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.     

State of heme in heme c systems: Cytochrome c and heme c models

Polarized resonance Raman spectra of horse heart ferricytochrome c as a function of pH in the range 1.0–12, in the presence of the extrinsic ligands imidazole, cyanide, and azide, and in 4 M urea, are reported, as are resonance Raman spectra of heme undecapeptide in the presence of imidazole, pH 6.8 and pH 2.0, and with cyanide at pH 6.8. The range of investigation is 140–1700 cm^?1, using the 5145-, 4880-, and 4579-Å excitations. The spectra have been analyzed in terms of complexity, sensitivity, and the conformation-heme energetics of the systems. The state of heme in various forms is analyzed with regard to heme energetics, core size, nature of planarity, and coordination configuration. All low-spin forms of heme c systems, cytochrome c, and heme models are concluded to be hexacoordinated, in-plane heme iron systems. The effect of the location of the heme in the protein environment is found to be a slight expansion of the porphyrin core, ～0.01 Å, while the covalent linkage of heme to protein and a mixed nature of axial coordination configuration seem to have little effect on the energetics of the heme group. Complex formation with extrinsic ligand, imidazole, cyanide, or azide, results in a slight contraction of the heme core. The formation of cytochrome c form IV, the alkaline form, is shown to follow a process with apK _a of about 8.4, and similarly, acidic form II is created following the prior formation of an intermediate form with apK _a of about 3.6. The precursor to form IV is interpreted as containing perturbation of the pyrrol rings, whereas the precursor to the acidic form seems to reflect alteration of the energetics of the C_αC_{m α} structures of the heme group. The acidic form of heme undecapeptide is a hexacoordinated high-spin heme with an estimated displacement of 0.25 Å from the heme plane. The pH 2 form of cytochrome c is also a hexacoordinated high-spin form with two weak axial ligands, but iron is in the plane of the porphyrin ring.     

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.     

Carbon monoxide-driven reduction of ferric heme and heme proteins

Oxidized cytochrome c oxidase in a carbon monoxide atmosphere slowly becomes reduced as shown by changes in its visible spectra and its reactivity toward oxygen. The "auto-reduction" of cytochrome c oxidase by this procedure has been used to prepare mixed valence hybrids. We have found that this process is a general phenomenon for oxygen-binding heme proteins, and even for isolated hemin in basic aqueous solution. This reductive reaction may have physiological significance. It also explains why oxygen-binding heme proteins become oxidized much more slowly and appear to be more stable when they are kept under a CO atmosphere. Oxidized alpha and beta chains of human hemoglobin become reduced under CO much more slowly than does cytochrome c oxidase, where the CO-binding heme is coupled with another electron accepting metal center. By observing the reaction in both the forward and reverse direction, we have concluded that the heme is reduced by an equivalent of the water-gas shift reaction (CO + H2O----CO2 + 2e- + 2H+). The reaction does not require molecular oxygen. However, when the CO-driven reduction of cytochrome c oxidase occurs in the presence of oxygen, there is a competition between CO and oxygen for the reduced heme and copper of cytochrome alpha 3. Under certain conditions when both CO and oxygen are present, a peroxide adduct derived from oxygen reduction can be observed. This "607 nm complex," described in 1981 by Nicholls and Chanady (Nicholls, P., and Chanady, G. (1981) Biochim. Biophys. Acta 634, 256-265), forms and decays with kinetics in accord with the rate constants for CO dissociation, oxygen association and reduction, and dissociation of the peroxide adduct. In the absence of oxygen, if a mixture of cytochrome c and cytochrome c oxidase is incubated under a CO atmosphere, auto-reduction of the cytochrome c as well as of the cytochrome c oxidase occurs. By our proposed mechanism this involves a redistribution of electrons from cytochrome alpha 3 to cytochrome alpha and cytochrome c.     

