Catalytic mechanism of heme oxygenase through EPR and ENDOR of cryoreduced oxy-heme oxygenase and its Asp 140 mutants

Heme oxygenase (HO) catalyzes the O(2)- and NADPH-cytochrome P450 reductase-dependent conversion of heme to biliverdin, Fe, and CO through a process in which the heme participates both as a prosthetic group and as a substrate. In the present study, we have generated a detailed reaction cycle for the first monooxygenation step of HO catalysis, conversion of the heme to alpha-meso-hydroxyheme. We employed EPR (using both (16)O(2) and (17)O(2)) and (1)H, (14)N ENDOR spectroscopies to characterize the intermediates generated by 77 K radiolytic cryoreduction and subsequent annealing of wild-type oxy-HO and D140A, F mutants. One-electron cryoreduction of oxy-HO yields a hydroperoxoferri-HO with g-tensor, g = [2.37, 2.187, 1.924]. Annealing of this species to 200 K is accompanied by spectroscopic changes that include the appearance of a new (1)H ENDOR signal, reflecting rearrangements in the active site. Kinetic measurements at 214 K reveal that the annealed hydroperoxoferri-HO species, denoted R, generates the ferri-alpha-meso-hydroxyheme product in a first-order reaction. Disruption of the H-bonding network within the distal pocket of HO by the alanine and phenylalanine mutations of residue D140 prevents product formation. The hydroperoxoferri-HO (D140A) instead undergoes heterolytic cleavage of the O-O bond, ultimately yielding an EPR-silent compound II-like species that does not form product. These results, which agree with earlier suggestions, establish that hydroperoxoferri-HO is indeed the reactive species, directly forming the alpha-meso-hydroxyheme product by attack of the distal OH of the hydroperoxo moiety at the heme alpha-carbon.     

Compound I of naked heme (iron protoporphyrin IX)

Gaseous iron protoporphyrin IX (heme) ions, Fe(PP-IX)+, obtained by electrospray ionization of a methanol solution of hemin chloride, are allowed to react with ozone, forming a species that is tentatively assigned the structure of an oxo complex, namely, an oxo iron(IV) protoporphyrin IX radical-cation species, (PP-IX).+FeIV=O. This species, representing the naked core of the putative active oxidant (compound I) of heme enzymes, is characterized by its reactivity behavior in Fourier transform ion cyclotron resonance mass spectrometry, performing as an active O-atom donor. A quite distinct reactivity is displayed by an isomeric species, holding the additional oxygen on the porphyrin frame, Fe(PP-IX(O))+. This isomer undergoes a ligand addition process, as was previously observed for Fe(PP-IX)+.     

Compound I in heme thiolate enzymes: a comparative QM/MM study

This study directly compares the active species of heme enzymes, so-called Compound I (Cpd I), across the heme-thiolate enzyme family. Thus, sixty-four different Cpd I structures are calculated by hybrid quantum mechanical/molecular mechanical (QM/MM) methods using four different cysteine-ligated heme enzymes (P450(cam), the mutant P450(cam)-L358P, CPO and NOS) with varying QM region sizes in two multiplicities each. The overall result is that these Cpd I species are similar to each other with regard to many characteristic features. Hence, using the more stable CPO Cpd I as a model for P450 Cpd I in experiments should be a reasonable approach. However, systematic differences were also observed, and it is shown that NOS stands out in most comparisons. By analyzing the electrical field generated by the enzyme on the QM region, one can see that (a) the protein exerts a large influence and modifies all the Cpd I species compared with the gas-phase situation and (b) in NOS this field is approximately planar to the heme plane, whereas it is approximately perpendicular in the other enzymes, explaining the deviating results on NOS. The calculations on the P450(cam) mutant L358P show that the effects of removing the hydrogen bond between the heme sulfur and L358 are small at the Cpd I stage. Finally, Mossbauer parameters are calculated for the different Cpd I species, enabling future comparisons with experiments. These results are discussed in the broader context of recent findings of Cpd I species that exhibit large variations in the electronic structure due to the presence of the substrate.     

