The ferric-hydroperoxo complex of chloroperoxidase

The hydroperoxo-ferric complex, or Compound 0 (Cpd 0), is an unstable transient intermediate common for oxygen activating heme enzymes such as the cytochromes P450, nitric oxide synthases, and heme oxygenases, as well as the peroxidases and catalases which utilize hydrogen peroxide as a source of oxygen and reducing equivalents. Detailed understanding of the mechanism of oxygen activation and formation of the higher valent catalytically active intermediates in heme enzyme catalysis requires the structural and spectroscopic characterization of this immediate precursor, Cpd 0. Using the method of cryoradiolytic reduction of the oxy-ferrous heme complex, we have prepared and characterized hydroperoxo-ferric complex in chloroperoxidase (CPO) and compared this to the same intermediate generated in cytochrome P450 CYP101. Optical absorption spectrum of Cpd 0 in CPO has a Soret band at 449 nm and poorly resolved α, β bands at 576 and 546 nm.     

Formation and decay of hydroperoxo-ferric heme complex in horseradish peroxidase studied by cryoradiolysis

Using radiolytic reduction of the oxy-ferrous horseradish peroxidase (HRP) at 77 K, we observed the formation and decay of the putative intermediate, the hydroperoxo-ferric heme complex, often called "Compound 0." This intermediate is common for several different enzyme systems as the precursor of the Compound I (ferryl-oxo pi-cation radical) intermediate. EPR and UV-visible absorption spectra show that protonation of the primary intermediate of radiolytic reduction, the peroxo-ferric complex, to form the hydroperoxo-ferric complex is completed only after annealing at temperatures 150-180 K. After further annealing at 195-205 K, this complex directly transforms to ferric HRP without any observable intervening species. The lack of Compound I formation is explained by inability of the enzyme to deliver the second proton to the distal oxygen atom of hydroperoxide ligand, shown to be necessary for dioxygen bond heterolysis on the "oxidase pathway," which is non-physiological for HRP. Alternatively, the physiological substrate H2O2 brings both protons to the active site of HRP, and Compound I is subsequently formed via rearrangement of the proton from the proximal to the distal oxygen atom of the bound peroxide.     

Unique features of recombinant heme oxygenase of Drosophila melanogaster compared with those of other heme oxygenases studied.

We cloned a cDNA for a Drosophila melanogaster homologue of mammalian heme oxygenase (HO) and constructed a bacterial expression system of a truncated, soluble form of D. melanogaster HO (DmDeltaHO). The purified DmDeltaHO degraded hemin to biliverdin, CO and iron in the presence of reducing systems such as NADPH/cytochrome P450 reductase and sodium ascorbate, although the reaction rate was slower than that of mammalian HOs. Some properties of DmHO, however, are quite different from other known HOs. Thus DmDeltaHO bound hemin stoichiometrically to form a hemin-enzyme complex like other HOs, but this complex did not show an absorption spectrum of hexa-coordinated heme protein. The absorption spectrum of the ferric complex was not influenced by changing the pH of the solution. Interestingly, an EPR study revealed that the iron of heme was not involved in binding heme to the enzyme. Hydrogen peroxide failed to convert it into verdoheme. A spectrum of the ferrous-CO form of verdoheme was not detected during the reaction from hemin under oxygen and CO. Degradation of hemin catalyzed by DmDeltaHO yielded three isomers of biliverdin, of which biliverdin IXalpha and two other isomers (IXbeta and IXdelta) accounted for 75% and 25%, respectively. Taken together, we conclude that, although DmHO acts as a real HO in D. melanogaster, its active-site structure is quite different from those of other known HOs.     

