Abstract
The molecular structure and dynamic properties of the active site environment of HmuO, a heme oxygenase (HO) from the pathogenic bacterium Corynebacterium diphtheriae, have been investigated by (1)H NMR spectroscopy using the human HO (hHO) complex as a homology model. It is demonstrated that not only the spatial contacts among residues and between residues and heme, but the magnetic axes that can be related to the direction and magnitude of the steric tilt of the FeCN unit are strongly conserved in the two HO complexes. The results indicate that very similar contributions of steric blockage of several meso positions and steric tilt of the attacking ligand are operative. A distal H-bond network that involves numerous very strong H-bonds and immobilized water molecules is identified in HmuO that is analogous to that previously identified in hHO (Li, Y., Syvitski, R. T., Auclair, K., Wilks, A., Ortiz de Montellano, P. R., and La Mar, G. N. (2002) J. Biol. Chem. 277, 33018-33031). The NMR results are completely consistent with the very recent crystal structure of the HmuO.substrate complex. The H-bond network/ordered water molecules are proposed to orient the distal water molecule near the catalytically key Asp(136) (Asp(140) in hHO) that stabilizes the hydroperoxy intermediate. The dynamic stability of this H-bond network in HmuO is significantly greater than in hHO and may account for the slower catalytic rate in bacterial HO compared with mammalian HO.
Highlights
Heme oxygenase (HO)[1] is an ␣-helical enzyme that carries out the highly stereoselective conversion of hemin to ␣-biliverdin, iron, and CO, excising CO from exclusively the ␣-meso position (1)
To provide a broader comparison with the NMR data on human HO (hHO) complexes (19 –22), we explore in parallel both the disordered HmuO1⁄7PH1⁄7CN complex and the homogeneous HmuO1⁄7DMDH1⁄7CN complex to show that this bacterial HO exhibits remarkable conservation of the distal steric effects on the axial ligand and distal H-bond network relative to hHO
PH Orientation—The orientation of PH in both hHO (19, 20) and HmuO complexes is rotationally disordered about the ␣/␥-meso axis in the initially formed complex, with very similar ϳ3:1 ratios at equilibrium and with the same heme orientation dominating in each complex in solution
Summary
Orientation of ligated azide in the rat HO1⁄7heme1⁄7N3 complex confirms such a steric influence (23). Functional (26, 27) and spectroscopic (27, 28) studies, as well as mutagenesis (29, 30), have confirmed the same mechanism and stereospecificity as for mammalian HOs, the turnover rate is slower (27); the enzyme has been crystallized (31), and the structure of the substrate hemin complex has been refined to 1.4-Å resolution.[2] Our interests are to establish the degree to which the available extensive NMR data on hHO1⁄7DMDH1⁄7CN (19 –22) and the crystal structure of hHO1⁄7PH1⁄7H2O (15) can be used to assign the resonances and to structurally interpret the NMR spectral parameters (32) of HmuO1⁄7PH1⁄7CN in terms of the orientation of the FeCN vector and the presence or absence of a distal H-bond network similar to that reported for hHO1⁄7DMDH1⁄7CN (21, 22) This 216-residue soluble HmuO enzyme has His[20] as its axial ligand (30) and exhibits extensive sequence homology to the distal helix and the four fragments of HO shown (21) to participate in the H-bond network in. To provide a broader comparison with the NMR data on hHO complexes (19 –22), we explore in parallel both the disordered HmuO1⁄7PH1⁄7CN complex and the homogeneous HmuO1⁄7DMDH1⁄7CN complex to show that this bacterial HO exhibits remarkable conservation of the distal steric effects on the axial ligand and distal H-bond network relative to hHO
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