The phthalate dioxygenase system, which catalyzes the dihydroxylation of phthalate to form its cis-dihydrodiol (DHD), has two components: phthalate dioxygenase (PDO), a multimer with one Rieske-type [2Fe-2S] and one Fe(II) center per monomer, and phthalate dioxygenase reductase (PDR), which contains flavin mononucleotide (FMN) and a plant-like ferredoxin [2Fe-2S] center. PDR is responsible for transferring electrons from NADH to the Rieske center of PDO, and the Rieske center supplies electrons to the mononuclear center for the oxygenation of substrate. Reduced PDO (PDO(red)) that lacks Fe(II) at the mononuclear metal site (PDO-APO) reacts slowly with O(2) (1.4 x 10(-3) s(-1) at 125 microM O(2) and 22 degrees C), presumably in a direct reaction with the Rieske center. Binding of phthalate and/or PDR(ox) to reduced PDO-APO increases the reactivity of the Rieske center with O(2). When no PDR or phthalate is present, the oxidation of the Rieske center in native PDO(red) [which contains Fe(II) at the mononuclear site] occurs in two phases (approximately 1 and 0.1 s(-1) at 125 mM O(2), 23 degrees C), both much faster than in the absence of Fe(II), presumably because in this case O(2) reacts at the mononuclear Fe(II). Addition of PDR(ox) to native PDO(red) resulted in a large fraction of the Rieske center being oxidized at 5 s(-1), and the addition of phthalate resulted in about 70% of the reaction proceeding at 42 s(-1). With both PDR(ox) and phthalate present, most of the PDO(red) (approximately 80-85%) oxidizes at 42 s(-1), with the remaining oxidizing at approximately 5 s(-1). Thus, the binding of phthalate or PDR(ox) to PDO(red) each results in greater reactivity of PDO with O(2). The presence of both the substrate and PDR was synergistic, making PDO fully catalytically active. A model that explains the observed effects is presented and discussed in terms of PDO subunit cooperativity. It is proposed that, during oxidation of reduced PDO, each of two Rieske centers on separate subunits transfers an electron to the Fe(II) mononuclear center on a third subunit. This explanation is consistent with the observed multiphasic kinetics of the oxidation of the Rieske center and is being further tested by product analysis experiments.

The phthalate dioxygenase system, a Rieske non-heme iron dioxygenase, catalyzes the dihydroxylation of phthalate to form the 4,5-dihydro-cis-dihydrodiol of phthalate (DHD). It has two components: phthalate dioxygenase (PDO), a multimer with one Rieske-type [2Fe-2S] and one mononuclear Fe(II) center per monomer, and a reductase (PDR) that contains flavin mononucleotide (FMN) and a plant-type ferredoxin [2Fe-2S] center. This work shows that product formation in steady-state reactions is tightly coupled to electron delivery, with 1 dihydrodiol (DHD) of phthalate formed for every 2 electrons delivered from NADH. However, in reactions of reduced PDO with O(2), only about 0.5 DHD is formed per Rieske center that becomes oxidized. Although the product forms rapidly, its release from PDO is slow in these reactions with oxygen that do not include reductase and NADH. EPR data show that, at the completion of the oxidation, iron in the mononuclear center remains in the ferrous state. In contrast, naphthalene dioxygenase (NDO) [Wolfe, M. D., Parales, J. V., Gibson, D. T., and Lipscomb, J. D. (2001) J. Biol. Chem. 276, 1945-1953] and benzoate dioxygenase (BZDO) [Wolfe, M. D., Altier, D. J., Stubna, A., Popescu, C. V., Munck, E., and Lipscomb, J. D. (2002) Biochemistry, 41, 9611-9626], related Rieske non-heme iron dioxygenases, form 1 DHD per Rieske center oxidized, and the mononuclear center iron ends up ferric. Thus, both electrons from reduced NDO and BZDO monomers are used to form the product, whereas only the reduced Rieske centers in PDO become oxidized during production of DHD. This emphasizes the importance of PDO subunit interaction in catalysis. Electron redistribution was practically unaffected by the presence of oxidized PDR. A scheme is presented that emphasizes some of the differences in the mechanisms involved in substrate hydroxylation employed by PDO and either NDO or BZDO.

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.

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...

The structural basis of the regulation of microsomal cytochrome P450 (P450) activity was investigated by mutating the highly conserved heme binding motif residue, Phe429, on the proximal side of cytochrome P450 2B4 to a histidine. Spectroscopic, pre-steady-state and steady-state kinetic, thermodynamic, theoretical, and structural studies of the mutant demonstrate that formation of an H-bond between His429 and the unbonded electron pair of the Cys436 axial thiolate significantly alters the properties of the enzyme. The mutant lost >90% of its activity; its redox potential was increased by 87 mV, and the half-life of the oxyferrous mutant was increased ∼37-fold. Single-crystal electronic absorption and resonance Raman spectroscopy demonstrated that the mutant was reduced by a small dose of X-ray photons. The structure revealed that the δN atom of His429 forms an H-bond with the axial Cys436 thiolate whereas the εN atom forms an H-bond with the solvent and the side chain of Gln357. The amide of Gly438 forms the only other H-bond to the tetrahedral thiolate. Theoretical quantification of the histidine–thiolate interaction demonstrates a significant electron withdrawing effect on the heme iron. Comparisons of structures of class I–IV P450s demonstrate that either a phenylalanine or tryptophan is often found at the location corresponding to Phe429. Depending on the structure of the distal pocket heme, the residue at this location may or may not regulate the thermodynamic properties of the P450. Regardless, this residue appears to protect the thiolate from solvent, oxidation, protonations, and other deleterious reactions.

