The “Bridging” Aspartate 178 in Phthalate Dioxygenase Facilitates Interactions between the Rieske Center and the Iron(II)−Mononuclear Center

Abstract

Phthalate dioxygenase (PDO) and its reductase are parts of a two-component Rieske dioxygenase system that initiates the aerobic breakdown of phthalate by forming cis-4,5-dihydro-4,5-dihydroxyphthalate (DHD). Aspartate D178 in PDO, located near its ferrous mononuclear center, is highly conserved among Rieske dioxygenases. The analogous aspartate has been implicated in electron transfer between the mononuclear iron and Rieske center in naphthalene dioxygenase (Parales, R.E. et. al. (1999) J Bacteriol 181, 1831-1837 and in substrate binding and oxygen reactivity in anthranilate dioxygenase (Beharry, Z.M. et. al (2003), Biochemistry 42, 13625-13636). The effects of substituting D178 in PDO with alanine or asparagine on the reactivity of the Rieske centers, phthalate hydroxylation, and coupling of Rieske center oxidation to DHD formation were studied previously (Pinto, A., Tarasev, M., and Ballou, D. P. (2006) Biochemistry in press). This work describes effects that D178N and D178A substitutions have on the interactions between the Rieske and mononuclear centers in PDO. The mutations affected protonation of the Rieske center histidine and conformation of subunits within the PDO multimer to create a more open structure with more solvent-accessible Rieske centers. When the Rieske centers in PDO were oxidized, D178N and D178A substitutions disrupted communication between the Rieske and Fe-mononuclear centers. This was shown by the lack of perturbations of the UV-vis spectra on phthalate binding to the D178N and D178A variants, as opposed to that observed in WT PDO. However, when the Rieske center was in the reduced state, communication between the centers was not disrupted. Phthalate binding similarly affected the rates of oxidation of the reduced Rieske center in both WT and mutant PDO. Nitric Oxide binding at the Fe(II) mononuclear center, as detected by EPR spectrometry of the Fe(II) nitrosyl complex, was regulated by the redox state of the Rieske center. When the Rieske center was oxidized in either WT or D178N PDO, NO bound to the mononuclear iron in the presence or absence of phthalate. However, when the Rieske center was reduced, NO bound only when phthalate was present. These findings are discussed in terms of the "communication functions" performed by the bridging Asp-178.Phthalate dioxygenase (PDO) and its reductase are parts of a two-component enzyme system (PDS) in Burkholderia cepacia DB01 that initiates the aerobic breakdown of phthalate by forming cis-4,5-dihydro-4,5-dihydroxyphthalate (DHD). PDS comprises the monomeric phthalate dioxygenase reductase (PDR), an enzyme that contains both FMN and a plant-type [2Fe-2S] ferredoxin, and a Rieske dioxygenase (PDO), an α 6 multimer that contains Rieske and ferrous mononuclear centers. Mononuclear iron and Rieske centers on each subunit in Rieske oxygenases are shown to be separated by more than 40 Å (2), making direct electron *To whom correspondence should be addressed. Email: dballou@umich.edu NIH Public Access transfer unfavorable (3). However, as s...

Characterization of 3-Ketosteroid 9α-Hydroxylase, a Rieske Oxygenase in the Cholesterol Degradation Pathway of Mycobacterium tuberculosis

“…This includes conserved Asp 178 , whose carboxylate bridges the metallocenters by forming hydrogen bonds with each of the following: the N⑀2 atom of His 89 (2.8 Å) and the N␦1 atom of His 181 (3.1 Å). In other ROs, this residue has been implicated in catalysis, although its precise role is unclear (20,55). The conformation of this aspartate is similar in all previous RO structures, but in KshA, the 1 angle differs by ϳ150°, such that the bond between the ␣-and ␤-carbons points toward the catalytic domain rather than the Rieske domain of the adjacent subunit (Fig.…”

Distinguishing Protonation States of Histidine Ligands to the Oxidized Rieske Iron–Sulfur Cluster through 15N Vibrational Frequency Shifts

“…[4] The Rieske cluster in these enzymesi sp roposed to pass electrons to am ononuclear iron complext hat comprises the enzyme's catalytic core. [5] Structural studies [6] and measurements of pHdependentr edox potentials [7] imply protonation of one His ligand of the Rieske cluster upon its reduction (for possible protonation states of the two His ligands to the Rieske ironsulfur cluster,s ee the captiont oF igure 1). Then, an Asp side chain hydrogenb onds to the protonated His and mediates electront ransfer to the catalytic, mononuclear iron center.T his mechanism implies that the His ligand is deprotonated in the cluster'so xidized state.…”

Substrate Specificities and Conformational Flexibility of 3-Ketosteroid 9α-Hydroxylases

“…The three KshA subunits are arranged in a head-to-tail fashion such that the [2Fe-2S] cluster and the mononuclear iron of adjoining subunits are 12 Å apart. Histidine ligands from adjacent metallocenters are bridged by a conserved aspartate that appears to mediate redox-dependent conformational changes during the RO catalytic cycle (20). Remarkably, the substrate access channel of KshA is angled at ϳ90°with respect to that of other ROs characterized to date and is significantly longer (ϳ28 Å) as measured from the mononuclear iron to the surface of the protein (19).…”