A new understanding on how heme metabolism occurs in heme oxygenase: water-assisted oxo mechanism

Heme metabolism by heme oxygenase (HO) is investigated with quantum mechanical/molecular mechanical (QM/MM) calculations. A mechanism assisted by water is proposed: (1) an iron-oxo species and a water molecule are generated by the heterolytic cleavage of the O-O bond of an iron-hydroperoxo species in a similar way to P450-mediated reactions, (2) a hydrogen atom abstraction by the iron-oxo species from the generated water molecule and the C-O bond formation between the water molecule and the α-meso carbon take place simultaneously. The water molecule is hydrogen-bonded to the oxo ligand and to the water cluster in the active site of HO. The water cluster can control the position of the generated water molecule to ensure the regioselective oxidation of heme at the α-meso position, at the same time, can facilitate the oxidation by stabilizing a positive charge on the water molecule in the transition state. A key difference between HO and P450 is observed in the structure of the active site; Thr252 in P450 blocks the access of the water molecule to the α-meso position, and can thus suppress the undesired heme oxidation for P450.     

Oxidation of heme to beta- and delta-biliverdin by Pseudomonas aeruginosa heme oxygenase as a consequence of an unusual seating of the heme

The origin of the unusual regioselectivity of heme oxygenation, i.e. the oxidation of heme to delta-biliverdin (70%) and beta-biliverdin (30%), that is exhibited by heme oxygenase from Pseudomonas aeruginosa (pa-HO) has been studied by (1)H NMR, (13)C NMR, and resonance Raman spectroscopies. Whereas resonance Raman indicates that the heme-iron ligation in pa-HO is homologous to that observed in previously studied alpha-hydroxylating heme oxygenases, the NMR spectroscopic studies suggest that the heme in this enzyme is seated in a manner that is distinct from that observed for all other alpha-hydroxylating heme oxygenase enzymes for which a structure is known. In pa-HO, the heme is rotated in-plane approximately 110 degrees, so the delta-meso-carbon of the major orientational isomer is located within the HO-fold in the place where the alpha-hydroxylating enzymes typically place the alpha-meso-carbon. The unusual heme seating displayed by pa-HO places the heme propionates so that these groups point in the direction of the solvent-exposed heme edge and appears to originate in large part from the absence of stabilizing interactions between the polypeptide and the heme propionates, which are typically found in alpha-hydroxylating heme oxygenase enzymes. These interactions typically involve Lys-16 and Tyr-112, in Neisseriae meningitidis HO, and Lys-16 and Tyr-134, in human and rat HO-1. The corresponding residues in pa-HO are Asn-19 and Phe-117, respectively. In agreement with this hypothesis, we found that the Asn-19 Lys/Phe-117 Tyr double mutant of pa-HO exists as a mixture of molecules exhibiting two distinct heme seatings; one seating is identical to that exhibited by wild-type pa-HO, whereas the alternative seating is very similar to that typical of alpha-hydroxylating heme oxygenase enzymes and is related to the wild-type seating by approximately 110 degrees in-plane rotation of the heme. Furthermore, each of these heme seatings in the pa-HO double mutant gives rise to a subset of two heme isomeric orientations that are related to each other by 180 degrees rotation about the alpha-gamma-meso-axis. The coexistence of these molecules in solution, in the proportions suggested by the corresponding area under the peaks in the (1)H NMR spectrum, explains the unusual regioselectivity of heme oxygenation observed with the double mutant, which we found produces alpha- (55%), delta- (35%), and beta-biliverdin (10%). Alpha-biliverdin is obtained by oxidation of the heme seated similar to that of alpha-hydroxylating enzymes, whereas beta- and delta-biliverdin are formed from the oxidation of heme seated as in wild-type pa-HO.     

