Spectroscopic studies of protein-heme interactions accompanying the allosteric transition in methemoglobins

Resonance Raman, optical absorption, and circular dichroism spectroscopic techniques have been used to examine the effect of the addition of inositol hexaphosphate (IHP) to a series of carp and human methemoglobin derivatives. Markers of spin equilibrium in the high-frequency region (1450-1650 cm-1) of the resonance Raman spectrum yield high/low-spin ratios consistent with direct magnetic susceptibility measurements. Changes in the low-frequency region (100-600 cm-1) of the resonance Raman spectrum appear to correlate with the quaternary structure transition. Changes in the ultraviolet absorption spectra and the circular dichroism spectra also appear to be related to the quaternary structure change. By using the resonance Raman spin markers, we find that those derivatives of carp methemoglobin which are in spin equilibrium have a larger ratio of high-spin to low-spin populations than the corresponding derivatives of human methemoglobin. Upon the addition of IHP to the methemoglobins the spin equilibrium is shifted toward a larger high-spin population. This change in equilibrium is larger for the carp protein than for the human protein. We obtain an IHP-induced change in the free energy difference between the high-spin and low-spin states of 300 cal/mol for those human methemoglobins in which a quaternary structure change occurs and 600 cal/mol for carp methemoglobins. Our data are consistent with a quaternary structure change induced by IHP in all the carp methemoglobins studied (F-, H2O, SCN-, NO2-, N3-, and CN-) and in the F-, H2O, and SCN- derivatives of the human protein but not in the NO2-, N3-, and CN- derivatives. The Fe-CN stretching mode has been identified by isotopic substitution and found to be unchanged in frequency in carp CN- metHb when the quaternary structure is changed. On the basis of our results we conclude that the protein forces at the heme due to the addition of IHP do not significantly affect the position of the iron atom with respect to the heme plane. Rather, the changes in spin equilibrium may be caused by protein-induced changes in the orientation of the proximal histidine or tertiary structure changes in the heme pocket which affect the porphyrin macrocycle. Either of these changes, or a combination thereof, leads to changes in the iron d orbital energies and concomitant changes in the spin equilibrium.     

Structure of cyanide methemoglobin

X-ray difference Fourier analysis at 2.8 Å resolution shows that the tertiary structures of horse cyanide methemoglobin and methemoglobin differ significantly. The conformations of the heme groups and their interactions with the globin are altered. Short contacts with globin side chains affect cyanide binding to the hemes, and the changes in globin-ligand contact upon substitution of cyanide for water in turn directly affect globin structure. Although the ligand peaks lie off the heme axes, the atoms FeCN may still lie on a straight line (as they do in small iron cyanide complexes), with this line not normal to the mean heme plane. This linear binding configuration is consistent with the observed motion and deformation of the porphyrin. Although motion of the iron atoms is not directly apparent, there is evidence that some changes in tertiary structure are induced by shortening of the iron-pyrrol nitrogen bond lengths. This and other studies suggest that the structural changes responsible for co-operativity in hemoglobin may be initiated not merely by an alteration in the covalent porphyrin-proximal histidine linkage, but by changes in the noncovalent interactions of the globin with the ligand and porphyrin as well.     

Comparison of the peroxidatic activity of cytochrome P-450 with other hemoproteins and model compounds.

The H2O2 dependent catalysis of cytochrome P-450 was compared with the catalytic mechanism of horse radish peroxidase, methemoglobin and iron protoporphyrin complexes. A relatively stable intermediate being comparable to compound I of horse radish peroxidase is formed in the case of iron porphyrin complexes, methemoglobin and probably cytochrome P-450. In the case of peroxidase compound II is the more stable intermediate. This could be the reason for the different catalytic properties of peroxidase on the one hand and iron porphyrin complexes, methemoglobin and cytochrome P-450 on the other hand.     