Hydrophobic modulation of heme properties in heme protein maquettes

We have investigated the properties of the two hemes bound to histidine in the H10 positions of the uniquely structured apo form of the heme binding four-helix bundle protein maquette [H10H24-L6I,L13F](2), here called [I(6)F(13)H(24)](2) for the amino acids at positions 6 (I), 13 (F) and 24 (H), respectively. The primary structure of each alpha-helix, alpha-SH, in [I(6)F(13)H(24)](2) is Ac-CGGGEI(6)WKL.H(10)EEF(13)LKK.FEELLKL.H(24)EERLKK.L-CONH(2). In our nomenclature, [I(6)F(13)H(24)] represents the disulfide-bridged di-alpha-helical homodimer of this sequence, i.e., (alpha-SS-alpha), and [I(6)F(13)H(24)](2) represents the dimeric four helix bundle composed of two di-alpha-helical subunits, i.e., (alpha-SS-alpha)(2). We replaced the histidines at positions H24 in [I(6)F(13)H(24)](2) with hydrophobic amino acids incompetent for heme ligation. These maquette variants, [I(6)F(13)I(24)](2), [I(6)F(13)A(24)](2), and [I(6)F(13)F(24)](2), are distinguished from the tetraheme binding parent peptide, [I(6)F(13)H(24)](2), by a reduction in the heme:four-helix bundle stoichiometry from 4:1 to 2:1. Iterative redesign has identified phenylalanine as the optimal amino acid replacement for H24 in the context of apo state conformational specificity. Furthermore, the novel second generation diheme [I(6)F(13)F(24)](2) maquette was related to the first generation diheme [H10A24](2) prototype, [L(6)L(13)A(24)](2) in the present nomenclature, via a sequential path in sequence space to evaluate the effects of conservative hydrophobic amino acid changes on heme properties. Each of the disulfide-linked dipeptides studied was highly helical (>77% as determined from circular dichroism spectroscopy), self-associates in solution to form a dimer (as determined by size exclusion chromatography), is thermodynamically stable (-DeltaG(H)2(O) >18 kcal/mol), and possesses conformational specificity that NMR data indicate can vary from multistructured to single structured. Each peptide binds one heme with a dissociation constant, K(d1) value, tighter than 65 nM forming a series of monoheme maquettes. Addition of a second equivalent of heme results in heme binding with a K(d2) in the range of 35-800 nM forming the diheme maquette state. Single conservative amino acid changes between peptide sequences are responsible for up to 10-fold changes in K(d) values. The equilibrium reduction midpoint potential (E(m7.5)) determined in the monoheme state ranges from -156 to -210 mV vs SHE and in the diheme state ranges from -144 to -288 mV. An observed heme-heme electrostatic interaction (>70 mV) in the diheme state indicates a syn global topology of the di-alpha-helical monomers. The heme affinity and electrochemistry of the three H24 variants studied identify the tight binding sites (K(d1) and K(d2) values <200 nM) having the lower reduction midpoint potentials (E(m7.5) values of -155 and -260 mV) with the H10 bound hemes in the parent tetraheme state of [H10H24-L6I,L13F](2), here called [I(6)F(13)H(24)](2). The results of this study illustrate that conservative hydrophobic amino acid changes near the heme binding site can modulate the E(m) by up to +/-50 mV and the K(d) by an order of magnitude. Furthermore, the effects of multiple single amino acid changes on E(m) and K(d) do not appear to be additive.     

Improvement of heme oxygenase-1-based heme sensor for quantifying free heme in biological samples

We recently reported a novel heme sensor using fluorescently labeled heme oxygenase-1; however, its inherent enzyme activity would be a potential obstacle in quantifying heme in biological samples. Here, we found that mutation of the catalytically important residue, Asp140, with histidine in the sensor not only diminished the heme degradation activity but also increased heme binding affinity. The sensor with a visible fluorophore was also found to be beneficial to avoid background emission from endogenous substance in biological samples. By using the improved heme sensor, we succeeded in quantifying free heme in rat hepatic samples for the first time.     

The role of the heme propionates in heme biochemistry

There are numerous studies, relying on both experimental and theoretical observations, illustrating the active role of the heme propionates in regulating electron delivery to the iron center as well as biochemical properties of the heme. Evidences for this come from a wide variety of heme containing systems: cytochromes, heme peroxidases, globins, etc. Here, we shortly summarize these studies and revisit previous theoretical calculations (V. Guallar, M.H. Baik, S.J. Lippard, R.A. Friesner, Proc. Natl. Acad. Sci. USA 100 (2003) 6998-7002) where the propionate groups induced the delocalization of the spin density in the cytochrome P450cam putative active species, Compound I. We introduce novel data, obtained by means of mixed quantum mechanics and molecular mechanics methods, indicating a larger electron delocalization into the protein. We also present novel results based on the recent migration of spin density observed by Barrows et al. (T.P. Barrows, T.L. Poulos, Biochemistry 44 (2005) 14062-68) on an ascorbate peroxidase mutant. All this data strongly supports the importance of the propionate groups in tuning the heme electronic properties.     

