Paramagnetic Resonance of Metallobiomolecules - American

Downloaded by PENNSYLVANIA STATE UNIV on September 2, 2012 | http://pubs.acs.org Publication Date: August 19, 2003 | doi: 10.1021/bk-2003-0858.ch005

Chapter 5

EPR Characterization of the Heme Oxygenase Reaction Intermediates and Its Implication for the Catalytic Mechanism 1,3

2,3

MasaoIkeda-Saito and Hiroshi Fujii 1

Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Katahira, Aoba, Sendai 980-8577, Japan Institute for Molecular Science, Okazaki 444-8585, Japan Currentaddress:Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44106-4970 2

3

Heme oxygenase (HO) catalyzes the regiospecific degradation of heme to biliverdin by using three O molecules and seven electrons. The enzyme binds one equivalent of heme to form the heme complex, and electron donation initiates the three stepwise oxygenase reactions through the two novel heme derivatives, α-hydroxyheme and verdoheme, during which CO andfreeFe are also produced. EPR has been used to study electronic and coordination structures of the HO catalytic intermediates, including the ferric hydroperoxo active species generated by one-electron reduction of the ferrous oxy form. A combination of the novel characteristics of the reaction intermediates and the protein environment are responsible for the unique HO enzyme catalytic mechanism. 2

© 2003 American Chemical Society In Paramagnetic Resonance of Metallobiomolecules; Telser, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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98 Biological heme catabolism is conducted by a family of enzymes termed as heme oxygenase (HO) which catalyzes oxidative degradation of iron protoporphyrin IX (heme hereafter) to biliverdin, iron and CO in the presence of electron donors (1). In mammalian systems where electrons are supplied by NADPH through NADPH-cytochrome P450 reductase (2), HO is the enzyme responsible for excess hemin excretion and iron recycling (3). The product CO has been implicated as a messenger molecule in vasodilation and neuronal transmission by activating soluble guanylyl cyclase (4-6) and in regulation of circadian rhythm by binding to a transcription factor, NAPS2 (7). In pathogenic bacteria which have to acquire ironfromtheir hosts for their own survival, HO is the key component in the heme-based iron acquisition pathway so as to circumvent the low concentration offreeextra-cellular iron (8-9), In its catalytic cycle, HOfirstbinds 1 equivalent of heme to form a hemeHO complex (l of Figure 1). Thefirstelectron donatedfromthe reducing equivalent reduces the ferric iron to the ferrous state (2). Then 0 binds to form a meta-stable oxy complex (3). Further electron donation to the oxy complex initiates the three-step conversion of oxyheme to Fe(IH)-biliverdin (7) through ferric hydroperoxo heme (4), a-meso-hydroxyheme (5) and ferrous verdoheme (6) intermediates. Thefinalstep involves electron donation to convert Fe(III)biliverdin (7) to the ferrous complex, and free iron and biliverdin ($) are liberatedfromthe HO protein. HO is not a heme protein per se but utilizes heme as both a prosthetic group and a substrate. The salient aspect of HO catalysis is that die one electron reduction of the oxy form generates a ferric hydroperoxo species that leads to site-selective hydroxylation of the a-mesocarbon of the porphyrin ring (10, 11). This is differentfromthe ferryl species, commonly found as an active catalytic intermediate in peroxidase and P450 enzymes (12). While major work has been conducted on the isoform-1 of mammalian HO termed as HO-1, recent studies have demonstrated that the bacterial counterparts convert heme to biliverdin by the same mechanism (1314). In this article, we describe the active site structures of HO intermediates derivedfromour paramagnetic resonance studies and their implications for HO catalysis. The objective is to demonstrate the power of EPR spectroscopy, when combined with other techniques, in elucidating the HO reaction mechanism by providing detailed active site structures of reaction intermediates beyond the resolution currently attained by X-ray crystallography on the HO proteins. 2

Active Site Structure of the Heme-HO Complex Thefirstintermediate of HO catalysis, the ferric heme-enzyme complex (1), exhibits an EPR spectrum of high spin axial symmetry (Figure 2A). The g = 2 signal of the spectrum is broadened when measured in buffered H 0 (Figure 2A, inset). The broadening is due to the unresolved hyperfine interaction with / 17

