Abstract
In nature, heme is a prosthetic group that is universally used as a cofactor for heme proteins. It is necessary for the execution of fundamental biological processes including electron transfer, oxidation and metabolism. However, free heme is toxic to cells, because of its capability to enhance oxidative stress, hence its cellular concentration is strictly regulated through multiple mechanisms. Heme oxygenase (HO) serves as an irreplaceable member in the heme degradation system. It is a ubiquitous protein, existing in many species including mammals, higher plants, and interestingly, certain pathogenic bacteria. In the HO reaction, HO catalyzes oxidative cleavage of heme to generate biliverdin and release carbon monoxide and ferrous iron. Because of the beneficial effects of these heme catabolism products, HO plays a key role in iron homeostasis and in defense mechanism against oxidative stress. HO is composed of an N-terminal structured region and a C-terminal membrane-bound region. Furthermore, the soluble form of HO, which is obtainable by excision of the membrane-bound region, retains its catalytic activity. Here, we present the backbone resonance assignments of the soluble form (residues 1–232) of HO-1 in the free and Zn(II) protoporphyrin IX (ZnPP)-bound states, and analyzed the structural differences between the states. ZnPP is a potent enzyme inhibitor, and the ZnPP-bound structure of HO-1 mimics the heme-bound structure. These assignments provide the structural basis for a detailed investigation of the HO-1 function.
Similar content being viewed by others
Biological context
Heme, a complex of iron with protoporphyrin IX, is ubiquitous in almost all living cells and serves as a cofactor for numerous heme proteins, such as hemoglobin, cytochromes, NADPH oxidase and myeloperoxidases. Heme is involved in a diverse range of biological functions such as electron transfer, oxygen transport, and drug metabolism. Free heme, however, is toxic to cells and leads to the production of reactive oxygen species possibly causing cellular injury (Khan and Quigley 2011). For the removal of excess heme, mammals possess unique enzymes, heme oxygenase (HO), which catalyzes the oxidation of heme to biliverdin with concomitant release of iron and carbon monoxide (CO); the reducing equivalents consumed in this oxidation are provided from the biological partner enzyme cytochrome P450 reductase. Uniquely, the heme-bound HO behaves transitory as a heme protein, in which heme serves as both a prosthetic group and a substrate. In mammals, heme is exclusively cleaved at the α position to yield α-biliverdin. Biliverdin is rapidly converted to bilirubin by biliverdin reductase, and bilirubin subsequently conjugated to glucuronic acid is excreted in the bile. Both biliverdin and bilirubin have antioxidant properties. The second metabolite, CO is nowadays known as a gaseous messenger molecule, and seems to acts as a potent neurovascular regulator and anti-inflammatory molecule (Khan and Quigley 2011; Verma et al. 1993). The third product released is ferrous iron, which is captured and stored in ferritin and contributes to iron homeostasis. Because of the diverse biological roles of HO, many studies of HO have been performed from both the molecular and clinical points of view.
Genes encoding HO has been isolated from various organisms including mammals, higher plants, red algae, and cyanobacteria. In mammalian systems, HO has two isoforms, designated as HO-1 and HO-2. HO-1 is known as a stress inducible protein and primarily in charge of heme catabolism in the liver and spleen, while HO-2 is expressed constitutively in the central nervous system, and is proposed to function as a CO generator. Both isoforms are highly conserved. Intact HOs are membrane proteins anchored to endoplasmic reticulum, consisting of an N-terminal structured region that faces cytoplasm and a C-terminal membrane-bound region. Since the soluble form of HO-1 lacking the C-terminal transmembrane region yet retains its catalytic activity, it has been widely used for the structural studies of HO-1. A large number of crystal structures of the water-soluble domain of HO-1 have been reported for many species including human and rat. Those structures exhibit a high degree of similarity; almost all HO-1 proteins are monomeric and well-folded alpha-helical proteins.
