SOLUTION H-1-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

The 265-residue soluble and completely active portion of inducible rec ombinant human heme oxygenase-l (hHO), the enzyme responsible for heme catabolism, has been investigated by H-1 NMR to elucidate the molecul ar 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 bu t reappear as new signals in the low-spin, cyanide-inhibited hHO-hemin complex. Unambiguous assignment of the heme and axial His25 signals i n the latter complex allows placement of the aromatic clusters, as wel l as other TOCSY-detected side chains, into proximal, distal, or perip heral positions over specific pyrroles based on dipolar contacts and/o r relaxation effects. The three aromatic clusters are located one on t he proximal side adjacent to the axial His and the other two periphera l and distal to the pyrrole I/II junction, the site of heme oxidation. The density of heme methyl dipolar contacts, when compared to those o f globins or peroxidases, reflects an ''open'' pocket, where hemin bin ds in the preformed aromatic cluster of hHO with pyrrole rings I and L I and parts of pyrrole ring IV buried in the protein, with pyrrole rin g III largely exposed to the solvent, and with the proximal side orien ted 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 similar to 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 O-2. Based on previous reports of inconsequential effec ts on HO activity upon mutating the conserved, non-heme-ligated His, t he NMR detection of the conserved distal base in H132A-hHO-CN, togethe r with the available NMR spectral parameters, we identify the distal b ase as a Tyr. The highly conserved His132 is found as one of two His g roups involved in a strong, pseudosymmetric hydrogen-bonding network t hat dynamically stabilizes the active-site structure.