SOLUTION 1H NMR INVESTIGATION OF THE MOLECULAR AND ELECTRONIC STRUCTURE OFTHE 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 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 contact...