For rats, the crystal structures of water-soluble HO-1 (residues 1–267) have been reported in various states during the catalytic cycle (PDB code: 1IRM, 1DVE, 2ZVU and 1J2C for the free, heme-bound, verdoheme-bound and biliverdin-iron-bound states, respectively). In the free state crystal, almost 30 amino acids in the N-terminus were invisible in the electron density map (Sugishima et al. 2002). In contrast, the corresponding region existed in both free and heme-bound crystal structures of human HO-1 (Schuller et al. 1999). Solution NMR studies have also been performed for human HO-1, and backbone 1H and 15N chemical shifts of the free and cyanide-inhibited complex states of the soluble region are available (Li et al. 2002, 2004). However, not all of the side chain signals were assigned. For rat HO-1, no resonance assignments have been published to date, although many crystal structures have been solved. To analyze the solution structure of rat HO-1, we report here the backbone 1H, 13C and 15N chemical shift assignments for a 232 residue fragment of HO-1, corresponding to the region that was observed in the crystal structure of the free state (Sugishima et al. 2000, 2002) and the Zn(II) protoporphyrin IX (ZnPP)-bound state, which is known to be the inhibited state.
Methods and experiments
Protein expression and purification
Rat HO-1 (residues 1–232) was expressed and purified according to previously reported procedures, with the exceptions of using the expression vector pET21a(+) and BL21(DE3) host cells (Omata et al. 1998). 2H/13C/15N-labeled HO-1 were prepared by growing cells in minimum M9 medium in 99.9 % 2H2O supplemented with 15N ammonium chloride, 15N ammonium sulfate and 13C6 glucose as sources of nitrogen and carbon, respectively. An overnight preculture was used to inoculate 1 L M9 media and the cells were further grown overnight at 303 K. The protein expression was induced by addition of isopropyl β-d-thiogalactopyranoside to a final concentration of 0.2 mM. The induced cultures were then incubated overnight and harvested by centrifugation. The cell pellet was stored at 193 K until required.
Spheroplasted cells were prepared as described, and HO-1 protein was purified according to a previous report using sodium ammonium sulfate precipitation followed by anion exchange chromatography, size exclusion chromatography, and hydroxyapatite chromatography (Omata et al. 1998). The purity of HO-1 was confirmed by SDS-PAGE analysis. The ZnPP-bound state of HO-1 was prepared as heme-bound HO-1 except that the ZnPP-bound state was maintained in the dark during following preparation (Sugishima et al. 2000). Protein concentrations were determined spectrophotometrically using a molecular extinction coefficient of 25,900 M−1 cm−1 at 280 nm for the free state and using the Bradford method for the ZnPP-bound state.
NMR spectroscopy
NMR experiments were performed on AVANCE DMX750 equipped with a 5-mm cryo TXI probe (Bruker Biospin) at 298 K using 0.4–1.1 mM 2H/13C/15N-labeled samples dissolved in 50 mM potassium phosphate (pH 7) containing 5 % D2O. 3D transverse relaxation optimized spectroscopy (TROSY) spectra of HNCO, HN(CA)CO, HNCA, HN(CO)CA, HNCACB, HN(CO)CACB, CC(CO)NH, and 15N-NOESY with 100 ms mixing time were measured for sequential assignments of backbone 1H, 13C and 15N chemical shifts of HO-1 in both the free and ZnPP-bound states. NMR data were processed using the program NMRPipe (Delaglio et al. 1995), and signal assignments were performed using the programs KUJIRA (Kobayashi et al. 2007).
Assignment and data deposition
In the free state HO-1, 1HN and 15N backbone assignments for all non-proline residues (E2 to Q232), except for two residues (E2 and S159), were assigned and a total of 98.7, 97.3 and 97.8 % chemical shifts assignments for 13Cα, 13Cβ and 13CO resonances were obtained. The 1H–15N TROSY–HSQC spectrum of HO-1 in the free state with sequence specific assignments is shown in Fig. 1. In the case of the ZnPP-bound state, 6 residues (E2, L54, I57, Y58, S142, and S159) of 1HN and 15N assignments were not obtained because severe signal line broadening was observed for residues around I57. A total of 98.3, 96.8, and 96.1 % resonances for 13Cα, 13Cβ and 13CO atoms were assigned for the ZnPP-bound state. Most of the missing assignments corresponded to the residues locating next to Pro, or to those locating at the kink in the F helix, whose flexibility caused signal line broadening. The 1H–15N TROSY–HSQC spectrum of HO-1 in the ZnPP-bound state with sequence specific assignments is shown in Supplementary Fig. 1. Signals of R85, A110, E127, A165 and F166 show unusual downfield chemical shifts (10.5–12.7 ppm). Since these chemical shifts were similar to those of human HO-1 (Li et al. 2004), the downfield chemical shifts are likely due to the effects of ring current shifts or strong hydrogen bonds between the amide proton and immobilized bound water as observed in human HO-1 (Li et al. 2002, 2004).