Efficiency of methemoglobin, hemin and ferric citrate in catalyzing protein tyrosine nitration, protein oxidation and lipid peroxidation in a bovine serum albumin-liposome system: Influence of pH

Protein tyrosine nitration, protein oxidation and lipid peroxidation are nitrative/oxidative modification of protein and lipids. In this paper, a BSA (bovine serum albumin)-lecithin liposome system was used to study the nature of different forms of iron, including methemoglobin, hemin and ferric citrate, in catalyzing H₂O₂-nitrite system to oxidize protein and lipid as well as nitrate protein. It was found that in pH range of 5.0-9.0, in pure BSA solution or pure liposome solution, hemin and methemoglobin catalyzed protein tyrosine nitration and lipid peroxidation were decreased with the increasing of pH, while hemin and methemoglobin catalyzed protein oxidation was significantly and moderately increased, respectively. Lipid completely inhibited hemin catalyzed protein tyrosine nitration but only partially inhibited methemoglobin catalyzed protein tyrosine nitration, and its inhibitory effect on hemin induced protein oxidation was also more pronounced. In addition, BSA showed more efficient in inhibiting hemin and ferric citrate induced lipid peroxidation. At the same condition, ferric citrate was relatively ineffective in all tests. Considering protein tyrosine nitration, protein oxidation and lipid oxidation as overall oxidative damage, these results indicated that methemoglobin is more toxic than hemin and ferric citrate, the degradation procedure of heme containing macromolecules, e.g. hemoglobin to hemin and finally to low molecular weight bounded iron, is step by step detoxification. These results provide fundamental knowledge on oxidative/nitrative of biomolecules in lipid-protein coexistence system.     

Enzymatic reduction of synthetic polymer-bound hemin derivatives: efficient method to prepare a heme-oxygen adduct in cooled aqueous medium

Synthetic polymer-bound hemin (iron(III) protoporphyrin IX) derivatives were effectively reduced by ferredoxin and ferredoxin-NADP reductase system. The resultant polymer-bound heme (iron(II) protoporphyrin IX) derivatives formed oxygen adducts with a lifetime of ca. 1 hr in aqueous solution at -30 degrees C. The reduction rate is discussed in terms of the structure of the hemin derivatives.     

Heme utilization in Campylobacter jejuni

A putative iron- and Fur-regulated hemin uptake gene cluster, composed of the transport genes chuABCD and a putative heme oxygenase gene (Cj1613c), has been identified in Campylobacter jejuni NCTC 11168. Mutation of chuA or Cj1613c leads to an inability to grow in the presence of hemin or hemoglobin as a sole source of iron. Mutation of chuB, -C, or -D only partially attenuates growth where hemin is the sole iron source, suggesting that an additional inner membrane (IM) ABC (ATP-binding cassette) transport system(s) for heme is present in C. jejuni. Genotyping experiments revealed that Cj1613c is highly conserved in 32 clinical isolates. One strain did not possess chuC, though it was still capable of using hemin/hemoglobin as a sole iron source, supporting the hypothesis that additional IM transport genes are present. In two other strains, sequence variations within the gene cluster were apparent and may account for an observed negative heme utilization phenotype. Analysis of promoter activity within the Cj1613c-chuA intergenic spacer region revealed chuABCD and Cj1613c are expressed from separate iron-repressed promoters and that this region also specifically binds purified recombinant Fur(Cj) in gel retardation studies. Absorbance spectroscopy of purified recombinant His(6)-Cj1613c revealed a 1:1 heme:His(6)-Cj1613c binding ratio. The complex was oxidatively degraded in the presence of ascorbic acid as the electron donor, indicating that the Cj1613c gene product functions as a heme oxygenase. In conclusion, we confirm the involvement of Cj1613c and ChuABCD in heme/hemoglobin utilization in C. jejuni.     

Interaction of human adult methemoglobin in low-spin state with inositol hexaphosphate. A proton magnetic resonance study

The hyperfine-shifted proton nuclear magnetic resonance (NMR) spectra of the low-spin complexes of human adult methemoglobin were found to be much altered by the addition of inositol hexaphosphate (IHP). The stoichiometry and pH-dependence of IHP binding, and the spin equilibrium of azide methemoglobin are parallel to those of high-spin human methemoglobin and of carp methemoglobin, both of which are proposed to be switched from the R to T states with IHP. The present NMR results show that IHP affects the structure of human methemoglobin regardless of the spin state of the heme iron, suggesting that there is no correspondence between quaternary structure and the spin state of ferric heme iron.     

Roles of porphyrins and host iron transport proteins in regulation of growth of Porphyromonas gingivalis W50.

