Modulation of the axial water hydrogen-bonding properties by chemical modification of the substrate in resting state, substrate-bound heme oxygenase from Neisseria meningitidis; coupling to the distal H-bond network via ordered water molecules.

The hydrogen bonding of ligated water in ferric, high-spin, resting-state substrate complexes of heme oxygenase from Neisseria meningitidis has been systematically perturbed by variable electron-withdrawing substituents on the hemin periphery. The pattern of 1H NMR-detected dipolar shifts due to the paramagnetic anisotropy is strongly conserved among the four complexes, with the magnitude of dipolar shifts or anisotropy increasing in the order of substituent formyl < vinyl < methyl. The magnetic anisotropy is axial and oriented by the axial Fe-His23 bond, and while individual anisotropies have uncertainties of approximately 5%, the relative values of deltachi (and the zero-field splitting constant, D proportional, variant deltachi(ax)) are defined to 1%. The unique changes in the axial field strength implied by the variable zero-field splitting are in accord with expectations for the axial water serving as a stronger H-bond donor in the order of hemin substituents formyl > vinyl > methyl. These results establish the axial anisotropy (and D) as a sensitive probe of the H-bonding properties of a ligated water in resting-state, substrate complexes of heme oxygenase. Correction of observed labile proton chemical shifts for paramagnetic influences indicates that Gln49 and His53, some approximately 10 angstroms from the iron, sense the change in the ligated water H-bonding to the three nonligated ordered water molecules that link the two side chains to the iron ligand. The present results augur well for detecting and characterizing changes in distal water H-bonding upon mutagenesis of residues in the distal network of ordered water molecules and strong H-bonds.