Solution1H NMR Investigation of the Molecular and Electronic Structure of the Active Site of Substrate-Bound Human Heme Oxygenase: the Nature of the Distal Hydrogen Bond Donor to Bound Ligands

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Abstract

The 265-residue soluble and completely active portion of inducible recombinant human heme oxygenase-1 (hHO), the enzyme responsible for heme catabolism, has been investigated by 1H NMR to elucidate the molecular and electronic structure of the substrate-bound complex. 2D NMR of substrate-free hHO reveals a cluster of nine mobile aromatic residues whose signals are largely “bleached” upon binding high-spin hemin but reappear as new signals in the low-spin, cyanide-inhibited hHO-hemin complex. Unambiguous assignment of the heme and axial His25 signals in the latter complex allows placement of the aromatic clusters, as well as other TOCSY-detected side chains, into proximal, distal, or peripheral positions over specific pyrroles based on dipolar contacts and/or relaxation effects. The three aromatic clusters are located one on the proximal side adjacent to the axial His and the other two peripheral and distal to the pyrrole I/II junction, the site of heme oxidation. The density of heme methyl dipolar contacts, when compared to those of globins or peroxidases, reflects an “open” pocket, where hemin binds in the preformed aromatic cluster of hHO with pyrrole rings I and II and parts of pyrrole ring IV buried in the protein, with pyrrole ring III largely exposed to the solvent, and with the proximal side oriented toward the protein surface and the distal site toward the protein interior. A distal labile proton has been located which serves as the H-bond donor to cyanide, is the likely origin of the spectroscopically detected pK of ∼7.6 in the hHO-hemin complex, and probably arises from the same distal residue that serves as the H-bond donor to the activated O2. Based on previous reports of inconsequential effects on HO activity upon mutating the conserved, non-heme-ligated His, the NMR detection of the conserved distal base in H132A-hHO-CN, together with the available NMR spectral parameters, we identify the distal base as a Tyr. The highly conserved His132 is found as one of two His groups involved in a strong, pseudosymmetric hydrogen-bonding network that dynamically stabilizes the active-site structure.

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