Porphyromonas gingivalis (Bacteroides gingivalis) requires iron in the form of hemin for growth and virulence in vitro, but the contributions of the porphyrin ring structure, porphyrin-associated iron, host hemin-sequestering molecules, and host iron-withholding proteins to its survival are unknown. Therefore, the effects of various porphyrins, host iron transport proteins, and inorganic iron sources on the growth of P. gingivalis W50 were examined to delineate the various types of iron molecules used for cellular metabolism. Cell envelope-associated hemin and iron stores contributed to the growth of P. gingivalis in hemin-free culture, and depletion of these endogenous reserves required eight serial transfers into hemin-free medium for total suppression of growth. Comparable growth of P. gingivalis was observed with 7.7 microM equivalents of hemin as hemoglobin (HGB), methemoglobin, myoglobin, hemin-saturated serum albumin, lactoperoxidase, cytochrome c, and catalase. Unrestricted growth was recorded in the presence of haptoglobin-HGB and hemopexin-hemin complexes, indicating that these host defense proteins do not sequester HGB and hemin from P. gingivalis. The iron chelator 2,2'-bipyridyl functionally chelated hemin-associated iron, resulting in dose-dependent inhibition of growth in hemin-restricted cultures at 1 to 25 microM 2,2'-bipyridyl concentrations. In the absence of an exogenous iron source, protoporphyrin IX did not support P. gingivalis growth. These findings suggest that the iron atom in the hemin molecule is the critical constituent for growth and that the tetrapyrrole porphyrin ring structure may represent an important vehicle for delivery of iron into the P. gingivalis cell. P. gingivalis does not have a strict requirement for porphyrins, since growth occurred with nonhemin iron sources, including high concentrations (200 muM) of ferric, ferrous, and nitrogenous inorganic iron, and P. gingivalis exhibited unrestricted growth in the presence of host transferrin, lactoferrin, and serum albumin. The diversity of iron substrates utilized by P. gingivalis and the observation that growth was not affected by the bacteriostatic effects of host iron-withholding proteins, which it may encounter in the periodontal pocket, may explain why P. gingivalis is such a formidable pathogen in the periodontal disease process.     

Circular dichroism studies on cytochrome c peroxidase from baker's yeast (Saccharomyces cerevisiae).

Circular dichroism spectra of cytochrome c peroxidase from baker's yeast, those of the reduced enzyme, the carbonyl, cyanide and fluoride derivatives and the hydrogen peroxide compound, Compound I, have been recorded in the wavelength range 200 to 660 nm. All derivatives show negative Soret Cotton effects. The results suggest that the heme group is surrounded by tightly packed amino acid sidechains and that there is a histidine residue bound to the fifth coordination site of the heme iron. The native ferric enzyme is probably pentacoordinated. The circular dichroism spectra of the ligand compounds indicate that the ligands form a nonlinear bond to the heme iron as a result of steric hindrance in the vicinity of the heme. The spectrum of Compound I shows no perturbation of the porphyrin symmetry. The dichroic spectrum of the native enzyme in the far-ultraviolet wave-length region suggests that the secondary structure consists of roughly equal amounts of alpha-helical, beta-structure and unordered structure. After the removal of the heme group no great changes in the secondary structure can be observed.     

Genetics and regulation of heme iron transport in Shigella dysenteriae and detection of an analogous system in Escherichia coli O157:H7.

Shigella species can use heme as the sole source of iron. In this work, the heme utilization locus of Shigella dysenteriae was cloned and characterized. A cosmid bank of S. dysenteriae serotype 1 DNA was constructed in an Escherichia coli siderophore synthesis mutant incapable of heme transport. A recombinant clone, pSHU12, carrying the heme utilization system of S. dysenteriae was isolated by screening on iron-poor medium supplemented with hemin. Transposon insertional mutagenesis and subcloning identified the region of DNA in pSHU12 responsible for the phenotype of heme utilization. Minicell analysis indicated that a 70-kDa protein encoded by this region was sufficient to allow heme utilization in E. coli. Synthesis of this protein, designated Shu (Shigella heme uptake), was induced by iron limitation. The 70-kDa protein is located in the outer membrane and binds heme, suggesting it is the S. dysenteriae heme receptor. Heme iron uptake was found to be TonB dependent in E. coli. Transformation of an E. coli hemA mutant with the heme utilization subclone, pSHU262, showed that heme could serve as a source of porphyrin as well as iron, indicating that the entire heme molecule is transported into the bacterial cell. DNA sequences homologous to shu were detected in strains of S. dysenteriae serotype 1 and E. coli O157:H7.     

