Crystal Structure of Rat Heme Oxygenase-1 in Complex with Heme Bound to Azide

Abstract

Heme oxygenase (HO) catalyzes physiological heme degradation consisting of three sequential oxidation steps that use dioxygen molecules and reducing equivalents. We determined the crystal structure of rat HO-1 in complex with heme and azide (HO-heme-N 3 ؊ ) at 1.9-Å resolution. The azide, whose terminal nitrogen atom is coordinated to the ferric heme iron, is situated nearly parallel to the heme plane, and its other end is directed toward the ␣-meso position of the heme. Based on resonance Raman spectroscopic analysis of HO-heme bound to dioxygen, this parallel coordination mode suggests that the azide is an analog of dioxygen. The azide is surrounded by residues of the distal F-helix with only the direction to the ␣-meso carbon being open. This indicates that regiospecific oxygenation of the heme is primarily caused by the steric constraint between the dioxygen bound to heme and the F-helix. The azide interacts with Asp-140, Arg-136, and Thr-135 through a hydrogen bond network involving five water molecules on the distal side of the heme. This network, also present in HO-heme, may function in dioxygen activation in the first hydroxylation step. From the orientation of azide in HO-heme-N 3 ؊ , the dioxygen or hydroperoxide bound to HO-heme, the active oxygen species of the first reaction, is inferred to have a similar orientation suitable for a direct attack on the ␣-meso carbon.

Crystal Structures of the NO- and CO-bound Heme Oxygenase from Neisseriae meningitidis

“…The picture that emerges is very similar to that of human and rat HO-1 (36). It now appears that a common feature of HOs is to establish a rigid network of distal pocket H-bonded water molecules that ensures proper proton delivery to the distal O atom of the iron bound O 2 ligand.…”

Solution 1H NMR Investigation of the Active Site Molecular and Electronic Structures of Substrate-bound, Cyanide-inhibited HmuO, a Bacterial Heme Oxygenase fromCorynebacterium diphtheriae

“…Nevertheless, the pattern of dipolar shifts for the protons on the proximal helix allowed determination of the orientation of the major magnetic axis, which could be correlated with a ϳ20°tilt of the FeCN in the direction of the ␣-meso position (19,20). The orientation of ligated azide in the rat HO⅐heme⅐N 3 complex confirms such a steric influence (23).…”

“…2) among TOCSY-detected side chains locate two helical fragments (I and II) for which numerous side chains exhibit moderate-to-large hyperfine shifts. Fragment I is hHO (20,21), indicates that Phe 201 is slightly shifted away from Ala 23 . Finally, note that 3-CH 3 in HmuO⅐PH⅐CN exhibits weak NOESY cross-peaks to Phe 208 (which also exhibits strong NOESY cross-peaks to the 2-vinyl group) (data not shown), whereas the small Phe 214 cross-peak to 3-CH 3 is not seen in hHO⅐PH⅐CN (20,21) (20,21) are absent in the HmuO complex (Fig.…”

“…The existence of a network of water molecules that includes water molecules near the catalytically critical distal helix Asp 136 / Asp 140 (12,14) supports the notion that water provides the stabilizing H-bond to the novel hydroperoxy intermediate. Interaction of Asp 140 with the distal ligand via two water molecules has been recently characterized in the crystal structure of the rat HO⅐PH⅐N 3 complex (23). The presence of a water/Hbond network that extends from the distal pocket through the enzyme to its surface on the opposite side from the substratebinding pocket (22) 2 suggests that the channel may funnel the required nine protons to the active site in a controlled manner.…”

Involvement of NADP(H) in the Interaction between Heme Oxygenase-1 and Cytochrome P450 Reductase

“…The His 25 side chain in the A helix contributes the proximal heme ligand. The distal heme pocket, a relatively narrow cavity, is responsible for the strict discrimination between the distal heme ligands (20 -22) and accommodates a hydrogen-bonded network that plays an important role in catalysis (23)(24)(25)(26). The distal and proximal helices are flexible or disordered in the heme-free state (27,28) and, to a lesser degree, in the product-bound state (29).…”