A novel mechanism of heme degradation to biliverdin studied by QM/MM and QM calculations.

Heme degradation by heme oxygenase enzymes is important for maintaining iron homeostasis and prevention of oxidative stress. Previous studies have reported that heme degradation proceeds through three consecutive steps of O2 activation: the regiospecific self-hydroxylation of heme, the conversion of hydroxyheme to verdoheme and CO, and the cleavage of the verdoheme macrocycle to release biliverdin and free ferrous iron. Our results indicate that in the second step of heme degradation, not only verdoheme is generated but ring opening and biliverdin production also occur. We have performed QM-cluster and QM/MM calculations, which show that calculations with H2O as the axial ligand of Fe give the lowest barrier. In the QM-cluster calculation, the reaction is exothermic by -85 kcal mol-1 and the rate-limiting barrier is 5 kcal mol-1, whereas the corresponding QM/MM calculations give a slightly lower barrier of 3 kcal mol-1, owing to strong hydrogen bonds and the protein environment.

Density functional theory studies on the conversion of hydroxyheme to iron-verdoheme in the presence of dioxygen.

Detailed insight into the second step of heme degradation by heme oxygenase, oxophlorin to verdoheme and biliverdin, is presented. Density functional theory methods are reported for the conversion of oxophlorin to verdoheme. Since it is currently unclear whether dioxygen binding to iron oxophlorin is followed by a reduction or not, in this work we have focused on the difference in reactivity between [(Im)(O2˙)FeIII(PO˙)] (PO˙ is the oxophlorin dianion radical) and [(Im)(O2˙)FeIII(PO)]- (PO is the oxophlorin trianion). Thus, we have shown that in [(Im)(O2˙)FeIII(PO˙)] and [(Im)(O2˙)FeIII(PO)]-, the mechanisms are stepwise with an initial C-O bond activation to form a ring-structure where the oxophlorin is distorted from planarity. This is followed by homolytic dioxygen bond breaking that directly leads to iron-oxo verdoheme products. The [(Im)(O2˙)FeIII(PO˙)] mechanism proceeds via two-state-reactivity patterns on the adjacent doublet and quartet spin state surfaces, whereas the [(Im)(O2˙)FeIII(PO)]- route shows single-state-reactivity on a triplet spin state surface. In both, the rate determining step is the C-O bond activation, with substantially lower barriers on the [(Im)(O2˙)FeIII(PO˙)] surface of 12.15 kcal mol-1 in the gas phase compared to 22.55 kcal mol-1 for the intermediate-spin of [(Im)(O2˙)FeIII(PO)]-. The complete active space self-consistent-field wave functions with second-order multi-reference perturbation theory were also studied. Finally, the effects of the solvent and the medium on the reaction barriers were tested and shown to be considerable.

Chameleonic nature of hydroxyheme in heme oxygenase and its reactivity: a density functional theory study.

Iron hydroxyheme is an intermediate in heme degradation that binds to HO-1 in a five-coordinated fashion wherein the fifth ligand is His25. The structure and reactivity of hydroxyheme have been investigated using the B3LYP*, OPBE, and CASSCF methods with the 6-31+G* and 6-311+G** basis sets. Hydroxyheme [(Im)Fe(II)(POH)] (POH is the hydroxyporphyrin) is readily oxidized to oxophlorin [(Im)Fe(III)(PO)] (PO is the oxophlorin trianion) in the protein heme oxygenase. A computational study in the gas phase has shown that (6)[(Im)Fe(II)(POH)] loses one electron from its a2u orbital in the presence of O2 and produces [(Im)Fe(II)(PO(•))]a2u(PO(•) is the oxophlorin dianion radical) in the sextet ground state with a ferrous keto π-neutral radical structure and dxy(2)a2u(1)dyz(1)dxz(1)σ*z2(1)d(x(2)-y(2))(1) electronic configuration. There is a closely lying exited state accompanying this ferrous keto π-neutral radical that has a high-spin ferric keto anion form of (6)[(Im)Fe(III)(PO)]xy with a2u(2)dxy(1)dyz(1)dxz(1)σ*z2(1)d(x(2)-y(2))(1) electronic configuration. In the protein environment with a dipole moment larger than 5.7, the ground state is reversed and (6)[(Im)Fe(III)(PO)]xy is at least 1.47 kcal/mol lower than the ferrous π-neutral radical of (6)[(Im)Fe(II) (PO(•))]a2u. The interaction of H2O, O2, and CO with iron oxophlorin will shift the electronic structure toward the formation of a keto π-neutral radical resonance form in the following order: CO > O2 > H2O.

Structure and redox behavior of iron oxophlorin and role of electron transfer in the heme degradation process.

Iron-oxophlorin is an intermediate in heme degradation, and the metal oxidation number can alter spin, electron distribution, and the reactivity of the metal and the oxophlorin ring. The role of electron transfer in the structure and reactivity of [(Py)(2)Fe(III)(PO)] (PO is the oxophlorin trianion) in different redox states has been investigated using the B3LYP and OPBE methods with the 6-31+G* and 6-311+G** basis sets. A computation study has shown that [(py)(2) Fe(III)(PO)] loses one electron from its a(2u) orbital. Thus the oxidized species, [(Py)(2)Fe(III)(PO(•))](+) (where PO(•) is the oxophlorin dianion radical), has an open-shell-singlet ground state with a d(xy)(2) d(xz)(2) a(2u)(1) d(yz)(1) electronic configuration with closely lying triplet and quintet states which are populated at ambient temperature. The aforementioned complex is highly reactive toward O(2). The reduced species [(Py)(2)Fe(II)(POH)] (where POH is the hydroxyheme) has the closed-shell-singlet ground state (π(xz) π(yz))(4) a(2u)(2) d(xy)(2) electronic configuration in which pyridines have a more π-accepting character and, thus, are tightly bound to iron. This reduced form is considerably less reactive toward O(2). The axial ligands effects (Im, t-BuNC) have also been studied in redox reactions of iron oxophlorin complexes. Complex [(Im)(2)Fe(III)(PO)] shows facile oxidation to form a cation radical and a reduction to form hydroxy while the [(t-BuNC)(2)Fe(II)(PO(•))] has high positive oxidation potential.

