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).

Fig. 1
figure 1

1H–15N TROSY–HSQC spectra of 2H/13C/15N-labeled HO-1 (residues 1–232) in the free state at pH 7.0, 298 K in 95 % H2O/5 % D2O. Signal assignments for backbone amides are indicated. a The full spectrum and b an expansion of the region of the full spectrum enclosed by the dashed box are shown, respectively

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).

Fig. 2
figure 2

Averaged chemical shift differences of amide proton and nitrogen atoms \(\left( {{\Delta \delta }_{\text{avg}} = \sqrt {{\Delta \delta }_{{{}^{1}{\text{H}}}}^{2} + \left( {{{{\Delta \delta }_{{{}^{15}{\text{N}}}} } \mathord{\left/ {\vphantom {{{\Delta \delta }_{{{}^{15}{\text{N}}}} } 5}} \right. \kern-0pt} 5}} \right)^{2} } } \right)\) between the free and ZnPP-bound states are plotted on the heme-bound HO-1 crystal structure. Heme is represented as a sphere model. The Δδavg values larger than the average chemical shift differences and larger than the average plus 1 standard deviation are color coded in yellow and red, respectively. The letters A–H represent α helical secondary structures

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.