Identification of histidine 45 as the axial heme iron ligand of heme oxygenase-2.

A truncated, soluble, and enzymatically active form of human heme oxygenase-2 (DeltaHHO2) was expressed in Escherichia coli. To identify the axial heme ligand of HO-2, His-45 to Ala (DeltaH45A) and His-152 to Ala (DeltaH152A) mutants have been prepared using this expression system. DeltaH45A could form a 1:1 complex with hemin but was completely devoid of the heme degradation activity. A 5-coordinate-type ferrous NO EPR spectrum was observed for the heme-DeltaH45A complex. The DeltaH152A mutant was expressed as an inclusion body and was recovered from the lysis pellet by dissolution in urea followed by dialysis. The solubilized fraction obtained, however, was composed of a mixture of a functional enzyme and an inactive fraction. The inactive fraction was removed by Sephadex G-75 column chromatography since it eluted out of the column at the void volume. The gel filtration-purified DeltaH152A exhibited spectroscopic and enzymatic properties identical to those of wild-type. We conclude, in contrast to the previous reports (McCoubrey and Maines (1993) Arch. Biochem. Biophys. 302, 402-408; McCoubrey, W. K., Jr., Huang, T. J., and Maines, M. (1997) J. Biol. Chem. 272, 12568-12574), that His-45, but not His-152, in heme oxygenase isoform-2 is the proximal ligand of the heme and is essential for the heme degradation activity of the enzyme. His-152 appears to play a structural role in stabilization of the heme oxygenase protein.

The oxygen and carbon monoxide reactions of heme oxygenase.

The O2 and CO reactions with the heme, alpha-hydroxyheme, and verdoheme complexes of heme oxygenase have been studied. The heme complexes of heme oxygenase isoforms-1 and -2 have similar O2 and CO binding properties. The O2 affinities are very high, KO2 = 30-80 microM-1, which is 30-90-fold greater than those of mammalian myoglobins. The O2 association rate constants are similar to those for myoglobins (kO2' = 7-20 microM-1 s-1), whereas the O2 dissociation rates are remarkably slow (kO2 = 0.25 s-1), implying the presence of very favorable interactions between bound O2 and protein residues in the heme pocket. The CO affinities estimated for both isoforms are only 1-6-fold higher than the corresponding O2 affinities. Thus, heme oxygenase discriminates much more strongly against CO binding than either myoglobin or hemoglobin. The CO binding reactions with the ferrous alpha-hydroxyheme complex are similar to those of the protoheme complex, and hydroxylation at the alpha-meso position does not appear to affect the reactivity of the iron atom. In contrast, the CO affinities of the verdoheme complexes are >10,000 times weaker than those of the heme complexes because of a 100-fold slower association rate constant (kCO' approximately 0. 004 microM-1 s-1) and a 300-fold greater dissociation rate constant (kCO approximately 3 s-1) compared with the corresponding rate constants of the protoheme and alpha-hydroxyheme complexes. The positive charge on the verdoporphyrin ring causes a large decrease in reactivity of the iron.

Histidine-132 does not stabilize a distal water ligand and is not an important residue for the enzyme activity in heme oxygenase-1.

Heme oxygenase is a key enzyme in the oxygen-dependent heme catabolism pathway. In order to clarify the role of highly conserved His132 in heme oxygenase isoform-1, we have prepared 30 kDa truncated rat heme oxygenase mutants in which His132 has been replaced by Ala, Gly, and Ser. The expressed recombinant mutant proteins were isolated in inclusion bodies and were recovered from the lysis pellet by dissolution in urea followed by dialysis. The solubilized fraction obtained, however, was composed of a mixture of a functional enzyme and an inactive fraction. The inactive fraction was removed by Sephadex G-75 gel filtration column chromatography, as it eluted out of the column at the void volume. The gel filtration-purified heme oxygenase mutants have spectroscopic and enzymatic properties identical to those of wild type. The hemin complex of the H132A mutant exhibits a transition between a high-spin acid form and a low-spin alkaline form with a pKa value of 7.6 identical to that in the wild-type complex. These results demonstrate that His132 in heme oxygenase does not link to the coordinated water molecule and is not an essential residue for the enzyme activity. These results are in accordance with our previous preliminary results [Ito-Maki, M., Ishikawa, K., Mansfield Matera, K., Sato, M., Ikeda-Saito, M., & Yoshida, T. (1995) Arch. Biochem. Biophys. 317, 253-258] but contradict a recent report that His132 is the distal base linked to the coordinated water molecule and an important residue for enzyme catalysis [Wilks, A., Ortiz de Montellano, P. R., Sun, J., & Loehr, T. M. (1996) Biochemistry 35, 930-936]. Prolonged storage of the solubilized fraction from the inclusion bodies of the mutants, H132S in particular, results in an increase in the void volume fraction with a concomitant decrease of the 30 kDa fraction. We infer that His132 plays a structural role in stabilization of the heme oxygenase protein.

Resonance Raman spectroscopic characterization of alpha-hydroxyheme and verdoheme complexes of heme oxygenase.

