Solution of helium-1 NMR studies of heme oxygenase from the pathogenic bacterium Corynebacterium diphtheriae

Heme oxygenase, HO, an enzyme utilizes heme as both substrate and cofactor, catalyzes the stereoselective reaction that cleaves heme to biliverdin, carbon monoxide, and free iron. HO from the pathogenic bacterium Corynebacterium diphtheriae, HmuO, functions to secure iron from mammalian hosts. Three forms of the enzyme were studied: the low-spin cyano- and high-spin aquo- paramagnetic ferric complexes, and diamagnetic apo-HmuO. Our goals were to: define and compare the active site structure to the crystals; quantitate the paramagnetic susceptibility tensor; assay distal steric influences through the Fe-CN tilt; compare the extended H-bond network and its associated ordered water molecules, and the dynamic stability, among the three derivatives and with other HOs.;The diamagnetic chemical shifts for low-field biased labile protons reveal an H-bond network which, with two exceptions, exhibits H-bond strengths independent of the ligand or substrate loss. Both Arg132 NϵH and Tyr53 OH exhibit greater H-bond strength when the axial ligand is an acceptor (cyanide) than donor (H2O). The positions of the major fraction of the ordered water molecules observed in the distal pocket and detected by NMR appear unaffected by the distal ligand or substrate loss. Dynamic stability, probed through the exchange of labile protons, is only weakly, and highly regionally, perturbed by the axial ligand, but more broadly and strongly decreased upon the substrate loss. It is demonstrated that apo-HmuO is superior diamagnetic references for paramagnetic complexes than shifts generated by available computer programs.;1H 2D NMR provided sufficient assignments to generate magnetic axes, and to show a major portion of the structure is retained upon the substrate loss. For the cyanide complex, we demonstrated a sizable tilt of the Fe-CN unit (which mimics Fe-OO) that confirms steric effects to facilitate the α-meso cleavage. We used dipolar shifts and relaxation as constraints to show that the distal helix in the cyanide complex lies approximately mid-way between the positions in the crystals of the aquo- and oxy-complexes. The axial anisotropy was determined for the aquo- complex and shown to be larger than in HO from N. meningitidis (HemO), indicating the ligated water is a better H-bond donor in HemO.