Kinetic isotope effects on the rate-limiting step of heme oxygenase catalysis indicate concerted proton transfer/heme hydroxylation

Heme oxygenase (HO) catalyzes the O2 and NADPH/cytochrome P450 reductase-dependent conversion of heme to biliverdin, free iron ion, and CO through a process in which the heme participates as both dioxygen-activating prosthetic group and substrate. We earlier confirmed that the first step of HO catalysis is a monooxygenation in which the addition of one electron and two protons to the HO oxy-ferroheme produces ferric-alpha-meso-hydroxyheme (h). Cryoreduction/EPR and ENDOR measurements further showed that hydroperoxo-ferri-HO converts directly to h in a single kinetic step without formation of a Compound I. We here report details of that rate-limiting step. One-electron 77 K cryoreduction of human oxy-HO and annealing at 200 K generates a structurally relaxed hydroperoxo-ferri-HO species, denoted R. We here report the cryoreduction/annealing experiments that directly measure solvent and secondary kinetic isotope effects (KIEs) of the rate-limiting R --> h conversion, using enzyme prepared with meso-deuterated heme and in H2O/D2O buffers to measure the solvent KIE (solv-KIE), and the secondary KIE (sec-KIE) associated with the conversion. This approach is unique in that KIEs measured by monitoring the rate-limiting step are not susceptible to masking by KIEs of other processes, and these results represent the first direct measurement of the KIEs of product formation by a kinetically competent reaction intermediate in any dioxygen-activating heme enzyme.The observation of both solv-KIE(298) = 1.8 and sec-KIE(298) = 0.8 (inverse) indicates that the rate-limiting step for formation of h by HO is a concerted process: proton transfer to the hydroperoxo-ferri-heme through the distal-pocket H-bond network, likely from a carboxyl group acting as a general acid catalyst, occurring in synchrony with bond formation between the terminal hydroperoxo-oxygen atom and the alpha-meso carbon to form a tetrahedral hydroxylated-heme intermediate. Subsequent rearrangement and loss of H2O then generates h.     

Dioxygen reactivity of meso -hydroxylated hemes: Intermediates in heme degradation process catalyzed by heme oxygenase

Heme oxygenase (HO) is the only enzyme in mammals known to catalyse the physiological degradation of unwanted heme into biliverdin, Fe ion and CO. The process involves introduction of the hydroxyl group at one of itsmeso-positions as the first fundamental step of the heme cleavage process. It was also found thatmeso-amino heme undergoes similar ring-cleavage process while reacting with dioxygen in presence of pyridine as an axial ligand. The present paper briefly reviews the reactions of modelmeso-hydroxylated heme and its analogues with dioxygen, and their relevance in the heme degradation process.     

The hydrogen-bonding network in heme oxygenase also functions as a modulator of enzyme dynamics: chaotic motions upon disrupting the H-bond network in heme oxygenase from Pseudomonas aeruginosa

Relaxation compensated Carr-Purcell-Meiboom-Gill (rc-CPMG) NMR experiments have been used to investigate micros-ms motions in heme oxygenase from Pseudomonas aeruginosa (pa-HO) in its ferric state, inhibited by CN- (pa-HO-CN) and N3- (pa-HO-N3), and in its ferrous state, inhibited by CO (pa-HO-CO). Comparative analysis of the data from the three forms indicates that the nature of the coordinated distal ligand affects the micros-ms conformational freedom of the polypeptide in regions of the enzyme far removed from the heme iron and distal ligand. Interpretation of the dynamical information in the context of the crystal structure of resting state pa-HO shows that residues involved in the network of structural hydrogen-bonded waters characteristic of HOs undergo micros-ms motions in pa-HO-CN, which was studied as a model of the highly paramagnetic S = 5/2 resting state form. In comparison, similar motions are suppressed in the pa-HO-CO and pa-HO-N3 complexes, which were studied as mimics of the obligatory oxyferrous and ferric hydroperoxide intermediates, respectively, in the catalytic cycle of heme degradation. These findings suggest that in addition to proton delivery to the nascent Fe(III)-OO(-) intermediate during catalysis, the hydrogen-bonding network serves two additional roles: (i) propagate the electronic state (reactive state) in each of the distinct steps of the catalytic cycle to key but remote sections of the polypeptide via small rearrangements in the network of hydrogen bonds and (ii) modulate the conformational freedom of the enzyme, thus allowing it to adapt to the demanding changes in axial coordination state and substrate transformations that take place during the catalytic cycle. This idea was probed by disrupting the hydrogen-bonding network in pa-HO by replacing R80 with L. NMR spectroscopic studies conducted with R80L-pa-HO-N3 and R80L-pa-HO-CO revealed that the mutant exhibits nearly global conformational disorder, which is absent in the equivalent complexes of the wild type enzyme. The "chaotic" disorder in the R80L mutant is likely related to its significantly lower efficiency to hydroxylate heme in the presence of H2O2, relative to the wild type enzyme.     

