Coupling of the distal hydrogen bond network to the exogenous ligand in substrate-bound, resting state human heme oxygenase.

Mammalian heme oxygenase (HO) possesses catalytically implicated distal ordered water molecules within an extended H-bond network, with one of the ordered water molecules (#1) providing a bridge between the iron-coordinated ligand and the catalytically critical Asp140, that, in turn, serves as an acceptor for the Tyr58 OH H-bond. The degree of H-bonding by the ligated water molecule and the coupling of this water molecule to the H-bond network are of current interest and are herein investigated by (1)H NMR. Two-dimensional NMR allowed sufficient assignments to provide both the H-bond strength and hyperfine shifts, the latter of which were used to quantify the magnetic anisotropy in both the ferric high-spin aquo and low-spin hydroxo complexes. The anisotropy in the aquo complex indicates that the H-bond donation to water #1 is marginally stronger than in a bacterial HO, while the anisotropy for the hydroxo complex reveals a conventional (d(xz), d(yz))(1) ground state indicative of only moderate to weak H-bond acceptance by the ligated hydroxide. Mapping out the changes of the H-bond strengths in the network during the ligated water --> hydroxide conversion by correcting for the effects of magnetic anisotropy reveals a very substantial change in H-bond strength for Tyr58 OH and lesser effects on nearby H-bonds. The effect of pH on the H-bonding network in human HO is much larger and transmitted much further from the iron than in a pathogenic bacterial HO. The implications for the HO mechanism of the H-bond of Tyr58 to Asp140 are discussed.