W0058

1.75 Å Crystal Structure of Human Myeloperoxidase and Halide Binding Site Characterization. T.J. Fiedler and R.E. Fenna, Dept. of Biochemistry & Molecular Biology, Univ. of Miami Medical School, Miami, FL 33101

The cryogenic X-ray crystal structure of human myeloperoxidase (MPO) was determined to 1.75 Å resolution. Initial phases were obtained from the 2.25 Å room temperature structure (Fenna et al., Arch. Biochem. Biophys. 316: 653-6, 1995). The current model was refined to a crystallographic R-factor of 21.8% with an R-free of 26.1% for 128108 unique reflections in the resolution range of 35 to 1.75 Å. The r.m.s. deviations from ideality for bond lengths and angles were 0.012 Å and 1.26º. The model confirmed 3 covalent heme-protein bonds: two ester linkages (D94 to ring C methyl, E 242 to ring A methyl) and a sulfonium ion linkage (M243 to ring A vinyl terminal carbon). The heme iron is displaced out of plane toward the proximal non-catalytic H336. The previously ambiguous H-bonding pattern for 4 water molecules in the distal cavity has now been resolved. Three sites of N-linked glycosylation (N189, N225, and N317) have been modeled; N317 at the dimer interface was observed with a full complement of (GlcNAC)₂ - (Man)₃ + Fuc (1,6 to initial GlcNAc). No direct interaction occurs between carbohydrate and protein of the same monomer. A 3-4 [sigma] electron density peak at the N-terminus of Helix 8 has been characterized as a chloride ion, 18 Å from the heme iron, although its role in catalysis remains unknown. The chloride was confirmed by difference Fourier analysis [(F_pI-) - (F_pCl-)] of a crystal soaked in 100 mM KI pH 5.5 where increased density at this location indicated replacement by iodide. Human MPO crystals belong to space group P2₁ (a=92.23 Å, b=63.41 Å, c=111.08 Å, beta=97.47º).