Binding Specificity of the Porphyromonas gingivalis Heme and Hemoglobin Receptor HmuR, Gingipain K, and Gingipain R1 for Heme, Porphyrins, and Metalloporphyrins

Previous genetic and biochemical studies have confirmed that hemoglobin and hemin utilization in Porphyromonas gingivalis is mediated by the outer membrane hemoglobin and heme receptor HmuR, as well as gingipain K (Kgp), a lysine-specific cysteine protease, and gingipain R1 (HRgpA), one of two arginine-specific cysteine proteases. In this study we report on the binding specificity of the recombinant P. gingivalis HmuR protein and native gingipains for hemoglobin, hemin, various porphyrins, and metalloporphyrins as assessed by spectrophotometric assays, by affinity chromatography, and by enzyme-linked immunosorbent assay. Protoporphyrin, mesoporphyrin, deuteroporphyrin, hematoporphyrin, and some of their iron, copper, and zinc derivatives were examined to evaluate the role of both the central metal ion and the peripheral substituents on binding to recombinant HmuR and soluble gingipains. Scatchard analysis of hemin binding to Escherichia coli cells expressing recombinant membrane-associated six-His-tagged HmuR yielded a linear plot with a binding affinity of 2.4 x 10(-5) M. Recombinant E. coli cells bound the iron, copper, and zinc derivatives of protoporphyrin IX (PPIX) with similar affinities, and approximately four times more tightly than PPIX itself, which suggests that the active site of HmuR contains a histidine that binds the metal ion in the porphyrin ring. Furthermore, we found that recombinant HmuR prefers the ethyl and vinyl side chains of the PPIX molecule to either the larger hydroxyethyl or smaller hydrogen side chains. Kgp and HRgpA were demonstrated to bind various porphyrins and metalloporphyrins with affinities similar to those for hemin, indicating that the binding of Kgp and HRgpA to these porphyrins does not require a metal within the porphyrin ring. We did not detect the binding of RgpB, the arginine-specific cysteine protease that lacks a C-terminal hemagglutinin domain, to hemoglobin, porphyrins, or metalloporphyrins. Kgp and HRgpA, but not RgpB, were demonstrated to bind directly to soluble recombinant six-His-tagged HmuR. Several possible mechanisms for the cooperation between outer membrane receptor HmuR and proteases Kgp and HRgpA in hemin and hemoglobin binding and utilization are discussed.     

Bacillus anthracis IsdG, a heme-degrading monooxygenase

Bacillus anthracis, the causative agent of anthrax, utilizes hemin and hemoglobin for growth in culture, suggesting that these host molecules serve as sources for the nutrient iron during bacterial infection. Bioinformatic analyses of the B. anthracis genome revealed genes with similarity to the iron-regulated surface determinant (isd) system responsible for heme uptake in Staphylococcus aureus. We show that the protein product of one of these genes, isdG, binds hemin in a manner resembling the heme binding of known heme oxygenases. Formation of IsdG:hemin complexes in the presence of a suitable electron donor, e.g., ascorbate or cytochrome P450 reductase, promotes catalytic degradation of hemin to biliverdin with concomitant release of iron. IsdG is required for B. anthracis utilization of hemin as a sole iron source, and it is also necessary for bacterial protection against heme-mediated toxicity. These data suggest that IsdG functions as a heme-degrading monooxygenase in B. anthracis.     

Effect of porphyrin ring ligands on the affinity of heme iron to axial ligands]

To clarify the influence of protein surrounding on the heme reactivity in heme proteins the effect of interaction between a porphyrin ring and pi-acceptor molecule, 1,2,4-trimethyl-pyridinium (TMP), on the affinity of deuteroheme to axial ligands (imidazole and cyanide) has been studied as a model system. It is shown that TMP induces the fourfold decrease in equilibrium constant of imidazole to deuteroheme. From the analysis of the two stages for cyanide binding it is concluded that TMP decreases the binding constant of the first cyanide by 40 times and does not apparently influence the second ligand binding. The effect of TMP on the reactivity of deuteroheme to axial ligands is interpreted as a result of a decrease in the electron density on the iron orbitals which is due to the altered pi-eleectron density in the porphyrin pi-system through the donor-acceptor interaction with TMP molecules. The possible significance of the contacts between the porphyrin and neighboring amino acid residues in determining heme affinity to axial ligands is discussed.     