Effect of axial ligand on the electronic configuration, spin states, and reactivity of iron oxophlorin.

Iron-oxophlorin is an intermediate in heme degradation, and the nature of the axial ligand can alter the spin, electron distribution, and reactivity of the metal and the oxophlorin ring. The structure and reactivity of iron-oxophlorin in the presence of imidazole, pyridine, and t-butyl isocyanide as axial ligands was investigated using the B3LYP and OPBE methods with the 6-31+G* and 6-311+G** basis sets. OPBE/6-311+G** has shown that the doublet state of [(Py)(2)Fe(III)(PO)] (where pyridines are in perpendicular planes and PO is the oxophlorin trianion) is 3.45 and 5.27 kcal/mol more stable than the quartet and sextet states, respectively. The ground-state electronic configuration of the aforementioned complex is π(xz)(2) π(yz)(2) a(2u)(2) d(xy)(1) at low temperatures and changes to π(xz)(2) π(yz)(2) d(xy)(2) a(2u)(1) at high temperatures. This latter electronic configuration is consistently seen for the [(t-BuNC)(2)Fe(II)(PO(•))] complex (where PO(•) is the oxophlorin dianion radical). The complex [(Im)(2)Fe(III)(PO)] adopted the d(xy)(2) (π(xz) π(yz))(3) ground state and has low-lying quartet excited state which is readily populated when the temperature is increased.

Theoretical investigation of the ring opening process of verdoheme to biliverdin in the presence of dioxygen.

The conversion of ferrous verdoheme to ferric biliverdin in the presence of O(2) was investigated using the B3LYP method. Both 6-31G and 6-31G (d) basis sets were employed for geometry optimization calculation as well as energy stabilization estimation. Three possible pathways for the conversion of iron verdoheme to iron biliverdin were considered. In the first route oxygen and reducing electron were employed. In this path formation of ferrous verdoheme-O(2) complex was followed by the addition of one electron to the ferrous-oxycomplex to produce ferric peroxide intermediate. The ferric peroxide intermediate experienced an intramolecular nucleophilic attack to the most positive position at 5-oxo carbons on the ring to form a closed ring biliverdin. Subsequently the ring opening process took place and the iron (III) biliverdin complex was formed. Closed ring iron biliverdin intermediate and open ring iron biliverdin formed as a product of verdoheme cleavage were respectively 13.20 and 32.70 kcal mol(-1) more stable than ferric peroxide intermediate. Barrier energy for conversion of ferric peroxide to closed ring Fe (III) biliverdin and from the latter to Fe (III) biliverdin were respectively 8.67 and 3.35 kcal mol(-1). In this path spin ground states are doublet except for iron (III) biliverdin in which spin state is quartet. In the second path a ferrous-O(2) complex was formed and, without going to a one electron reduction process, nucleophilic attack of iron superoxide complex took place followed by the formation of iron (III) biliverdin. This path is thermodynamically and kinetically less favorable than the first one. In addition, iron hydro peroxy complex or direct attack of O(2) to macrocycle to form an isoporphyrin type intermediate have shown energy surfaces less favorable than aforementioned routes.

Theoretical investigations of the hydrolysis pathway of verdoheme to biliverdin.

Conversion of iron(II) verdoheme to iron biliverdin in the presence of OH(-) was investigated using B3LYP method. Both 3-21G and 6-31G* basis sets were employed for geometry optimization calculation as well as energy stabilization estimation. Calculation at 6-31G* level was found necessary for a correct spin state estimation of the iron complexes. Two possible pathways for the conversion of iron verdoheme to iron biliverdin were considered. In one path the iron was six-coordinate while in the other it was considered to be five-coordinate. In the six-coordinated pathway, the ground state of bis imidazole iron verdoheme is singlet while that for open chain iron biliverdin it is triplet state with 4.86 kcal/mol more stable than the singlet state. The potential energy surface suggests that a spin inversion take place during the course of reaction after TS. The ring opening process in the six-coordinated pathway is in overall -2.26 kcal/mol exothermic with a kinetic barrier of 9.76 kcal/mol. In the five-coordinated pathway the reactant and product are in the ground triplet state. In this path, hydroxyl ion attacks the iron center to produce a complex, which is only 1.59 kcal/mol more stable than when OH(-) directly attacks the macrocycle. The activation barrier for the conversion of iron hydroxy species to the iron biliverdin complex by a rebound mechanism is estimated to be 32.68 kcal/mol. Large barrier for rebound mechanism, small barrier of 4.18 kcal/mol for ring opening process of the hydroxylated macrocycle, and relatively same stabilities for complexes resulted by the attack of nucleophile to the iron and macrocycle indicate that five-coordinated pathway with direct attack of nucleophile to the 5-oxo position of macrocycle might be the path for the conversion of verdoheme to biliverdin.