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Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants
Author(s) -
Moomaw Ellen W,
Hoffer Eric,
Moussatche Patricia,
Salerno John,
Grant Morgan,
Immelman Bridget,
Uberto Richard,
Ozarowski Andrew,
Angerhofer Alexander
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.789.9
Subject(s) - chemistry , oxalate , active site , protonation , catalysis , electron paramagnetic resonance , binding site , hydrogen peroxide , oxidase test , stereochemistry , enzyme , photochemistry , inorganic chemistry , biochemistry , organic chemistry , ion , physics , nuclear magnetic resonance
Ceriporiopsis subvermispora oxalate oxidase (CsOxOx) is a Mn‐dependent enzyme that catalyzes the oxygen‐dependent oxidation of oxalate to carbon dioxide in a reaction that is coupled with the formation of hydrogen peroxide. EPR spectra indicate that the Mn is present as Mn(II). EPR spin‐trapping experiments support the existence of an oxalate‐derived radical species formed during turnover. The dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated. The pH profile of the D241A CsOxOx mutant suggests that the protonation state of aspartic acid 241 is mechanistically significant and that catalysis takes place at the N‐terminal Mn binding site. The fact that the D241S CsOxOx eliminates Mn binding to both the N‐ and C‐ terminal Mn binding sites suggests that both sites must be intact for Mn incorporation into either site. Mutation of conserved arginine residues further support that catalysis takes place at the N‐terminal Mn binding site and that both sites must be intact for Mn incorporation into either site. This work was supported by the National Science Foundation (MCB‐1041912) to EWM.