CO-trapping site in heme oxygenase revealed by photolysis of its co-bound heme complex: mechanism of escaping from product inhibition

Heme oxygenase (HO) catalyzes physiological heme degradation using O(2) and reducing equivalents to produce biliverdin, iron, and CO. Notably, the HO reaction proceeds without product inhibition by CO, which is generated in the conversion reaction of alpha-hydroxyheme to verdoheme, although CO is known to be a potent inhibitor of HO and other heme proteins. In order to probe how endogenous CO is released from the reaction site, we collected X-ray diffraction data from a crystal of the CO-bound form of the ferrous heme-HO complex in the dark and under illumination by a red laser at approximately 35 K. The difference Fourier map indicates that the CO ligand is partially photodissociated from the heme and that the photolyzed CO is trapped in a hydrophobic cavity adjacent to the heme pocket. This hydrophobic cavity was occupied also by xenon, which is similar to CO in terms of size and properties. Taking account of the affinity of CO for the ferrous verdoheme-HO complex being much weaker than that for the ferrous heme complex, the CO derived from alpha-hydroxyheme would be trapped preferentially in the hydrophobic cavity but not coordinated to the iron of verdoheme. This structural device would ensure the smooth progression of the subsequent reaction, from verdoheme to biliverdin, which requires O(2) binding to verdoheme.     

Degradation of mesoheme and hydroxymesoheme catalyzed by the heme oxygenase system: involvement of hydroxyheme in the sequence of heme catabolism

Mesoheme bound to heme oxygenase protein was easily degraded to mesobiliverdin by incubation with NADPH-cytochrome c reductase and NADPH. The features of mesoheme degradation were very similar to those of protoheme degradation catalyzed by the heme oxygenase system; an intermediate compound having its absorption maximum at 660 nm appeared in the couse of mesoheme degradation and this compound is presumably equivalent to the 688 nm compound which appears in the course of protoheme degradation. Hydroxymesoheme was chemically prepared and a complex of hydroxymesoheme and heme oxygenase was prepared. The complex was fairly stable in air, but when the complex was incubated with the NADPH-cytochrome c reductase system, the hydroxymesoheme bound to heme oxygenase was readily converted to mesobiliverdin through the 660 nm compound as an intermediate. It is evident that hydroxyheme is a real intermediate of heme degradation in the heme oxygenase reaction and that the 688 nm compound (or the 660 nm compound in the mesoheme system) is located between hydroxyheme and the biliverdin-iron chelate. The ferrous state of heme-iron may also be necessary for the onset of further oxidation of hydroxyheme.     

Visible absorption spectra of quantum mixed-spin ferric heme proteins.

Recently, it has been shown that the magnetic data for Chromatium ferricytochrome c' at pH 7 are consistent with quantum mechanically (as distinguished from thermally) mixed mid-spin (S = 3/2) and high-spin (S = 5/2) heme. Visible absorption spectra of the protein measured at 77 degrees K and 293 degrees K, pH 7, show peaks at 400, 490, and 632 nm. The observation of a 630 nm band in quantum mixed-spin heme spectra, and the spin state-dependence of the band intensity, are discussed in the context of the iron-ligand structure for quantum mixed-spin heme inferred from magnetic data.     

Variation of the oxidation state of verdoheme in the heme oxygenase reaction

Heme oxygenase (HO) converts hemin to biliverdin, CO, and iron applying molecular oxygen and electrons. During successive HO reactions, two intermediates, α-hydroxyhemin and verdoheme, have been generated. Here, oxidation state of the verdoheme-HO complexes is controversial. To clarify this, the heme conversion by soybean and rat HO isoform-1 (GmHO-1 and rHO-1, respectively) was compared both under physiological conditions, with oxygen and NADPH coupled with ferredoxin reductase/ferredoxin for GmHO-1 or with cytochrome P450 reductase for rHO-1, and under a non-physiological condition with hydrogen peroxide. EPR measurements on the hemin-GmHO-1 reaction with oxygen detected a low-spin ferric intermediate, which was undetectable in the rHO-1 reaction, suggesting the verdoheme in the six-coordinate ferric state in GmHO-1. Optical absorption measurements on this reaction indicated that the heme degradation was extremely retarded at verdoheme though this reaction was not inhibited under high-CO concentrations, unlike the rHO-1 reaction. On the contrary, the Gm and rHO-1 reactions with hydrogen peroxide both provided ferric low-spin intermediates though their yields were different. The optical absorption spectra suggested that the ferric and ferrous verdoheme coexisted in reaction mixtures and were slowly converted to the ferric biliverdin complex. Consequently, in the physiological oxygen reactions, the verdoheme is found to be stabilized in the ferric state in GmHO-1 probably guided by protein distal residues and in the ferrous state in rHO-1, whereas in the hydrogen peroxide reactions, hydrogen peroxide or hydroxide coordination stabilizes the ferric state of verdoheme in both HOs.     