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In Paramagnetic Resonance of Metallobiomolecules; Telser, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


COOH

COOH

biliverdin (8)

COOH

COOH

a-meso-hydroxyheme (6)

COOH

ferrous heme (2)

COOH

COOH

e-/H

oxy heme (3)

COOH

+

COOH

COOH

ferric hydroperoxo heme (4)

Figure 1. Schematic diagram of the reaction intermediates of HO catalysis.

ferric biliverdin (7)

COOH

verdoheme (6)

COOH

ferric heme (1)

COOH


v©


100

ι 0

1

1

0.1

ι

j

1

0.2

1

0.3

1

1

1

0.4 _

1

.0.5

BQ (Tesla)

Figure 2. EPR spectra of the heme-HO-1 complex (A), the azide adduct of the heme-HO-1 complex (B), the ferric hydroperoxo spices (C), the annealed product of the ferric hydroperoxo species (D), and ferric a-mesohydroxyheme-HO-1 (E). The inset is the g = 2 region of the heme-HO complex measured in buffered H 0 (solid line) and H 0 (broken line). Spectra (A), (B), and (E) were recorded at X-band (9.45 GHZ) at 6 K, while (C) and (D) were at Q-band (35 GHz) at 2.1 K. The asterisk (*) denotes signals due to cavity impurities. 16

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I7

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101 = 5/2 of the water oxygen, indicating water coordination to the heme iron. EPR spectra of the ferrous NO complex of wild-type and histidine mutants show coordination of histidine as the proximal axial ligand (15-17). The axial coordination structure established by EPR has been confirmed by the crystal structures of both mammalian and bacterial HOs (18-20). The ferric heme-enzyme complex binds azide to form a low spin species with a dissociation constant of -10 μΜ for HO-1 at pH 7 and 20 °C. Different from low spin EPR spectra commonly seen in heme protein azide complexes (for example, g ~ 2.78,2.22, and 1. 73 for MbN ), the azide-bound HO-1 shows a so-called "large g^ EPR spectrum" (Figure 2B), g=3.64. The large g EPR spectrum is induced by the spin-orbit coupling of the t2 ground state, mainly from degeneration of iron d x z and d^ orbitals. The d x z and d^ orbital energies are changed by the orientations of the two axial ligands, the proximal histidine imidazole and the bound-azide in this case. When the imidazole plane and azide axis eclipse the porphyrin meso carbon-Fe axes, the π-orbitals of the imidazole and azide are orthogonal to the d^ and d ^ orbitals. This results in the degeneration of the d x z and d^ orbitals and exhibits the large g^ EPR spectrum. The large g^ spectra are seen in a heme model complex with plane axial ligands with relative perpendicular orientation eclipsing adjacent Fe-meso carbon axes (21). On the other hand, rotation of the imidazole and/or azide ligands allows the ligand π-orbitals to overlap with the iron d and d ^ orbitals. This d-π interaction leads to the split of the degeneration and gives a rhombic EPR spectrum as seen for most ferric low spin heme proteins, where the proximal histidine imidazole orients to overlap with the d ^ and d ^ oribitals. Hence, the large g^ EPR spectrum of the azide-bound HO-1 indicates that the proximal imidazole plane lays over the porphyrin meso carbon-Fe axis and the bound azide has its N=N=N vector perpendicular to the imidazole plane. The HO crystal structures (18-20) show that the imidazole plane of the proximal histidine closely eclipses the heme meso β-δ axis, and that the kinked distal helix located close to the heme plane sterically controls direction of the bound ligand pointing towards the α-meso carbon of the porphyrin ring. These structural features predict that the projection of the bound N onto the porphyrin plane is perpendicular to the plane of the imidazole ligand trans to the bound azide. The crystal structure of the azide-bound heme-HO-1 complex reported recently confirms the aforementioned axial ligand geometry of the HO azide complex (22). This coordination structure of HO-1 also explains the

Paramagnetic Resonance of Metallobiomolecules - American

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