly an antiparallel six-stranded β-sheet. Based on this structure, we propose modes of binding for NAD(P)H and biliverdin, and a possible mechanism for the enzyme.

Posttranslational and direct integration of heme oxygenase into microsomes

Rat liver heme oxygenase has a large cytoplasmically exposed domain containing the N-terminus that can be cleaved from the membranes by a low concentration of trypsin, indicating that heme oxygenase is embedded in membranes with an insertion sequence near its C-terminal portion. Heme oxygenase synthesized in a cell-free system or purified from microsomes after detergent-solubilization was integrated into microsomal membranes posttranslationally and directly, like cytochrome b5.

Importance of histidine residue 25 of rat heme oxygenase for its catalytic activity

A truncated, soluble, and enzymatically active rat heme oxygenase lacking its membrane-associative, C-terminal segment was expressed in E. coli strain JM109. The roles of its four histidine residues were examined by determining the enzymatic activities of mutant enzymes in which each of these residues in turn was replaced by alanine. Mutation of histidine residue 25 to alanine resulted in marked decrease in activity for heme breakdown, indicating that this histidine residue has an important role in the heme oxygenase reaction.

Hypoxia reduces the expression of heme oxygenase-2 in various types of human cell lines. A possible strategy for the maintenance of intracellular heme level.

Heme oxygenase consists of two structurally related isozymes, heme oxygenase‐1 and and heme oxygenase‐2, each of which cleaves heme to form biliverdin, iron and carbon monoxide. Expression of heme oxygenase‐1 is increased or decreased depending on cellular microenvironments, whereas little is known about the regulation of heme oxygenase‐2 expression. Here we show that hypoxia (1% oxygen) reduces the expression levels of heme oxygenase‐2 mRNA and protein after 48u2003h of incubation in human cell lines, including Jurkat T‐lymphocytes, YN‐1 and K562 erythroleukemia, HeLa cervical cancer, and HepG2 hepatoma, as judged by northern blot and western blot analyses. In contrast, the expression level of heme oxygenase‐1 mRNA varies under hypoxia, depending on the cell line; it was increased in YN‐1 cells, decreased in HeLa and HepG2 cells, and remained undetectable in Jurkat and K562 cells. Moreover, heme oxygenase‐1 protein was decreased in YN‐1 cells under the conditions used, despite the induction of heme oxygenase‐1 mRNA under hypoxia. The heme oxygenase activity was significantly decreased in YN‐1, K562 and HepG2 cells after 48u2003h of hypoxia. To explore the mechanism for the hypoxia‐mediated reduction of heme oxygenase‐2 expression, we showed that hypoxia shortened the half‐life of heme oxygenase‐2 mRNA (from 12u2003h to 6u2003h) in YN‐1 cells, without affecting the half‐life of heme oxygenase‐1 mRNA (9.5u2003h). Importantly, the heme contents were increased in YN‐1, HepG2 and HeLa cells after 48u2003h of incubation under hypoxia. Thus, the reduced expression of heme oxygenase‐2 may represent an important adaptation to hypoxia in certain cell types, which may contribute to the maintenance of the intracellular heme level.

Protein expressed by the ho2 gene of the cyanobacterium Synechocystis sp. PCC 6803 is a true heme oxygenase. Properties of the heme and enzyme complex.

Two isoforms of a heme oxygenase gene, ho1 and ho2, with 51% identity in amino acid sequence have been identified in the cyanobacterium Synechocystis sp. PCC 6803. Isoform‐1, Syn HO‐1, has been characterized, while isoform‐2, Syn HO‐2, has not. In this study, a full‐length ho2 gene was cloned using synthetic DNA and Syn HO‐2 was demonstrated to be highly expressed in Escherichia coli as a soluble, catalytically active protein. Like Syn HO‐1, the purified Syn HO‐2 bound hemin stoichiometrically to form a heme–enzyme complex and degraded heme to biliverdin IXα, CO and iron in the presence of reducing systems such as NADPH/ferredoxin reductase/ferredoxin and sodium ascorbate. The activity of Syn HO‐2 was found to be comparable to that of Syn HO‐1 by measuring the amount of bilirubin formed. In the reaction with hydrogen peroxide, Syn HO‐2 converted heme to verdoheme. This shows that during the conversion of hemin to α‐meso‐hydroxyhemin, hydroperoxo species is the activated oxygen species as in other heme oxygenase reactions. The absorption spectrum of the hemin–Syn HO‐2 complex at neutral pH showed a Soret band at 412u2003nm and two peaks at 540u2003nm and 575u2003nm, features observed in the hemin‐Syn HO‐1 complex at alkaline pH, suggesting that the major species of iron(III) heme iron at neutral pH is a hexa‐coordinate low spin species. Electron paramagnetic resonance (EPR) revealed that the iron(III) complex was in dynamic equilibrium between low spin and high spin states, which might be caused by the hydrogen bonding interaction between the distal water ligand and distal helix components. These observations suggest that the structure of the heme pocket of the Syn HO‐2 is different from that of Syn HO‐1.