The Interplay between Molten Globules and Heme Disassociation Defines Human Hemoglobin Disassembly

Hemoglobin functions as a tetrameric oxygen transport protein, with each subunit containing a heme cofactor. Its denaturation, either in vivo or in vitro, involves autoxidation to methemoglobin, followed by cofactor loss and globin unfolding. We have proposed a global disassembly scheme for human methemoglobin, linking hemin (ferric protoporphyrin IX) disassociation and apoprotein unfolding pathways. The model is based on the evaluation of circular dichroism and visible absorbance measurements of guanidine-hydrochloride-induced disassembly of methemoglobin and previous measurements of apohemoglobin unfolding. The populations of holointermediates and equilibrium disassembly parameters were estimated quantitatively for adult and fetal hemoglobins. The key stages are characterized by hexacoordinated hemichrome intermediates, which are important for preventing hemin disassociation from partially unfolded, molten globular species during early disassembly and late-stage assembly events. Both unfolding experiments and independent small angle x-ray scattering measurements demonstrate that heme disassociation leads to the loss of tetrameric structural integrity. Our model predicts that after autoxidation, dimeric and monomeric hemichrome intermediates occur along the disassembly pathway inside red cells, where the hemoglobin concentration is very high. This prediction suggests why misassembled hemoglobins often get trapped as hemichromes that accumulate into insoluble Heinz bodies in the red cells of patients with unstable hemoglobinopathies. These Heinz bodies become deposited on the cell membranes and can lead to hemolysis. Alternatively, when acellular hemoglobin is diluted into blood plasma after red cell lysis, the disassembly pathway appears to be dominated by early hemin disassociation events, which leads to the generation of higher fractions of unfolded apo subunits and free hemin, which are known to damage the integrity of blood vessel walls. Thus, our model provides explanations of the pathophysiology of hemoglobinopathies and other disease states associated with unstable globins and red cell lysis and also insights into the factors governing hemoglobin assembly during erythropoiesis.     

A new thioether-ligated iron porphyrin as a model of a protonated form of P450 active site

Thioether-ligated iron porphyrin (complex 1) was synthesized as a model of the protonated form of P450 to explore the possible involvement of the protonated form in the catalytic cycle, and ether-ligated iron porphyrin (complex 2) was also synthesized for comparison. The thioether and ether ligands enhanced heterolytic O-O bond cleavage of peroxy acid-iron porphyrin complex even in highly hydrophobic media without the assistance of acid or base, using mCPPAA as an oxidant. Competitive oxidation of cyclooctane/cyclooctene catalyzed by iron porphyrins showed that complexes 1 and 2 are less effective than heme thiolate (P450 and a synthetic heme thiolate (SR complex)) in oxidizing alkane. The possibility that thiol-ligated heme, which is a protonated form of heme thiolate, is not involved in the active intermediate structure of P450 is indicated by this result. This is the first report concerning the oxidizing ability of a thioether-ligated iron porphyrin.     

Proton NMR study of the myoglobin reconstituted with meso-tetra(n-propyl)hemin

Spectrophotometric titration of meso-tetra(n-propyl)hemin with sperm-whale apomyoglobin revealed their 1:1 complex formation. The purified reconstituted metmyoglobin bound with an equal molar amount of CN- and the second CN- ligation was not evidenced, suggesting that the hemin is not loosely attached to the globin surface, but incorporated into the heme pocket. The hyperfine-shifted proton NMR spectrum of the deoxy myoglobin revealed the proximal imidazole NH resonance at 85.1 ppm to indicate the formation of the Fe-N(His-F8) bond. The eight pyrrole protons of the hemin of myoglobin in the absence of external ligand were observed as a single peak at -16 ppm. This indicates the electronic symmetry of the hemin and the low-spin configuration of the heme iron. The pyrrole-proton NMR patterns of the cyanide and deoxy myoglobins were found to be remarkably temperature-dependent, which was consistently explained in terms of the free rotation of the prosthetic group. The NMR results suggest that introduction of meso-tetra(n-propyl)hemin totally disrupts the highly stereospecific heme-globin contacts, making the prosthetic group mobile in the heme cavity.     

