| Popis: |
1H NMR was used to investigate the molecular structure, and dynamic properties of soluble, recombinant, substrate-free human heme oxygenase (apohHO) on a comparative basis with similar studies on the substrate complex. Limited but crucial sequence-specific assignments identify five conserved secondary structural elements, and the detection of highly characteristic dipolar or H-bond interactions among these elements together with insignificant chemical shift differences confirm a strongly conserved folding topology of helices C-H relative to that of substrate complexes in either solution or the crystal. The correction of the chemical shifts for paramagnetic and porphyrin ring current influences in the paramagnetic substrate complex reveals that the strength of all but one of the numerous relatively robust H-bonds are conserved in apohHO, and similar ordered water molecules are located near these H-bond donors as observed in the substrate complexes. The unique and significant weakening of the Tyr(58) OH hydrogen bond to the catalytically critical Asp(140) carboxylate in apohHO is suggested to arise from the removal of the axial H-bond acceptor ligand rather than the loss of substrate. The interhelical positions of the conserved strong H-bonds argue for a structural role in maintaining a conserved structure for helices C-H upon loss of substrate. While the structure and H-bond network are largely conserved upon loss of substrate, the variably increased rate of NH lability dictates a significant loss of dynamic stability in the conserved structure, particularly near the distal helix F. |