Implication for using heme methyl hyperfine shifts as indicators of heme seating as related to stereoselectivity in the catabolism of heme by heme oxygenase: in-plane heme versus axial his rotation

The triple mutant of the solubilized, 265-residue construct of human heme oxygenase, K18E/E29K/R183E-hHO, has been shown to redirect the exclusive alpha-regioselectivity of wild-type hHO to primarily beta,delta-selectivity in the cleavage of heme (Wang, J., Evans, J. P., Ogura, H., La Mar, G. N., and Ortiz de Montellano, P. R. (2006) Biochemistry 45, 61-73). The 1H NMR hyperfine shift pattern for the substrate and axial His CbetaH's and the substrate-protein contacts of the cyanide-inhibited protohemin and 2,4-dimethyldeuterohemin complexes of the triple mutant have been analyzed in detail and compared to data for the WT complex. It is shown that protein contacts for the major solution isomers for both substrates in the mutant dictate approximately 90 degrees in-plane clockwise rotation relative to that in the WT. The conventional interpretation of the pattern of substrate methyl hyperfine shifts, however, indicates substrate rotations of only approximately 50 degrees . This paradox is resolved by demonstrating that the axial His25 imidazole ring also rotates counterclockwise with respect to the protein matrix in the mutant relative to that in the WT. The axial His25 CbetaH hyperfine shifts are shown to serve as independent probes of the imidazole plane orientation relative to the protein matrix. The analysis indicates that the pattern of heme methyl hyperfine shifts cannot be used alone to determine the in-plane orientation of the substrate as it relates to the stereospecificity of heme cleavage, without explicit consideration of the orientation of the axial His imidazole plane relative to the protein matrix.     

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.     

Control of intracellular heme levels: Heme transporters and heme oxygenases

Heme serves as a co-factor in proteins involved in fundamental biological processes including oxidative metabolism, oxygen storage and transport, signal transduction and drug metabolism. In addition, heme is important for systemic iron homeostasis in mammals. Heme has important regulatory roles in cell biology, yet excessive levels of intracellular heme are toxic; thus, mechanisms have evolved to control the acquisition, synthesis, catabolism and expulsion of cellular heme. Recently, a number of transporters of heme and heme synthesis intermediates have been described. Here we review aspects of heme metabolism and discuss our current understanding of heme transporters, with emphasis on the function of the cell-surface heme exporter, FLVCR. Knockdown of Flvcr in mice leads to both defective erythropoiesis and disturbed systemic iron homeostasis, underscoring the critical role of heme transporters in mammalian physiology.     

Measurement of heme efflux and heme content in isolated developing chloroplasts

Hemes destined for cytosolic hemoproteins must originate in one of the cellular compartments which have the capacity for heme synthesis, namely the chloroplast or the mitochondria. Since developing chloroplasts from greening cucumber (Cucumis sativus, cv. Sumter) cotyledons are known to contain complete heme and chlorophyll biosynthetic pathways, they were tested for their capacity export hemes. Picomole quantities of heme were measured by reconstitution of the heme with apo-peroxidase and subsequent determination of peroxidase activity. The assay method was sensitive (as little as 0.7 picomole of heme could be detected in a volume of 100 microliters) and was linear with heme concentration. When intact plastids were incubated with apo-peroxidase, a steady-state rate of efflux between 0.12 and 0.45 picomole heme/minute/milligram plastid protein was measured. The efflux rate was not due to plastid breakage and could be enhanced by incubating with the heme precursor, δ-aminolevulinic acid. Cold acetone extraction removed 47 ± 17 picomoles heme/milligram plastid protein from the total b-type heme pool in the chloroplasts (166 ± 9 picomoles heme/milligram protein, by acid-acetone extraction). The reconstitution technique provided a similar estimate of readily exchangeable heme in the plastid, 37 ± 8 picomoles heme/milligram protein (or 6 micromolar in the plastids). These values may be indicative of a `free heme pool' which exists in the chloroplast.     

Photochemical generation and reactions of heme cation radicals in heme proteins

A novel approach is described for generating reactive oxidizing centers in heme proteins, with zinc hemoglobin (Zn Hb) and zinc cytochrome c (Zn cyt c) used as examples. The reaction of 3Zn* Hb with [CoIII(NH3)5 Cl]2+, and of 3Zn* cyt c with methyl viologen are described. In the case of Zn Hb the cation radical produced decays with a rate constant of k3 = 2400s-1. Using this value the rate of the reaction (formula; see text) can be calculated to be 4500s-1.     