Molecular oxygen oxidizes the porphyrin ring of the ferric α-hydroxyheme in heme oxygenase in the absence of reducing equivalent

Heme oxygenase catalyzes the regiospecific oxidative degradation of iron protoporphyrin IX (heme) to biliverdin, CO and Fe, utilizing molecular oxygen and electrons donated from the NADPH-cytochrome P-450 reductase. The catalytic conversion of heme proceeds through two known heme derivatives, alpha-hydroxyheme and verdoheme. In order to assess the requirement of reducing equivalents in the second stage of heme degradation, from alpha-hydroxyheme to verdoheme, we have prepared the alpha-hydroxyheme complex with rat heme oxygenase isoform-1 and examined its reactivity with molecular oxygen in the absence of added electrons. Upon reaction with oxygen, the majority of the alpha-hydroxyheme in heme oxygenase is altered to a species which exhibits an optical absorption spectrum with a broad Soret band, along with the minority which is converted to verdoheme. The major product species, which is electron paramagnetic resonace-silent, can be recovered to the original alpha-hydroxyheme by addition of sodium dithionite. We have also found that oxidation of the alpha-hydroxyheme-heme oxygenase complex by ferricyanide or iridium(IV) chloride yields a species which exhibits an optical absorption spectrum and reactivity similar to those of the main product of the oxygen reaction. We infer that the oxygen reaction with the ferric alpha-hydroxyheme-heme oxygenase complex forms a ferric-porphyrin cation radical. We conclude that in the absence of reducing agents, the oxygen molecule functions mainly as an oxidant for the porphyrin ring and has no role in the oxygenation of alpha-hydroxyheme. This result corroborates our previous conclusion that the catalytic conversion of alpha-hydroxyheme to verdoheme by heme oxygenase requires one reducing equivalent along with molecular oxygen.

Rat liver mitochondrial and cytosolic fumarases with identical amino acid sequences are encoded from a single mRNA with two alternative in-phase AUG initiation sites.

By use of anticytosolic fumarase antibody, a cDNA clone was isolated from a rat liver cDNA library in the expression vector lambda gt11 and in the pBR 322 vector. A clone with an insert of about 1.7 kbp was isolated. Nucleotide sequence analysis of the insert revealed that the cDNA contained a noncoding region composed of 25 nucleotides in the 5 terminus, the coding region composed of 1,521 nucleotides, and the 3 nontranslated region composed of 43 nucleotides followed by a poly(A)+ tail. The open reading frame encoded a polypeptide of 507 amino acid residues (predicted Mr = 54,462), which contained an additional sequence composed of 41 amino acid residues on the N-terminus of the mitochondrial mature fumarase (the presequence). Thus, this reading frame was concluded to encode the precursor of mitochondrial fumarase. The amino acid sequence predicted from the nucleotide sequence contained all the amino acid sequences of 12 proteolytic polypeptides obtained by digestion of purified mitochondrial fumarase with V8 protease. The total amino acid sequence of the mitochondrial fumarase also contained all the sequences of 14 proteolytic peptides prepared from the cytosolic fumarase, indicating that the amino acid sequences of these two isozymes are identical. Furthermore, the results obtained by hybrid-selected translation, Northern blot and primer-extension analyses using appropriate cDNA segments prepared from fumarase cDNA (1.7 kbp) as the probe or primer suggested a possibility that both precursors of the mitochondrial and cytosolic fumarases were synthesized with one species of mRNA having base sequence coding presequence of the mitochondrial fumarase by unknown post-transcriptional mechanism(s). Rat liver cells may contain a specific RNA(18S) modulating the translational activity of mRNA for fumarase. This RNA(s), which was contained in poly(A)- fraction, was partially purified by high-performance gel filtration. The partially purified RNA(s) suppressed the translational activity of the cytosolic fumarase, whereas the translational activity of the mitochondrial one was accelerated by this RNA(s).

Primary structure of rat brain protein carboxyl methyltransferase deduced from cDNA sequence.

Two cDNA clones for protein carboxyl methyltransferase were isolated from a rat brain cDNA library in lambda gt 11 with synthetic oligonucleotides as probes. The two clones differ in size, but the nucleotide sequence including the whole coding region of the shorter cDNA is completely identical with the corresponding sequence of the longer cDNA. The open reading frame encodes a polypeptide of 227 amino acid residues, with a molecular weight of 24,626. This molecular weight is comparable to those reported for other protein carboxyl methyltransferases from several animals, which were determined by gel filtration chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Spectroscopic characterization of a higher plant heme oxygenase isoform-1 from Glycine max (soybean) −coordination structure of the heme complex and catabolism of heme

Heme oxygenase converts heme into biliverdin, CO, and free iron. In plants, as well as in cyanobacteria, heme oxygenase plays a particular role in the biosynthesis of photoreceptive pigments, such as phytochromobilins and phycobilins, supplying biliverdin IXα as a direct synthetic resource. In this study, a higher plant heme oxygenase, GmHO‐1, of Glycine max (soybean), was prepared to evaluate the molecular features of its heme complex, the enzymatic activity, and the mechanism of heme conversion. The similarity in the amino acid sequence between GmHO‐1 and heme oxygenases from other biological species is low, and GmHO‐1 binds heme with 1u2003:u20031 stoichiometry at His30; this position does not correspond to the proximal histidine of other heme oxygenases in their sequence alignments. The heme bound to GmHO‐1, in the ferric high‐spin state, exhibits an acid–base transition and is converted to biliverdin IXα in the presence of NADPH/ferredoxin reductase/ferredoxin, or ascorbate. During the heme conversion, an intermediate with an absorption maximum different from that of typical verdoheme–heme oxygenase or CO–verdoheme–heme oxygenase complexes was observed and was extracted as a bis‐imidazole complex; it was identified as verdoheme. A myoglobin mutant, H64L, with high CO affinity trapped CO produced during the heme degradation. Thus, the mechanism of heme degradation by GmHO‐1 appears to be similar to that of known heme oxygenases, despite the low sequence homology. The heme conversion by GmHO‐1 is as fast as that by SynHO‐1 in the presence of NADPH/ferredoxin reductase/ferredoxin, thereby suggesting that the latter is the physiologic electron‐donating system.