title: Substitutions of the "bridging" aspartate 178 result in profound changes in the reactivity of the Rieske center of phthalate dioxygenase.
abstract: Phthalate dioxygenase (PDO) and its reductase (PDR) are parts of a two-component Rieske oxygenase system that initiates the aerobic breakdown of phthalate by forming cis-4,5-dihydro-4,5-dihydroxyphthalate. Aspartate D178 in PDO, which lies between the Rieske [2Fe-2S] center of one subunit and the mononuclear center of the adjacent subunit, is highly conserved among the Rieske dioxygenases. The analogous aspartate has been implicated in electron transfer in naphthalene dioxygenase and in substrate binding and oxygen reactivity in anthranilate dioxygenase. Substitution of D178 with alanine or asparagine in PDO resulted in proteins with significantly increased Fe(II) dissociation constants. The rates of oxidation of the reduced Rieske centers in D178A and D178N were decreased by more than 10(4)-fold; only part of the loss of activity can be attributed to depletion of iron from the mononuclear centers. Reduction of PDO by reduced PDR was also slower in the D178A and D178N variants. Observed decreases in turnover rates of D178A and D178N compared to that of wild-type (WT) PDO (>10(2)-fold) can be ascribed to the cumulative effect of the low intrinsic iron content of the D178A and D178N mutants and the combination of the decreased rates of Rieske center reduction and oxidation. The coupling of dihydrodiol formation approached 100% in WT PDO but was only approximately 16% in D178A and approximately 7% in D178N. In single-turnover experiments, very small amounts of DHD were produced by D178A and D178N "as purified". The presence of saturating amounts of ferrous ion improved coupling to nearly 100% for the D178N variant but only slightly improved coupling for D178A. Thus, although hydroxylation is still possible in the variants, the reactions are largely uncoupled due to slow intramolecular electron transfer rates and the apparent weak binding of iron at the mononuclear centers.
authors.affiliation: Department of Biological Chemistry, University of Michigan, 1301 Catherine Street, Ann Arbor, Michigan 48109-0606, USA.
authors.forename: Alex
authors.initials: A
authors.lastname: Pinto
authors.forename: Michael
authors.initials: M
authors.lastname: Tarasev
authors.forename: David P
authors.initials: DP
authors.lastname: Ballou
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D016956:Burkholderia cepacia / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D014450:Electron Transport Complex III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006900:Hydroxylation; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D010795:Phthalic Acids / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D010795:Phthalic Acids; C037989:Rieske iron-sulfur protein; D001224:Aspartic Acid; C032279:phthalic acid; D010088:Oxidoreductases; C049135:phthalate oxygenase reductase; D010105:Oxygenases; C044732:phthalate dioxygenase; D014450:Electron Transport Complex III