Title

Two-electron oxidation in myeloperoxidase probed by x-ray crystallography

Date of Award

2001

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Roger E. Fenna, Committee Chair

Abstract

Catalysis by myeloperoxidase (MPO) results in hypohalous acid formation via a two electron oxidation of halide ions. To investigate the mechanism of the two electron oxidation, five crystal structures of human MPO are reported herein. The crystal structure of human MPO has been solved using X-ray diffraction data to 1.8 A resolution collected at 85 K. The model (R = 0.197, R free = 0.239) is in agreement with the previous 2.3 A room temperature structure regarding the covalent heme-protein bonds, secondary structure assignment, the proximal helix halide binding site, and structure of N-Linked carbohydrates. The oxidized Cys 150 has been modeled as a sulfenic acid, based on similar findings in the crystal structure of glutathione reductase (PDB entry IGSN; Becker et al., 1998). Detection of an additional water molecule (Water 957) in the distal heme cavity has resolved the previous ambiguity of solvent hydrogen bonding at the active site in the two halves of the molecule. Interactions of the hexasaccharide N-linked to Asn 317 at the dimer interface are described. The MPO-bromide complex (R = 0.243, Rfree = 0.296) indicates that this halide can replace chloride at the proximal helix halide binding site and also replace Water 843 in the distal cavity. To our knowledge, this is the first reported peroxidase crystal structure of a substrate halide bound directly in the heme cavity. The Water 843 site is proposed to be the site for halide inhibition since binding of a halide at this position would prevent interaction between the distal histidine and the incoming peroxide and would also favor protonation of the histidine Nepsilon. Halide-dependent spectral shifts also are proposed to result from halides binding in place of Water 843 on account of the proximity to the porphyrin. A complex of MPO with cyanide (R = 0.172, Rfree = 0.215) indicates an in-plane movement of the heme iron and cyanide bound in the sixth coordination site in a bent geometry (mean Fe-C-N angle = 159°), near the site of Water 844 which is displaced upon cyanide binding. The geometry of the cyanide ligand appears to be maintained via hydrogen bonding interactions with the distal histidine Nepsilon and with Waters 843 and 845 in the distal cavity. The low pH (pH 4.0) MPO crystal structure (R = 0.204, Rfree = 0.248) suggests that a putative peroxide (H 2O2) binds in the distal cavity in place of Waters 843 and 844. (Abstract shortened by UMI.)

Keywords

Biology, Molecular; Chemistry, Biochemistry

Link to Full Text

http://access.library.miami.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3032371