Figure 2 shows the amide proton chemical shift differences between the free and the ZnPP-bound states of HO-1. As expected, large chemical shift differences were observed in the heme-binding site, including the proximal A helix and distal F helix. This result supports the conclusion that ZnPP binds HO-1 in a manner similar to that of heme (Fe protoporphyrin IX).
The chemical shift assignments for the free and ZnPP-bound states of HO-1 have been deposited in the BioMagResBank (BMRB http://www.bmrb.wisc.edu) under the accession numbers 18798 and 18800, respectively.
References
Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293. doi:10.1007/BF00197809
Khan AA, Quigley JG (2011) Control of intracellular heme levels: heme transporters and heme oxygenases. Biochem Biophys Acta 1813:668–682. doi:10.1016/j.bbamcr.2011.01.008
Kobayashi N, Iwahara J, Koshiba S, Tomizawa T, Tochio N, Güntert P, Kigawa T, Yokoyama S (2007) KUJIRA, a package of integrated modules for systematic and interactive analysis of NMR data directed to high-throughput NMR structure studies. J Biomol NMR 39:31–52. doi:10.1007/s10858-007-9175-5
Li Y, Syvitski RT, Auclair K, Ortiz de Montellano PR, La Mar GN (2002) Solution NMR characterization of an unusual distal H-bond network in the active site of the cyanide-inhibited, human heme oxygenase complex of the symmetric substrate, 2,4-dimethyldeuterohemin. J Biol Chem 277:33018–33031. doi:10.1074/jbc.M204216200
Li Y, Syvitski RT, Auclair K, Ortiz de Montellano PR, La Mar GN (2004) 1H NMR investigation of the solution structure of substrate-free human heme oxygenase: comparison to the cyanide-inhibited, substrate-bound complex. J Biol Chem 279:10195–10205. doi:10.1074/jbc.M308379200
Omata Y, Asada S, Sakamoto H, Fukuyama K, Noguchi M (1998) Crystallization and preliminary X-ray diffraction studies on the water soluble form of rat heme oxygenase-1 in complex with heme. Acta Crystallogr D54:1017–1019
Schuller DJ, Wilks A, Ortiz de Montellano PR, Poulos TL (1999) Crystal structure of human heme oxygenase-1. Nat Struct Biol 6:860–867. doi:10.1038/12319
Sugishima M, Omata Y, Kakuta Y, Sakamoto H, Noguchi M, Fukuyama K (2000) Crystal structure of rat heme oxygenase-1 in complex with heme. FEBS Lett 471:61–66
Sugishima M, Sakamoto H, Kakuta Y, Omata Y, Hayashi S, Noguchi M, Fukuyama K (2002) Crystal structure of rat apo-heme oxygenase-1 (HO-1): mechanism of heme binding in HO-1 inferred from structural comparison of the apo and heme complex forms. Biochemistry 41:7293–7300
Verma A, Hirsch DJ, Glatt CE, Ronnett GV, Snyder SH (1993) Carbon monoxide: a putative neural messenger. Science 259:381–384
Acknowledgments
We thank Naohiro Kobayashi for assistance in NMR chemical shift assignments. This work was supported by Grants-in-Aid for Scientific Research for KS, MS, JH, MN, and KF from the Ministry of Education, Culture, Sports and Technology of Japan.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Harada, E., Sugishima, M., Harada, J. et al. Backbone assignments of the apo and Zn(II) protoporphyrin IX-bound states of the soluble form of rat heme oxygenase-1. Biomol NMR Assign 9, 197–200 (2015). https://doi.org/10.1007/s12104-014-9573-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12104-014-9573-z