Heme oxygenase (HO) is the microsomal enzyme that catalyzes the oxidative degradation of protoheme (iron protoporphyrin IX) and the generation of carbon monoxide. The enzyme converts protoheme into biliverdin through two known heme derivatives, alpha-hydroxyheme and verdoheme. To gain insight into the degradation mechanisms of the two intermediates, the resonance Raman spectra were observed for alpha-hydroxyheme and verdoheme complexes of HO and compared with those of apomyoglobin (apo-Mb) complexes. The ferrous alpha-hydroxyheme complexed with both HO and apo-Mb shows a resonance Raman spectral pattern similar to that of the protoheme complexes. On the contrary, the ferric alpha-hydroxyheme and ferrous verdoheme complexes of HO and apo-Mb show atypical Raman patterns, which are interpreted as the result of the symmetry lowering of the porphyrin-conjugated pi-electron system. The comparison of the resonance Raman spectra of the verdoheme complexed with HO and apo-Mb with those of the five- and six-coordinate model complexes of verdoheme shows that the ferrous forms of the verdoheme-protein complexes are six-coordinate. The Fe-CO and Fe-CN stretching frequencies of ferrous verdoheme compounds are distinct from those of ferrous heme compounds. It is inferred that the positive charge of the verdoheme ring possesses some of the charge density on the iron atom, causing unique characteristics of the iron ligand stretching vibrations and altered ligand binding properties.

Oxygen and one reducing equivalent are both required for the conversion of alpha-hydroxyhemin to verdoheme in heme oxygenase.

Heme oxygenase is a central enzyme of heme degradation and associated carbon monoxide biosynthesis. We have prepared the alpha-hydroxyheme-heme oxygenase complex, which is the first intermediate in the catalytic reaction. The active site structure of the complex was examined by optical absorption, EPR, and resonance Raman spectroscopies. In the ferric form of the enzyme complex, the heme iron is five coordinate high spin and the alpha-hydroxyheme group in the complex assumes a structure of an oxophlorin where the alpha-meso hydroxy group is deprotonated. In the ferrous form, the alpha-hydroxy group is protonated and consequently the prosthetic group assumes a porphyrin structure. The alpha-hydroxyheme group undergoes a redox-linked conversion between a keto and an enol form. The ferric alpha-hydroxyheme reacts with molecular oxygen to form a radical species. Reaction of the radical species with a reducing equivalent yields the verdoheme-heme oxygenase complex. Reaction of the ferrous alpha-hydroxyheme-heme oxygenase complex with oxygen also yields the verdoheme-enzyme complex. We conclude that the catalytic conversion of ferric alpha-hydroxyheme to verdoheme by heme oxygenase requires molecular oxygen and one reducing equivalent.

Heme oxygenase-2. Properties of the heme complex of the purified tryptic fragment of recombinant human heme oxygenase-2.

Recombinant human microsomal heme oxygenase-2 was expressed in Escherichia coli. Tryptic digestion of the membrane fraction, in which the wild-type enzyme was localized, yielded a soluble tryptic peptide of 28 kDa, which retained the ability to accept electrons from NADPH-cytochrome P-450 reductase and the enzymatic activity for conversion of heme to biliverdin. The tryptic fragment, when purified to apparent homogeneity, bound one equivalent of heme to form a substrate-enzyme complex that had spectroscopic properties characteristic of heme proteins, such as myoglobin and hemoglobin. Optical absorption, Raman scattering, and EPR studies of the heme-tryptic fragment complex revealed that the ferric heme was six coordinate high spin at neutral pH and six coordinate low spin at alkaline pH, with a pK alpha value of 8.5. EPR and Raman scattering studies indicated that a neutral imidazole of a histidine residue served as the proximal ligand in the heme-heme oxygenase-2 fragment complex. The reaction with hydrogen peroxide converted the heme of the heme oxygenase-2 fragment complex into a verdoheme-like intermediate, while the reaction with m-chloroperbenzoic acid yielded a oxoferryl species. These spectroscopic properties are similar to those obtained for heme oxygenase-1, and thus the catalytic mechanism of heme oxygenase-2 appears to be similar to that of heme oxygenase-1.

Demonstration that histidine 25, but not 132, is the axial heme ligand in rat heme oxygenase-1.

A truncated, soluble rat heme oxygenase-1 lacking its C-terminal, membrane-anchoring segment, and its His25-->Ala and His132-->Ala mutants have been prepared by site-directed mutagenesis and expression in Escherichia coli. We found that wild-type enzyme can degrade heme to biliverdin, but its specific activity was about one-fifth that of the native, full-length enzyme, suggesting that the C-terminal segment is important for accepting electrons from NADPH cytochrome P450 reductase. His132-->Ala mutant had an enzyme activity comparable to that of the wild-type enzyme; hence, the highly conserved His132 is not essential for the display of the heme oxygenase activity. In contrast, His25-->Ala mutation completely abolished the enzyme's catalytic activity. A five-coordinate type ferrous NO EPR spectrum was observed for the heme-heme oxygenase H25A complex. Hence, we conclude that His25 is the proximal axial ligand of the heme iron and is essential for the heme degradation activity of the enzyme.