The majority of the active site residues of cyanideinhibited, substrate-bound human heme oxygenase have been assigned on the basis of two-dimensional NMR using the crystal structure of the water-ligated substrate complex as a guide (Schuller, D. J., Wilks, A., Ortiz de Montellano, P. R., and Poulos, T. L. (1999) Nat. Struct. Biol. 6, 860 -867). The proximal helix and the N-terminal portion of the distal helix are found to be identical to those in the crystal except that the heme for the major isomer (ϳ75-80%) in solution is rotated 180°a bout the ␣-␥-meso axis relative to the unique orientation in the crystal. The central portion of the distal helix in solution is translated slightly over the heme toward the distal ligand, and a distal four-ring aromatic cluster has moved 1-2 Å closer to the heme, which allows for strong hydrogen bonds between the hydroxyls of Tyr-58 and Tyr-137. These latter interactions are proposed to stabilize the closed pocket conducive to the high stereospecificity of the ␣-meso ring opening. The determination of the magnetic axes, for which the major axis is controlled by the Fe-CN orientation, reveals a ϳ20°tilt of the distal ligand from the heme normal in the direction of the ␣-meso bridge, demonstrating that the close placement of the distal helix over the heme exerts control of stereospecificity by both blocking access to the , ␥, and ␦-meso positions and tilting the axial ligand, a proposed peroxide, toward the ␣-meso position.
Heme oxygenase (HO)1 is a membrane-bound protein that carries out the NADPH-, O 2 -, and cytochrome P450 reductasedependent regiospecific catabolism of heme to iron, ␣-biliverdin, and carbon monoxide (1). There are two well-established mammalian isoforms, the 288-residue inducible HO1 (2, 3) and the 316-residue constitutive HO2 (4), that share significant identity and have a common substrate, mechanisms, and products. The sequence of HO fails to exhibit significant identity to any previously structurally characterized enzyme, and hence, in the absence of a molecular model, the early research on the soluble enzyme focused on functional (5-13), mutational (5, 11), and spectroscopic (5, 14 -18) investigations. The current understanding of HO is largely based on investigation of a recombinant, truncated 265-residue portion of HO1 from which the membrane-binding domain has been deleted, but which retains full activity (13). The mechanism of HO resembles that of cytochrome P450 in its ability to oxidize unactivated C-H bonds (19). However, in contrast to cytochrome P450 and the heme peroxidases, the action of HO does not pass through a ferryl intermediate (13,20) but appears rather to involve a peroxo ligand capable of attacking the ␣-meso bridge.Spectroscopic investigations agree on a normal iron-His (5-7, 14, 15) bond that more closely resembles that of Mb, with a neutral imidazole ligand, than that of the peroxidases, in which the axial ligand has significant imidazolate character (21). Mutational work identified His-25 as the axial ligand (5, 22). Whereas there...