How heme metabolism occurs in heme oxygenase: computational study of oxygen-donation ability of the oxo and hydroperoxo species

We report a density functional theory study on the heme metabolism in heme oxygenase using iron-hydroperoxo and -oxo models. The activation energies for heme oxidation at the alpha-carbon by the iron-hydroperoxo and -oxo species are calculated to be 42.9 and 39.9 kcal/mol, respectively. These high activation barriers lead us to reconsider the catalytic mechanism of heme oxygenase     

Alkyl peroxides reveal the ring opening mechanism of verdoheme catalyzed by heme oxygenase

Heme oxygenase (HO) catalyzes heme catabolism through three successive oxygenation steps where the substrate heme itself activates O2. Although a rate-determining step of the HO catalysis is considered as third oxygenation, the verdoheme degradation mechanism has been the least understood in the HO catalysis. In order to discriminate three possible pathways proposed for the verdoheme ring-opening, we have examined reactions of the verdoheme-HO-1 complex with alkyl peroxides, namely MeOOH. Under reducing conditions, the MeOOH reaction afforded two novel products whose absorption spectra are similar to but slightly different from that of biliverdin. HPLC, ESI-MS, and NMR analysis show that these products are 1- and 19-methoxy-deoxy-biliverdins. The addition of a methoxy group at one end of the linear tetrapyrrole unambiguously indicates transient formation of the Fe-OOMe intermediate and rearrangement of its terminal methoxy group to the alpha-pyrrole carbon. The corresponding OH transfer of the Fe-OOH species is highly probable in the H2O2-dependent verdoheme degradation and is likely to be the case in the O2-dependent reaction catalyzed by HO as well.     

Comparative effects of curcuminoids on endothelial heme oxygenase-1 expression: ortho-methoxy groups are essential to enhance heme oxygenase activity and protection

Recently, it has been reported that curcumin, which is known as a potent antioxidant, acts as a non- stressful and non-cytotoxic inducer of the cytoprotective heme oxygenase (HO)-1. In this study, naturally occurring curcuminoids, such as pure curcumin, demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC), were compared for their potential ability to modulate HO-1 expression and cytoprotective activity in human endothelial cells. All three curcuminoids could induce HO-1 expression and HO activity with differential levels. The rank order of HO activity was curcumin, DMC and BDMC. In comparison with endothelial protection against H2O2-induced cellular injury, cytoprotective capacity was found to be highest with curcumin, followed by DMC and BDMC. Interestingly, cytoprotective effects afforded by curcuminoids were considerably associated with their abilities to enhance HO activity. Considering that the main difference among the three curcuminoids is the number of methoxy groups (none for BDMC, one for DMC, and two for curcumin), the presence of methoxy groups in the ortho position on the aromatic ring was suggested to be essential to enhance HO-1 expression and cytoprotection in human endothelial cells. Our results may be useful in designing more efficacious HO-1 inducers which could be considered as promising pharmacological agents in the development of therapeutic approaches for the prevention or treatment of endothelial diseases caused by oxidative damages.     