The source of heme for vascular heme oxygenase II: de novo heme biosynthesis in rat aorta

Heme is an essential prosthetic group or substrate for many proteins, including hemoglobin, and hemo enzymes such as nitric oxide synthase, soluble guanylyl cyclase, and heme oxygenase (HO). HO is responsible for the breakdown of heme into equimolar amounts of biliverdin, iron, and carbon monoxide, the latter of which is thought to play a role in the regulation of vascular tone. It is not clear whether the source of heme for cardiovascular functions is derived from uptake from the extracellular milieu or synthesis. In this study, we tested the hypothesis that blood vessels obtain their supply of heme for HO through de novo synthesis. Adult male Sprague-Dawley rat aorta was incubated at 37 degrees C in Krebs' solution with 1 micro M [14C]delta-aminolevulinic acid (ALA). [14C]ALA uptake was linear for about 30 min and reached a plateau at approximately 100 min. The radioactivity was incorporated into porphyrins and heme as determined by esterification of 14C-labelled metabolites and thin-layer chromatography. The first and rate-limiting step of heme biosynthesis is catalyzed by ALA synthase (ALA-S), the activity of which was determined in rat aorta using a radiometric assay, approximately 250 nmol x (g wet mass)(-1) x h(-1). Inducing HO-1 in rat aorta with S-nitroso-N-acetylpenicillamine (500 micro M) did not increase ALA-S activity as compared with basal activity levels of the enzyme. It appears that there is a sufficient amount of heme available under basal ALA-S activity conditions to meet the increased demand for heme resulting from HO-1 induction. These observations indicate that the complete enzymatic pathway for de novo heme biosynthesis resides in rat aorta and furthermore indicate that de novo heme synthesis is capable of supplying a substantial portion of the heme substrate for HO in the aorta.     

Expression of heme oxygenase and its RNA in mouse liver after injection of heme and splenectomy.

Heme is known to activate the HO (heme oxygenase) gene in cultured cells, but little is known about the effect of heme on the HO gene in intact organisms. The expressions of HO and its RNA in mouse liver were measured using mouse HO cDNA and HO antibody after injection of heme or splenectomy. The antibody was prepared against a beta-galactosidase-HO hybrid protein made in Escherichia coli. The HO mRNA level increased to a maximum 15 h after heme injection. In contrast, expression of HO was maximal about 45 h after heme injection. Essentially the same results were obtained in mice after splenectomy. These results suggest that the HO gene in mouse liver was activated by the injection of heme and splenectomy.     

Crystal structures of ferrous and CO-, CN(-)-, and NO-bound forms of rat heme oxygenase-1 (HO-1) in complex with heme: structural implications for discrimination between CO and O2 in HO-1