Transmembrane movement of heme

Evidence for CO-heme partitioning into and across lipid bilayers was obtained by kinetic and chromatographic studies. Biphasic time courses were observed when CO-heme was rapidly mixed with unilamellar lipid vesicles in a stopped-flow spectrometer. The initial rapid phase depended linearly on lipid concentration and was assigned to heme partitioning between the external solvent phase and the outer lipid layer of the membranes. The rate of the second, much slower phase was independent of both heme and lipid concentration. The fraction of absorbance change associated with this slower phase increased with increasing heme to lipid ratios and reached a maximum of approximately 45%. A similar slow phase was observed when membrane-bound heme was reacted with apomyoglobin. In the presence of excess globin, all of the CO-heme was extracted from the membranes to form native CO myoglobin. Under these conditions, the fractional amount of absorbance change associated with the slow dissociation phase was approximately 45%, regardless of the heme to lipid ratio. These results suggest strongly that the slow phases represent transmembrane movement of heme, from the outer to the inner lipid layer in the association reactions and from the inner to the outer layer in dissociation reactions. The temperature dependence of the rate of CO-heme binding to the outer lipid layer was markedly different from that of transmembrane movement. The rate of the latter, slower process decreased greatly with increasing acyl chain length, whereas the rate of the initial binding process varied little with vesicle composition, as long as the membranes were examined above their melting temperatures. Finally, the two kinetically distinct bound heme fractions could be isolated directly by column chromatography.     

Enzymes of heme biosynthesis

 Heme is a necessary component in a variety of oxygen-binding proteins and electron-transfer proteins, and as such it occupies a central role in cellular and organismal metabolism. With only rare exceptions, organisms that utilize heme possess the entire biosynthetic pathway to produce this tetrapyrrole compound. The enzymes involved catalyze a variety of interesting reactions and utilize both common and unique cofactors and metals. Aminolevulinate dehydratase from all organisms and ferrochelatase from higher animals are both metalloenzymes, while 5-aminolevulinate synthase contains pyridoxal phosphate, and porphobilinogen deaminase possesses a unique dipyrrole cofactor. Two pathway enzymes catalyze multiple decarboxylations and yet have no cofactors, and one enzyme catalyzes a six-electron oxidation with a single FAD. To add additional scientific interest there exist biochemically and clinically distinct human genetic diseases for every step in this pathway. Received: 12 March 1997 / Accepted: 8 May 1997     

Characterization of heme ligation properties of Rv0203, a secreted heme binding protein involved in Mycobacterium tuberculosis heme uptake

The secreted Mycobacterium tuberculosis (Mtb) heme binding protein Rv0203 has been shown to play a role in Mtb heme uptake. In this work, we use spectroscopic (absorption, electron paramagnetic resonance, and magnetic circular dichrosim) methods to further characterize the heme coordination environments of His-tagged and native protein forms, Rv0203-His and Rv0203-notag, respectively. Rv0203-His binds the heme molecule through bis-His coordination and is low-spin in both ferric and ferrous oxidation states. Rv0203-notag is high-spin in both oxidation states and shares spectroscopic similarity with pentacoordinate oxygen-ligated heme proteins. Mutagenesis experiments determined that residues Tyr59, His63, and His89 are required for Rv0203-notag to efficiently bind heme, reinforcing the hypothesis based on our previous structural and mutagenesis studies of Rv0203-His. While Tyr59, His63, and His89 are required for the binding of heme to Rv0203-notag, comparison of the absorption spectra of the Rv0203-notag mutants suggests the heme ligand may be the hydroxyl group of Tyr59, although an exogenous hydroxide cannot be ruled out. Additionally, we measured the heme affinities of Rv0203-His and Rv0203-notag using stopped flow techniques. The rates for binding of heme to Rv0203-His and Rv0203-notag are similar, 115 and 133 μM(-1) s(-1), respectively. However, the heme off rates differ quite dramatically, whereby Rv0203-His gives biphasic dissociation kinetics with fast and slow rates of 0.0019 and 0.0002 s(-1), respectively, and Rv0203-notag has a single off rate of 0.082 s(-1). The spectral and heme binding affinity differences between Rv0203-His and Rv0203-notag suggest that the His tag interferes with heme binding. Furthermore, these results imply that the His tag has the ability to stabilize heme binding as well as alter heme ligand coordination of Rv0203 by providing an unnatural histidine ligand. Moreover, the heme affinity of Rv0203-notag is comparable to that of other heme transport proteins, implying that Rv0203 may act as an extracellular heme transporter.     

Deconstructing heme.

Heme degradation plays important biological roles, ranging from generating light-absorbing compounds in plants to facilitating iron homeostasis in mammals. The X-ray crystal structure of human heme oxygenase-1, which instigates the degradation process, reveals insights into the enzymatic mechanism of this important process.     