RBCs, even from similar donors, vary significantly in levels and changes of both AH and MF, the clinical significance of which must still be ascertained. While further study is needed, donors with severe hypertriglyceridemia may not be appropriate as blood donors due to the unacceptable level of hemolysis observed during storage of our affected study subject.

Phthalate dioxygenase (PDO), a hexamer with one Rieske-type [2Fe-2S] and one Fe (II) -mononuclear center per monomer, and its reductase (PDR), which contains flavin mononucleotide and a plant-type ferredoxin [2Fe-2S] center, are expressed by Burkholderia cepacia at ∼30 mg of crude PDO and ∼1 mg of crude PDR per liter of cell culture when grown with phthalate as the main carbon source. A high level expression system in Escherichia coli was developed for PDO and PDR. Optimization relative to Escherichia coli cell line, growth parameters, time of induction, media composition, and iron-sulfur additives resulted in yields of about 1 g/L for PDO and about 0.2 g/L for PDR. Protein expression was correlated to the increase in pH of the cell culture and exhibited a pronounced (variable from 5 to 20 hours) lag after the induction. The specific activity of purified PDO did not depend on the pH of the cell culture when harvested. However, when the pH of the culture reached 8.5-9, a large fraction of the PDR that was expressed lacked its ferredoxin domain, presumably because of proteolysis. Termination of growth while the pH of the cell culture was < 8 decreased the fraction of proteolyzed enzyme, whereas yields of the unclipped PDR were only marginally lower. Overall, changes in pH of the cell culture were found to be an excellent indicator of the overall level of native protein expression. Its monitoring allowed the real time tracking of the protein expression and made it possible to tailor the expression times to achieve a combination of high quality and high yield of protein.Among the various systems used for the expression of recombinant proteins, Escherichia coli has the advantage of being available in a wide array of mutant host strains, the ability to grow rapidly and to high density, of being better characterized genetically than other microorganisms, of having many compatible expression plasmids, and it gives good yields of target proteins. Considerable attention has been paid to the improvement of E. coli systems for expression of complex eukaryotic proteins (1,2). However, expression has been especially troublesome for proteins containing Fe-S clusters that are involved in a number of physiological processes including catalysis, electron transfer, biosynthesis, DNA repair and transcriptional regulation, and sensing for regulatory processes (3). In many cases supplementation of the growth media with Fe 2+ and S 2− was not sufficient for good expression (3). The formation of Fe-S clusters was found to depend on the so-called isc (iron-sulfur cluster) genes (4,5), all nine individual components of which are important for the expression of some active ). In a number of cases it was found that coexpression of the isc cluster was essential for achieving high yields of active recombinant Fe-S proteins (11-13). Cluster inactivation in E. coli resulted in a marked decrease in the production of native and recombinant iron-sulfur * To whom correspondence should be addressed. Email: dballou@umich.edu Publisher's Dis...

Mammalian cytochrome P450 2B4 (CYP2B4) is a phenobarbital-inducible rabbit hepatic monooxygenase that catalyzes the N-demethylation of benzphetamine and metabolism of numerous other compounds. To probe the interactions of the heme environment and bound benzphetamine with the dioxygen (O2) complex of CYP2B4, homogeneous O2 complexes of the wild-type enzyme and three mutants at sites of conserved amino acids, two on the heme distal side (T302A and E301Q) and one on the proximal side (F429H), have been prepared and stabilized at ~−50 °C in mixed solvents (60–70% v/v glycerol). We report that the magnetic circular dichroism and electronic absorption spectra of wild-type oxyferrous CYP2B4, in the presence and absence of substrate, are quite similar to those of the dioxygen complex of bacterial cytochrome P450-CAM (CYP101). However, the oxyferrous complexes of the T302A and E301Q CYP2B4 mutants have significantly perturbed electronic structure (~4 nm and ~3 nm red-shifted Soret features, respectively) compared to that of the wild-type oxyferrous complex. On the other hand, the heme proximal side mutant, CYP2B4 F429H, undergoes relatively facile conversion to a partially (~50%) denatured (P420) form upon reduction. The structural changes in the heme pocket environments of the CYP2B4 mutants that lead to the spectroscopic distinctions reported herein can be related to the differences in oxidation activities of wild-type CYP2B4 and its E301Q, T302A and F429H mutants.