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

摘要

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 approx 5%,the relative values of DELTAchi (and the zero-field splitting constant,D_(propor.to) DELTA_(chiax)) 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 labi.le proton chemical shifts for paramagnetic influences indicates that Gln49 and His53,some approx 10 A 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.