Heme oxygenase (HO) catalyzes heme degradation by utilizing O(2) and reducing equivalents to produce biliverdin IX alpha, iron, and CO. To avoid product inhibition, the heme[bond]HO complex (heme[bond]HO) is structured to markedly increase its affinity for O(2) while suppressing its affinity for CO. We determined the crystal structures of rat ferrous heme[bond]HO and heme[bond]HO bound to CO, CN(-), and NO at 2.3, 1.8, 2.0, and 1.7 A resolution, respectively. The heme pocket of ferrous heme-HO has the same conformation as that of the previously determined ferric form, but no ligand is visible on the distal side of the ferrous heme. Fe[bond]CO and Fe[bond]CN(-) are tilted, whereas the Fe[bond]NO is bent. The structure of heme[bond]HO bound to NO is identical to that bound to N(3)(-), which is also bent as in the case of O(2). Notably, in the CO- and CN(-)-bound forms, the heme and its ligands shift toward the alpha-meso carbon, and the distal F-helix shifts in the opposite direction. These shifts allow CO or CN(-) to bind in a tilted fashion without a collision between the distal ligand and Gly139 O and cause disruption of one salt bridge between the heme and basic residue. The structural identity of the ferrous and ferric states of heme[bond]HO indicates that these shifts are not produced on reduction of heme iron. Neither such conformational changes nor a heme shift occurs on NO or N(3)(-) binding. Heme[bond]HO therefore recognizes CO and O(2) by their binding geometries. The marked reduction in the ratio of affinities of CO to O(2) for heme[bond]HO achieved by an increase in O(2) affinity [Migita, C. T., Matera, K. M., Ikeda-Saito, M., Olson, J. S., Fujii, H., Yoshimura, T., Zhou, H., and Yoshida, T. (1998) J. Biol. Chem. 273, 945-949] is explained by hydrogen bonding and polar interactions that are favorable for O(2) binding, as well as by characteristic structural changes in the CO-bound form.     

Evidence that the heme regulatory motifs in heme oxygenase-2 serve as a thiol/disulfide redox switch regulating heme binding

Heme oxygenase (HO) catalyzes the O(2)- and NADPH-dependent conversion of heme to biliverdin, CO, and iron. The two forms of HO (HO-1 and HO-2) share similar physical properties but are differentially regulated and exhibit dissimilar physiological roles and tissue distributions. Unlike HO-1, HO-2 contains heme regulatory motifs (HRMs) (McCoubrey, W. K., Jr., Huang, T. J., and Maines, M. D. (1997) J. Biol. Chem. 272, 12568-12574). Here we describe UV-visible, EPR, and differential scanning calorimetry experiments on human HO-2 variants containing single, double, and triple mutations in the HRMs. Oxidized HO-2, which contains an intramolecular disulfide bond linking Cys(265) of HRM1 and Cys(282) of HRM2, binds heme tightly. Reduction of the disulfide bond increases the K(d) for ferric heme from 0.03 to 0.3 microm, which is much higher than the concentration of the free heme pool in cells. Although the HRMs markedly affect the K(d) for heme, they do not alter the k(cat) for heme degradation and do not bind additional hemes. Because HO-2 plays a key role in CO generation and heme homeostasis, reduction of the disulfide bond would be expected to increase intracellular free heme and decrease CO concentrations. Thus, we propose that the HRMs in HO-2 constitute a thiol/disulfide redox switch that regulates the myriad physiological functions of HO-2, including its involvement in the hypoxic response in the carotid body, which involves interactions with a Ca(2+)-activated potassium channel.     

Preparation and spectral characterization of the heme d1.apomyoglobin complex: an unusual protein environment for the substrate-binding heme of Pseudomonas cytochrome oxidase

