A nuclear Overhauser effect study of the active site of myeloperoxidase. Structural similarity of the prosthetic group to that on lactoperoxidase.
The low-spin, cyanide-ligated ferric complex of the intact bovine granulocyte myeloperoxidase (MPO-CN) has been studied by proton nuclear magnetic resonance utilizing the nuclear Overhauser effect (NOE). This is the largest globular protein (approximately 1.5 x 10(5) for the intact alpha 2 beta 2 tetrameric species) for which successful NOEs have been observed without serious interference of spin diffusion, and demonstrably confirms the utility of such studies on large paramagnetic as compared to diamagnetic proteins. The 1H NMR spectrum of MPO-CN is found to have a remarkable similarity in the number, resonance pattern, and metal ion-induced relaxation properties of the resolved, hyperfine-shifted resonances to those reported earlier for the analogous complex of bovine lactoperoxidase (LPO-CN); moreover, the interproton connectivities between pairs of hyperfine-shifted proton sets, as reflected by the NOEs, are also essentially the same (Thanabal, V., and La Mar, G. N. (1989) Biochemistry 28, 7038-7044). Since the extracted prosthetic group of lactoperoxidase is a porphyrin with proposed functionalization of the 8-methylene group (Nichol, A. W., Angel, L. A., Moon, T., and Clezy, P. S. (1987) Biochem. J. 247, 147-150), we interpret the resultant similarity in 1H NMR spectral parameters for LPO-CN and MPO-CN as indicating that the prosthetic groups in MPO and LPO are very similar, and hence likely both porphyrins with a similarly functionalized periphery that allows covalent linkage to the protein matrix. The hyperfine shift pattern of the broadest resolved lines lead to their assignment to the axial histidyl imidazole side chain. Two pairs of resonances are found to have similar relaxation properties and/or dipolar as similarly shifted resonances that arise from a distal His and Arg in horseradish peroxidase (as also found in LPO-CN), and suggest that MPO also possesses these catalytically functional residues in the distal heme pocket.
Dugad
L B
LB
Department of Chemistry, University of California, Davis 95616.
La Mar
G N
GN
Lee
H C
HC
Ikeda-Saito
M
M
Booth
K S
KS
Caughey
W S
WS
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003903:Deuterium; D017666:Deuterium Oxide; D006098:Granulocytes / Q000201:enzymology; D006859:Hydrogen; D007784:Lactoperoxidase / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009195:Peroxidase / Q000378:metabolism*; D010544:Peroxidases / Q000378:metabolism*; D014867:Water
D014867:Water; D006859:Hydrogen; D003903:Deuterium; D007784:Lactoperoxidase; D010544:Peroxidases; D009195:Peroxidase; D017666:Deuterium Oxide