Heme Oxygenase: Structure

The heme oxygenases are ubiquitiously expressed key enzymes playing a central role in heme turnover as they catalyze the oxidative degradation of heme to biliverdin (biliverdin IXα), carbon monoxide (CO), and ferrous iron (Fe²⁺) ¹. Two major isoforms are expressed in mammals: HO-1 and HO-2 that are encoded by two different functional protein-coding genes in humans (HMOX1, HMOX2) ⁸². While HO-2 is constitutively expressed under basic requirements in the majority of human tissues, HO-1 represents the inducible HO variant whose expression is highly up-regulated upon exposure to different kinds of stress ^2-4. HO-1 and HO-2 show contrast in amino acid sequence and biochemical/biophysical properties ^{13, 151, 175, 176}. Enzymologically, v_max of HO-2 and the K_m for heme are approximately one-tenth and three times that of HO-1 respectively ^{65, 66}. HO-1 is a 288 amino acid ER-associated protein which is embedded in the membrane of the smooth ER through a single hydrophobic C-terminal transmembrane segment (TMS) of 22 amino acid residues (aa265 – 287), while the residual part is cytoplasmic ⁵⁶. Using CD spectroscopy and molecular modeling, Hwang and colleagues anticipated that the secondary structure of the HO-1 TMS is an α-helix ⁶¹. The analysis of the crystal structure of the human HO-1 protein revealed a flexible heme-binding pocket that can open and close aiming at regulating the catalytic activity of the molecule ¹⁷⁷. HO-2 is highly homologous to HO-1 and embedded in the microsomal membrane via a similar C-terminal TMS ⁶⁷. Unlike HO-1, HO-2 bears additional 30 amino acid residues at the N-terminus as well as two Cys-Pro dipeptides (Cys265Pro266 and Cys282Pro283, respectively) near the C-terminus identified as heme regulatory motifs (HRMs; Figure 3). These HRMs act as additional heme ligation sites apart from the heme catalytic center ¹⁷⁸. These further heme ligation sites have been assumed to serve as a basin for CO and NO, thus fulfilling duties different from heme degradation ^178-181. Previous studies by the group of Stephen W. Ragsdale demonstrated that the cysteinyl residues within the HRMs in HO-2 might act as a thiol/disulfide redox switch regulating heme ligation ^{182, 183}, even though it is controversially discussed in the literature ¹⁸⁴. It has been shown previously that Cys265 can bind to ferric (Fe³⁺) heme under reducing conditions ¹⁸⁵. More recently, nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR), and X-ray absorption (XAS) analyses located the C-terminal HRMs in HO-2 to a disordered region ⁶³. The same study specified that reduction of the disulfide bond between Cys265 and Cys282 induces the interaction of the two HRMs with the catalytic center of HO-2 by ligating the crucial Cys to ferric heme. As a consequence, the cysteines in the HRMs directly interact with a second bound heme ⁶³. From these findings the authors speculate that reduced HO-2 binds two molecules of heme, one of them attached to the catalytic center and the other to the HRMs ⁶³. A novel splice variant of human HO-1, harboring a 164-aa deletion (aa49 – 212 of 32 kDa HO-1) and a molecular mass of 14 kDa, was identified in the cytosol of malignant cells ⁶. This variant is generated by alternative splicing of the corresponding HMOX1 mRNA, resulting in the deletion of exon 3 and the loss of HO-1’s heme-binding capacity.

 

HO-1 proteins were first purified and solubilized from porcine spleen ³⁶ and the livers of CoCl₂ or heme-induced rats ^{37, 38}. HO-1 and HO-2 belong to the alpha-only protein class and share a common secondary structure and a highly conserved catalytic site in the cytoplasmic domain ⁷⁶. Previous studies on rodent HO revealed that HO-1 and HO-2 share hydrophobic regions at the C-terminus and a highly conserved heme catalytic domain as well ^{186, 187}. The crystal structures of human HO-1, either in its free form and in complex with heme, provided important insights into the structural features of the enzyme. These studies demonstrated that HO can adopt different ligand-triggered conformations. Comparison of the human apo-HO-1 and heme-HO-1 structures proposed the apo-HO-1 active site coincides more closely with the open conformation than the closed conformation of heme-HO-1 ⁶⁰. In the absence of bound heme, the basic residues Lys179, Arg183, Lys18, and Lys22, implicated in the correct orientation of heme within the heme-binding pocket, have mutually disgusted each other in apo-HO-1 ⁶⁰.  Human HO-1 shows a flexible bi-helical architecture of the heme-binding pocket in which heme is sandwiched between the distal and proximal helices, involving the conserved His25 within the proximal helix as being the iron-binding ligand ^{57, 60}. Moreover, heme has been found to further interact with Gly139 and Gly143 in the distal helix domain of HO-1 ⁵⁷. A major structural characteristic of HO-1 represents the deformity of the distal helix, coating partly the distal heme surface and forming an intrinsic proportion of the oxygen-binding pocket ⁵⁷. In apo-HO-1, the distal helix has been found to exist in an open conformation ⁶⁰. Unlike heme-HO-1, the distal helix in the apo-HO-1 molecule is more open, implying that, irrespective of its flexible structure, the open/closed conformation noted in the heme-HO-1 complex most probably represents a transient state ⁶⁰. The work by Lad and colleagues also exhibited a critical implication of certain amino acid residues in the enzymatic catalysis of HO-1. Amongst them, Asp140 was described as being H-bonded and locked into a fixed area through an H-bonding network involving Asn210, Arg136, and a second group comprising Tyr58 and Try114. This network is identical in both, the apo-HO-1 and heme-HO-1 architecture ⁶⁰. Analysis of the open and closed heme-HO-1 conformations illustrate that the Asp140/H-bonded complex get sorted in the closed conformation. It has been hypothesized that the binding of heme and the concomitant closing of the active site consequently induce the partial formation of an essential Asp140/H-bonded network which may constitute the proton shuttle machinery necessary for oxygen activation ⁶⁰.

 

Human HO-1 and HO-2 share the same α-helical fold with the imidazole nitrogen of the conserved His in the catalytic domain (His25 in HO-1 and His45 in HO-2) serving as the proximal heme ligand ^{57, 62}. An accessary role in heme ligation has been attributed to His151 in HO-2 as well ^{188, 189}. While the conserved His132 facilitates heme oxidation in HO-1, this role is held by His151 in HO-2 ^{189, 190}. HO-1 has been assumed to exist as a monomer in cells due to the lack of cysteinyl residues required for the formation of disulfide bridges ⁷⁷. However, recent investigations revealed that HO-1 forms dimers/oligomers (Figure 2) in the ER via TMS/TMS interactions as a prerequisite for optimal structural and functional qualities of the molecule ⁶¹. Fluorescence resonance energy transfer (FRET) analysis revealed a crucial implication of Trp270 in the self-assembly of the HO-1 TMS ⁶¹.