The heme d1 prosthetic group isolated from Pseudomonas cytochrome oxidase combines with apomyoglobin to form a stable, optically well-defined complex. Addition of ferric heme d1 quenches apomyoglobin tryptophan fluorescence suggesting association in a 1:1 molar ratio. Optical absorption maxima for heme d1.apomyoglobin are at 629 and 429 nm before, and 632 and 458 nm after dithionite reduction; they are distinct from those of heme d1 in aqueous solution but more similar to those unobscured by heme c in Pseudomonas cytochrome oxidase. Cyanide, carbon monoxide and imidazole alter the spectrum of heme d1.apomyoglobin demonstrating axial coordination to heme d1 by exogeneous ligands. The cyanide-induced optical difference spectra exhibit isosbestic points, and a Scatchard-like analysis yields a linear plot with an apparent dissociation constant of 4.2 X 10(-5) M. However, carbon monoxide induces two absorption spectra with Soret maxima at 454 or 467 nm, and this duplicity, along with a shoulder that correlates with the latter before binding, suggests multiple carbon monoxide and possibly heme d1 orientations within the globin. The 50-fold reduction in cyanide affinity over myoglobin is more consistent with altered heme pocket interactions than the intrinsic electronic differences between the two hemes. However, stability of the heme d1.apomyoglobin complex is verified further by the inability to separate heme d1 from globin during dialysis and column chromatography in excess cyanide or imidazole. This stability, together with a comparison between spectra of ligand-free and -bound derivatives of heme d1-apomyoglobin and heme d1 in solution, implies that the prosthetic group is coordinated in the heme pocket through a protein-donated, strong-field ligand. Furthermore, the visible spectrum of heme d1.apomyoglobin varies minimally with ligand exchange, in contrast to the Soret, which suggests that much spectral information concerning heme d1 coordination in the oxidase is lost by interference from heme c absorption bands. A comparison of the absorption spectra of heme d1.apomyoglobin and Pseudomonas cytochrome oxidase, together with a critical examination of the previous axial ligand assignments from magnetic resonance techniques in the latter, implies that it is premature to accept the assignment of bishistidine heme d1 coordination in oxidized, ligand-free oxidase and other iron-isobacteriochlorin-containing enzymes.     

Molecular basis for the inability of an oxygen atom donor ligand to replace the natural sulfur donor heme axial ligand in cytochrome P450 catalysis. Spectroscopic characterization of the Cys436Ser CYP2B4 mutant

All cytochrome P450s (CYPs) contain a cysteinate heme iron proximal ligand that plays a crucial role in their mechanism of action. Conversion of the proximal Cys436 to Ser in NH₂-truncated microsomal CYP2B4 (ΔCYP2B4) transforms the enzyme into a two-electron NADPH oxidase producing H₂O₂ without monooxygenase activity [K.P. Vatsis, H.M. Peng, M.J. Coon, J. Inorg. Biochem. 91 (2002) 542–553]. To examine the effects of this ligation change on the heme iron spin-state and coordination structure of ΔC436S CYP2B4, the magnetic circular dichroism and electronic absorption spectra of several oxidation/ligation states of the variant have been measured and compared with those of structurally defined heme complexes. The spectra of the substrate-free ferric mutant are indicative of a high-spin five-coordinate structure ligated by anionic serinate. The spectroscopic properties of the dithionite-reduced (deoxyferrous) protein are those of a five-coordinate (high-spin) state, and it is concluded that the proximal ligand has been protonated to yield neutral serine (ROH-donor). Low-spin six-coordinate ferrous complexes of the mutant with neutral sixth ligands (NO, CO, and O₂) examined are also likely ligated by neutral serine, as would be expected for ferric complexes with anionic sixth ligands such as the hydroperoxo-ferric catalytic intermediate. Ligation of the heme iron by neutral serine vs. deprotonated cysteine is likely the result of the large difference in their acidity. Thus, without the necessary proximal ligand push of the cysteinate, although the ΔC436S mutant can accept two electrons and two protons, it is unable to heterolytically cleave the O–O bond of the hydroperoxo-ferric species to generate Compound I and hydroxylate the substrate.     

Purification and properties of heme oxygenase from rat liver microsomes.