Quantum mechanical/molecular mechanical study of mechanisms of heme degradation by the enzyme heme oxygenase: the strategic function of the water cluster

Heme degradation by heme oxygenase (HO) enzymes is important in maintaining iron homeostasis and prevention of oxidative stress, etc. In response to mechanistic uncertainties, we performed quantum mechanical/molecular mechanical investigations of the heme hydroxylation by HO, in the native route and with the oxygen surrogate donor H2O2. It is demonstrated that H2O2 cannot be deprotonated to yield Fe(III)OOH, and hence the surrogate reaction starts from the FeHOOH complex. The calculations show that, when starting from either Fe(III)OOH or Fe(III)HOOH, the fully concerted mechanism involving O-O bond breakage and O-C(meso) bond formation is highly disfavored. The low-energy mechanism involves a nonsynchronous, effectively concerted pathway, in which the active species undergoes first O-O bond homolysis followed by a barrier-free (small with Fe(III)HOOH) hydroxyl radical attack on the meso position of the porphyrin. During the reaction of Fe(III)HOOH, formation of the Por+*FeIV=O species, compound I, competes with heme hydroxylation, thereby reducing the efficiency of the surrogate route. All these conclusions are in accord with experimental findings (Chu, G. C.; Katakura, K.; Zhang, X.; Yoshida, T.; Ikeda-Saito, M. J. Biol. Chem. 1999, 274, 21319). The study highlights the role of the water cluster in the distal pocket in creating "function" for the enzyme; this cluster affects the O-O cleavage and the O-Cmeso formation, but more so it is responsible for the orientation of the hydroxyl radical and for the observed alpha-meso regioselectivity of hydroxylation (Ortiz de Montellano, P. R. Acc. Chem. Res. 1998, 31, 543). Differences/similarities with P450 and HRP are discussed.     

Theory favors a stepwise mechanism of porphyrin degradation by a ferric hydroperoxide model of the active species of heme oxygenase

The report uses density functional theory to address the mechanism of heme degradation by the enzyme heme oxygenase (HO) using a model ferric hydroperoxide complex. HO is known to trap heme molecules and degrade them to maintain iron homeostasis in the biosystem. The degradation is initiated by complexation of the heme, then formation of the iron-hydroperoxo species, which subsequently oxidizes the meso position of the porphyrin by hydroxylation, thereby enabling eventually the cleavage of the porphyrin ring. Kinetic isotope effect studies indicate that the mechanism is assisted by general acid catalysis, via a chain of water molecules, and that all the events occur in concert. However, previous theoretical treatments indicated that the concerted mechanism has a high barrier, much higher than an alternative mechanism that is initiated by O-O bond homolysis of iron-hydroperoxide. The present contribution studies the stepwise and concerted acid-catalyzed mechanisms using H(3)O(+)(H(2)O)(n)(), n = 0-2. The effect of the acid strength is tested using the H(4)N(+)(H(2)O)(2) cluster and a fully protonated ferric hydroperoxide. All the calculations show that a stepwise mechanism that involves proton relay and O-O homolysis, in the rate-determining step, has a much lower barrier (>10 kcal/mol) than the corresponding fully concerted mechanism. The best fit of the calculated solvent kinetic isotope effect, to the experimental data, is obtained for the H(3)O(+)(H(2)O)(2) cluster. The calculated alpha-deuterium secondary kinetic isotope effect is inverse (0.95-0.98), but much less so than the experimental value (0.7). Possible reasons for this quantitative difference are discussed. Some probes are suggested that may enable experiment to distinguish the stepwise from the concerted mechanism.     

The hydroxide complex of Pseudomonas aeruginosa heme oxygenase as a model of the low-spin iron(III) hydroperoxide intermediate in heme catabolism: 13C NMR spectroscopic studies suggest the active participation of the heme in macrocycle hydroxylation