The mechanism of heme transfer from the cytoplasmic heme binding protein PhuS to the delta-regioselective heme oxygenase of Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa has evolved two outer membrane receptor-mediated uptake systems (encoded by the phu and has operons) by which it can utilize the hosts heme and hemeproteins as a source of iron. PhuS is a cytoplasmic heme binding protein encoded within the phu operon and has previously been shown to function in the trafficking of heme to the iron-regulated heme oxygenase (pa-HO). While the heme association rate for PhuS was similar to that of myoglobin, a markedly higher rate of heme dissociation (approximately 10(5) s(-1)) was observed, in keeping with a function in heme-trafficking. Additionally, the transfer of heme from PhuS to pa-HO was shown to be specific and unidirectional when compared to transfer to the non-iron regulated heme oxygenase (BphO), in which heme distribution between the two proteins merely reflects their relative intrinsic affinities for heme. Furthermore, the rate of transfer of heme from holo-PhuS to pa-HO of 0.11 +/- 0.01 s(-1) is 30-fold faster than that to apo-myoglobin, despite the significant higher binding affinity of apo-myoglobin for heme (kH = 1.3 x 10(-8) microM) than that of PhuS (0.2 microM). This data suggests that heme transfer to pa-HO is independent of heme affinity and is consistent with temperature dependence studies which indicate the reaction is driven by a negative entropic contribution, typical of an ordered transition state, and supports the notion that heme transfer from PhuS to pa-HO is mediated via a specific protein-protein interaction. In addition, pH studies, and reactions conducted in the presence of cyanide, suggest the involvement of spin transition during the heme transfer process, whereby the heme undergoes spin change from 6-c LS to 6-c HS either in PhuS or pa-HO. On the basis of the magnitudes of the activation parameters obtained in the presence of cyanide, whereby both complexes are maintained in a 6-c LS state, and the biphasic kinetics of heme transfer from holo-PhuS to pa-HO-wt, supports the notion that the spin-state crossover occur within holo-PhuS prior to the heme transfer step. Alternatively, the lack of the biphasic kinetic with pa-HO-G125V, 6-c LS, and with comparable rate of heme transfer as pa-HO is supportive of a mechanism in which the spin-change could occur within pa-HO. The present data suggests either or both of the two pathways proposed for heme transfer may occur under the present experimental conditions. The dissection of which pathway is physiologically relevant is the focus of ongoing studies.     

Flavin-containing heme enzymes

There are many examples of oxidative enzymes containing both flavin and heme prosthetic groups that carry out the oxidation of their substrate. For the purpose of this article we have chosen five systems. Two of these, the l-lactate dehydrogenase flavocytochrome b₂ and cellobiose dehydrogenase, carry out the catalytic chemistry at the flavin group. In contrast, the remaining three require activation of dioxygen at the heme group in order to accomplish substrate oxidation, these being flavohemoglobin, a nitric oxide dioxygenase, and the mono-oxygenases nitric oxide synthase and flavocytochrome P450 BM3, which functions as a fatty acid hydroxylase. In the light of recent advances we will describe the structures of these enzymes, some of which share significant homology. We will also discuss their diverse and sometimes controversial catalytic mechanisms, and consider electron transfer processes between the redox cofactors in order to provide an overview of this fascinating set of enzymes.     

Cytochrome P-450 heme and the regulation of hepatic heme oxygenase activity

The degradation of cytochrome P-450 heme in the liver has been studied by a new approach. In rats, hepatic heme was labeled by administration of a tracer pulse of [5-¹⁴C]δ-aminolevulinic acid (ALA), and its degradation was analyzed in terms of labeled carbon monoxide (¹⁴CO) excretion, which is a specific degradation product of the labeled heme. Within minutes after administration of [5-¹⁴C]ALA, 14CO was detectable and increased after 2 h to an “early peak,” reflecting the elimination of labeled heme from a rapidly turning over pool in the liver. Beyond the early peak, the rate of ¹⁴CO production decreased in a log-linear manner, consistent with the degradation of heme in stable hepatic hemoproteins. From the rate at which ¹⁴CO production declined during this phase, from the predominant labeling of cytochrome P-450 heme by the administered [5-¹⁴C]ALA and from the known turnover characteristics of this hemoprotein in the liver, it could be inferred that production of ¹⁴CO—between 16 and 30 h after administration of labeled ALA—largely reflected degradation of cytochrome P-450 heme. This approach, which permits serial measurements in a single animal, was used to study the effect on cytochrome P-450 heme of administered heme or endotoxin, both of which are potent stimulators of hepatic heme oxygenase activity. Both of these substances caused marked acceleration of the degradation of cytochrome P-450 heme, the effect occurring over the same dose range as that for stimulation of hepatic heme oxygenase. The findings suggest that stimulation of this enzyme activity in the liver is closely related to the rate of degradation of cytochrome P-450 heme.