Heme oxygenase was purified to apparent homogeneity from liver microsomes of rats which had been treated with either cobaltous chloride or hemin to induce heme oxygenase in the liver and the purified preparations from either rats showed an apparent molecular weight of about 200,000 when estimated by gel filtration on a column of Sephadex G-200, and gave a minimum molecular weight of about 32,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hepatic heme oxygenase could bind heme to form a heme . heme oxygenase complex showing an absorption peak at 405 nm, and the extinction coefficient at 405 nm of the heme . heme oxygenase complex was 140 mM-1 cm-1. The heme bound to the hepatic heme oxygenase protein was easily converted to biliverdin when the complex was incubated with the NADPH-cytochrome c reductase system in air. The hepatic heme oxygenase appears to have characteristics essentially similar to those of the splenic heme oxygenase (Yoshida, T., and Kikuchi, G. (1978) J. Biol. Chem. 253, 4224 and 4230). The heme oxygenase preparation which was purified from the cobalt-treated rats contained a small amount of cobaltic protoporphyrin, indicating that cobalt protoporphyrin was synthesized in these rats.     

The source of heme for vascular heme oxygenase I: heme uptake in rat aorta

During the last decade, heme oxygenase (HO) and carbon monoxide (CO) have garnered substantial research interest in terms of cell and organ regulation, especially as they bear on the central nervous system, organ transplantation, and the cardiovascular system. While the enzymatic mechanism, substrates, and products of HO are well known, it is not clear whether the cardiovascular system derives its supply of the heme substrate through de novo synthesis or uptake from the extracellular milieu. The objective of the present study was to test the latter possibility in rat aorta and to determine the influence of plasma proteins that bind heme in vivo, viz. hemopexin and albumin. Aortic tissue was exposed to [14C]heme in vitro, and the concentration and time dependence of heme uptake was assessed. The presence of hemopexin or albumin in the incubation medium dramatically decreased heme uptake by the aorta. Heme uptake by aortic tissue was not altered after induction of HO-1, which would be expected to increase tissue heme demand. In summary, the rat, isolated aorta was capable of obtaining heme from its external milieu, but this was obtunded in the presence of the plasma proteins hemopexin or albumin. For normal physiological situations, heme uptake may not be a usual source of substrate for vascular HO and hemoenzymes such as nitric oxide synthase, soluble guanylyl cyclase, and cyclooxygenase.     

Interaction between heme oxygenase-1 and -2 proteins

The three isoforms of heme oxygenase (HO), the rate-limiting enzyme in heme degradation, are the products of different genes that show marked differences in regulation and expression. Why is there redundancy in the heme degradation pathway, and why are there differences in tissue expression of HO isoenzymes are unanswered questions? An interaction between HO-1 and HO-2 is suspected by the co-localization of these enzymes in the lung and regions of the brain. Using multiple models and assays, we demonstrated an interaction between HO-1 and HO-2 at amino acids 0-45 of HO-2 and amino acids 58-80 of HO-1. The latter corresponds to a highly conserved, hydrophilic, and exposed region of the protein. Furthermore, the observed activity of the HO-1.HO-2 complex was lower than that expected from the sum of HO-1- and HO-2-derived activities, suggesting that this interaction serves to limit HO enzymatic activity. We speculate that this HO-1.HO-2 protein interaction may promote non-enzymatic functions of HO.     

Heme oxygenase and heme degradation

The microsomal heme oxygenase system consists of heme oxygenase (HO) and NADPH-cytochrome P450 reductase, and plays a key role in the physiological catabolism of heme which yields biliverdin, carbon monoxide, and iron as the final products. Heme degradation proceeds essentially as a series of autocatalytic oxidation reactions involving heme bound to HO. Large amounts of HO proteins from human and rat can now be prepared in truncated soluble form, and the crystal structures of some HO proteins have been determined. These advances have greatly facilitated the understanding of the mechanisms of individual steps of the HO reaction. HO can be induced in animals by the administration of heme or several other substances; the induction is shown to involve Bach1, a translational repressor. The induced HO is assumed to have cytoprotective effects. An uninducible HO isozyme, HO-2, has been identified, so the authentic HO is now called HO-1. HOs are also widely distributed in invertebrates, higher plants, algae, and bacteria, and function in various ways according to the needs of individual species.