13C NMR spectroscopic studies have been conducted with the hydroxide complex of Pseudomonas aeruginosa heme oxygenase (Fe(III)-OH), where OH(-) has been used as a model of the OOH(-) ligand to gain insights regarding the elusive ferric hydroperoxide (Fe(III)-OOH) intermediate in heme catabolism at ambient temperatures. Analysis of the heme core carbon resonances revealed that the coordination of hydroxide in the distal site of the enzyme results in the formation of at least three populations of Fe(III)-OH complexes with distinct electronic configurations and nonplanar ring distortions that are in slow exchange relative to the NMR time scale. The most abundant population exhibits a spin crossover between S = (1)/(2) and S = (3)/(2) spin states, and the two less abundant populations exhibit pure, S = (3)/(2) and S = (1)/(2), (d(xy)())(1) electronic configurations. We propose that the highly organized network of water molecules in the distal pocket of heme oxygenase, by virtue of donating a hydrogen bond to the coordinated hydroxide ligand, lowers its ligand field strength, thereby increasing the field strength of the porphyrin (equatorial) ligand, which results in nonplanar deformations of the macrocycle. This tendency to deform from planarity, which is imparted by the ligand field strength of the coordinated OH(-), is likely reinforced by the flexibility of the distal pocket in HO. These findings suggest that if the ligand field strength of the coordinated OOH(-) in heme oxygenase is modulated in a similar manner, the resultant large spin density at the meso carbons and nonplanar deformations of the pophyrin ring prime the macrocycle to actively participate in its own hydroxylation.     

Solution 1H, 15N NMR spectroscopic characterization of substrate-bound, cyanide-inhibited human heme oxygenase: water occupation of the distal cavity

A solution NMR spectroscopic study of the cyanide-inhibited, substrate-bound complex of uniformly (15)N-labeled human heme oxygenase, hHO, has led to characterization of the active site with respect to the nature and identity of strong hydrogen bonds and the occupation of ordered water molecules within both the hydrogen bonding network and an aromatic cluster on the distal side. [(1)H-(15)N]-HSQC spectra confirm the functionalities of several key donors in particularly robust H-bonds, and [(1)H-(15)N]HSQC-NOESY spectra lead to the identification of three additional robust H-bonds, as well as the detection of two more relatively strong H-bonds whose identities could not be established. The 3D NMR experiments provided only a modest, but important, extension of assignments because of the loss of key TOCSY cross-peaks due to the line broadening from a dynamic heterogeneity in the active site. Steady-state NOEs upon saturating the water signal locate nine ordered water molecules in the immediate vicinity of the H-bond donors, six of which are readily identified in the crystal structure. The additional three are positioned in available spaces to account for the observed NOEs. (15)N-filtered steady-state NOEs upon saturating the water resonances and (15)N-filtered NOESY spectra demonstrate significant negative NOEs between water molecules and the protons of five aromatic rings. Many of the NOEs can be rationalized by water molecules located in the crystal structure, but strong water NOEs, particularly to the rings of Phe47 and Trp96, demand the presence of at least an additional two immobilized water molecules near these rings. The H-bond network appears to function to order water molecules to provide stabilization for the hydroperoxy intermediate and to serve as a conduit to the active site for the nine protons required per HO turnover.     

Oxidation of 10-undecenoic acid by cytochrome P450(BM-3) and its Compound I transient

Oxidations of 10-undecenoic acid by cytochrome P450(BM-3) and its Compound I transient were studied. The only product formed in Compound I oxidations was 10,11-epoxyundecanoic acid, whereas the enzyme under turnover conditions gave the epoxide and 9-hydroxy-10-undecenoic acid in a 10 : 90 ratio. Kinetic studies at 0 °C of oxidations by Compounds I formed by MCPBA oxidation and by a photo-oxidation pathway gave the same results, displaying saturation kinetics that yielded equilibrium binding constants and first-order oxidation rate constants that were experimentally indistinguishable. Oxidation of 10-undecenoic acid by Compound I from CYP119 generated by MCBPA oxidation also gave 10,11-epoxyundecanoic acid as the only product. CYP119 Compound I bound the substrate less strongly but reacted with a faster oxidation rate constant than P450(BM-3) Compound I. The kinetic parameters for oxidation of the substrate by P450(BM-3) under turnover conditions were similar to those of the Compound I transient even though the products differed.