Analysis of low temperature magnetic circular dichroism of high spin ferrihemoproteids in the near UV region

Magnetic circular dichroism spectra of fluoride complexes of metmyoglobin, methemoglobin, and horseradish peroxidase in the region of 300-450 nm at temperatures from 300 to 2.1 K were measured and analyzed. The temperature dependence of magnetic circular dichroism in the Soret region was found to be different from that of other paramagnetic forms and from the theoretically predicted dependence. The difference is explained by the superposition of the pi-->pi*-transition of porphyrin with one (peroxidase) or two charge transfer transitions and by substantially different temperature dependences of magnetic circular dichroism for the transitions of the two types. By minimization of differences between the expected and observed temperature dependences of magnetic circular dichroism, the parameters of its temperature dependence for charge transfer transitions and the parameter D of the zero-field splitting of the electronic ground state of the heme were found. The values of D for the fluoride complexes of metmyoglobin (5.8 cm-1) and methemoglobin (6.1 cm-1) agree well with those obtained by other methods. The D value for the fluoride complex of horseradish peroxidase (8.8 cm-1) was determined for the first time.     

In vitro antimalarial activity of ICL670: a further proof of the correlation between inhibition of β-hematin formation and of peroxidative degradation of hemin

Iron chelators such as deferiprone, deferoxamine (DFO) and ICL670 (deferasirox) have previously been shown to display in vitro and/or in vivo antimalarial activities. To gain further insight in their antimalarial mechanism of action, their activities on inhibition of β-hematin formation and on both peroxidative and glutathione (GSH)-mediated degradation of hemin were investigated. Neither deferiprone nor DFO were able to inhibit β-hematin formation while ICL670 activity nearly matched that of chloroquine (CQ). Peroxidative degradation of hemin was also only strongly inhibited by both CQ and ICL670, the latter being significantly more efficient at pH 5.2. All iron chelators displayed minor, if any, inhibitory activity on GSH-mediated degradation of hemin. Discrepancies in the results obtained for the three iron chelators show that iron chelation is not the main driving force behind interference with heme degradation. Deferiprone, DFO and ICL670 share little structural community but both ICL670 and antimalarial ursolic acid derivatives (previously shown to block β-hematin formation and the peroxidative degradation of hemin) have hydrophobic groups and hydroxyphenyl moieties. These similarities in structures and activities further back up a possible two-step mechanism of action previously proposed for ursolic acid derivatives (Mullié et al., 2010) implying (1) stacking of an hydrophobic structure to hemin and (2) additive protection of hemin ferric iron from H₂O₂ by hydroxyphenyl groups through steric hindrance and/or trapping of oxygen reactive species in the direct neighborhood of ferric iron. These peculiar antimalarial mechanisms of action for ICL670 warrant further investigations and development.     

Resonance Raman spectra of bovine liver catalase: enhancement of proximal tyrosinate vibrations

Resonance Raman spectra of native bovine liver ferri-catalase have been obtained in the 200-1800 cm-1 region. Excitation at a series of wavelengths ranging from 406.7 to 514.5 nm has been used and gives rise to distinct sets of resonance Raman bands. Excitation within the Soret and Q-bands of the heme group produces the expected set of polarized and nonpolarized porphyrin modes, respectively. The frequencies of the porphyrin skeletal stretching bands in the 1450-1700 cm-1 region indicate that catalase contains only five-coordinate, high-spin heme groups. In addition to the porphyrin modes, bovine liver catalase exhibits bands near 1612 and 1520 cm-1 that are attributable to ring vibrations of the proximal tyrosinate that are enhanced via resonance with a proximal tyrosinate----Fe(III) change transfer transition centered near 490 nm. Similar bands have been observed in mutant hemoglobins that have tyrosinate axial ligands and in other Fe(III)-tyrosinate proteins. No resonance Raman bands have been observed that can be attributed to degraded hemes. The spectra are relatively insensitive to pH over the range of 5-10, and the same spectra are observed for catalase samples that do and do not contain tightly bound NADPH. Resonance Raman spectra of the fluoride complex exhibit porphyrin skeletal stretching modes that show it to be six coordinate, high spin, while the cyanide complex is six coordinate, low spin. Both the azide and thiocyanate complexes, however, are spin-state mixtures with the high-spin form predominant.     

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.