Comparison of Apo- and Heme-bound Crystal Structures of a Truncated Human Heme Oxygenase-2

Abstract

Heme oxygenase (HO) catalyzes the first step in the heme degradation pathway. The crystal structures of apo-and heme-bound truncated human HO-2 reveal a primarily ␣-helical architecture similar to that of human HO-1 and other known HOs. Proper orientation of heme in HO-2 is required for the regioselective oxidation of the ␣-mesocarbon. This is accomplished by interactions within the heme binding pocket, which is made up of two helices. The iron coordinating residue, His 45 , resides on the proximal helix. The distal helix contains highly conserved glycine residues that allow the helix to flex and interact with the bound heme. Tyr 154 , Lys 199 , and Arg 203 orient the heme through direct interactions with the heme propionates. The rearrangements of side chains in heme-bound HO-2 compared with apoHO-2 further elucidate HO-2 heme interactions. Heme oxygenase (HO)2 catalyzes the degradation of heme to free iron, carbon monoxide, and biliverdin in the presence of NADPH-dependent cytochrome P450 reductase. Seven electrons are transferred, and three molecules of oxygen are consumed in the degradation of one molecule of heme to biliverdin (Fig. 1). Subsequently, biliverdin reductase reduces biliverdin to bilirubin (1). Present in both bacteria and eukaryotes, HO is the only known enzyme that can degrade heme. HO not only plays a critical role in heme and iron homeostasis, but the products of heme degradation have important physiological functions (1, 2). Iron is the essential catalytic center for heme and many nonheme metalloenzymes and has crucial effects on cytoprotection (3, 4). Although CO is toxic at levels above ϳ500 ppm, it also is a signaling molecule (5, 6). CO has anti-apoptotic, antiinflammation, and anti-proliferation properties (7-9). Bilirubin is an antioxidant that is involved in resistance to oxidative stress (10, 11).Two HO isoforms have been reported in mammalian cells, heme oxygenase-1 (HO-1) and heme oxygenase-2 (HO-2) (12). The mammalian HO-1 and HO-2 exhibit similar catalytic activities and share 55% identity and 76% similarity in humans (13,14). Another isoform, HO-3, was recently reported in rat; however, no activity was detected for this isoform, and it is considered a pseudogene (15). Plants contain several HOs, including one named HO-2, but these lack the C-terminal membranespanning region seen in mammalian HOs and are more closely related to the bacterial and insect HO-1 than to the mammalian HO-2 (13, 16).Although they are both membrane-bound proteins and their catalytic activities are similar, HO-1 and HO-2 are expressed in discrete tissues and exhibit very different patterns of expression (17). Although human HO-1 is an inducible protein whose expression is up-regulated by heat shock and oxidative stress conditions, HO-2 is constitutively expressed (17-22). HO-1 has been found in most tissues, with particularly high expression levels in liver and spleen; however, HO-2 is mainly found in the brain, testes, and carotid body (23-26).HO-1 and HO-2 exhibit two regions of sequence divergence: o...

Structural insights into human heme oxygenase-1 inhibition by potent and selective azole-based compounds

“…of the a-carbons ranging from 0.697 to 0.862 Å , for the various conformations determined thus far (i.e. open and closed conformations of the human HO-1 holoenzyme) [58]. The three-dimensional structures of both HOs are predominantly a-helical with the heme sandwiched between the distal and proximal helices (figure 2b,c) [58][59][60][61].…”

“…A third isozyme, HO-3, has been demarcated as a pseudogene and is inactive, despite sharing approximately 90 per cent sequence identity with HO-2 [56]. Sequence alignment between HO-1 and HO-2 shows 45 per cent identity between the full-length sequences, with 55 per cent identity in the conserved core region, especially that of the conserved heme pocket regions (figure 2a) [58]. Structural alignment of the crystal structures of the truncated, heme-conjugated human HO-1 and HO-2 shows remarkable structural conservation rsif.royalsocietypublishing.org J R Soc Interface 10: 20120697 with r.m.s.d.…”

“…open and closed conformations of the human HO-1 holoenzyme) [58]. The three-dimensional structures of both HOs are predominantly a-helical with the heme sandwiched between the distal and proximal helices (figure 2b,c) [58][59][60][61]. A number of conserved glycines in the distal helix provide flexibility to accommodate substrate binding and product release.…”

“…The major point of sequence divergence between HO-1 and HO-2 is in the C-terminal region (figure 2a), as well as the presence of three heme regulatory motifs (HRMs) in HO-2 with a characteristic Cys-Pro signature; while HRM3 is centred around Cys127, HRM1 and HRM2 reside in the C-terminus [58]. There is some controversy with respect to the precise role of these HRMs.…”

“…Moreover, identification of essential structural features critical for inhibition, both within the inhibitor as well as structural changes within the protein, provided valuable insights that enhanced the design of more effective HO-1 inhibitors. During the course of our study, the X-ray crystal structure of a truncated, human HO-2 derivative containing a Cys127Ala mutation was also determined [58], which provided more insights regarding potential structural differences that may be used in inhibitor design to enhance isozyme selectivity (figure 2c).…”