Subunit structure of oxygenase component in benzoate-1,2-dioxygenase system from Pseudomonas arvilla C-1.

Benzoate-1,2-dioxygenase system from Pseudomonas arvilla C-1 consists of two protein components, benzoate-1,2-dioxygenase reductase and benzoate-1,2-dioxygenase (Yamaguchi, M., and Fujisawa, H. (1980) J. Biol. Chem. 255, 5058-5063). Benzoate-1,2-dioxygenase exhibited two protein bands (alpha and beta) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their molecular weights were estimated to the 50,000 and 20,000, respectively. The intensities of protein staining on polyacrylamide gels suggested that these two subunits were present in equimolar quantities in benzoate-1,2-dioxygenase. Molecular weight of benzoate-1,2-dioxygenase was estimated to be 201,000 by sedimentation equilibrium (Yphantis method). The values of molecular weights of native enzyme and its subunits suggested that the subunit structure of benzoate-1,2-dioxygenase may be alpha 3 beta 3. Cross-linking experiments also suggested the same subunit structure. These two subunits were separated from each other by Ultrogel AcA44 chromatography in the presence of 6 M urea. Amino acid compositions of the two subunits were examined and compared with that of native enzyme. NH2-terminal amino acids of alpha and beta subunits were both serine, and isoelectric points of alpha and beta in the presence of 6 M urea were determined to be pH 5.6 and pH 4.8, respectively. The enzyme contained 8.2 mol of iron and 5.9 mol of labile sulfide/mol of enzyme, suggesting the presence of additional iron atoms besides iron-sulfur clusters. The isolated beta subunit did not contain any significant amounts of iron and labile sulfide, but the alpha subunit contained approximately 2 mol each of iron and labile sulfide and exhibited an absorption spectrum of binuclear iron cluster type.

* This research was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan, and a research grant from the Kuribayashi Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. subunits, the molecular weights of which were estimated to be 50,000 and 20,000, respectively. Furthermore, the ironsulfur cluster was shown to lie on the larger subunit.
Sedimentation Experiments-Ultracentrifuge measurements were carried out in a Hitachi 282 analytical ultracentrifuge. Prior to sedimentation analysis, the sample was applied to a column of Sephadex G-200 equilibrated with 50 rn Tris/HCI buffer, pH 6.8, containing 5% dimethyl sulfoxide, 0.1 M NaC1, and 1 m~ dithiothreitol, and eluted with the same buffer to remove the aggregated forms of the enzyme. Sedimentation equilibrium was carried out according to the method of Yphantis (10) in a three-channel centerpiece. After centrifugation a t 12,000-13,OOO rpm at 4 "C for 24 h, scanning was done a t 280 nm by ultraviolet optics.
Cross-linking Experiments-Cross-linking of benzoate-l,2-dioxygenase with DTBP was carried out in 0.2 M triethanolamine/HCl buffer, pH 8.5 ( l l ) , a t room temperature in a final volume of 100 pl. The reaction was quenched by the addition of 5 p1 of 1 M ammonium acetate solution (8). After incubation for 10 min, 4 pmol of N-ethylmaleimide (in 5 pi of ethanol) were added to prevent disulfide-sulfhy-dry1 exchange. For electrophoresis, 400 pl of 0.1 M sodium borate buffer, pH 8.5, containing 2% SDS, 10% glycerol, and 0.001% bromphenol blue, were added to the reaction mixture, and the mixture was heated for 3 min at 100 "C.
Disc Gel Electrophoresis-For determination of molecular weights of benzoate-1.2-dioxygenase subunits, SDS-polyacrylamide gel electrophoresis was performed at 6 mA/gel for 4 h according to the method of Weber and Osborn (12). The gels were stained with Amido black 10B or Coomassie brilliant blue (R-250) and then scanned at 580 nm by ISCO UA-5 absorbance monitor. For analysis of cross-linked enzyme, electrophoresis was performed at 8 mA/gel for 2 h on 5% polyacrylamide gels according to the procedure of Davies and Stark (11). Gels were stained with Coomassie brilliant blue (R-250).
Two-dimensional Gel Electrophoresis-Electrophoresis of the frrst dimension was performed on 5 8 polyacrylamide gels in the presence of 0.1% SDS in glass tubing (12 X 0.25 cm) essentially according to the procedure of Davies and Stark (11). After electrophoresis at 2 mA/gel for 3 h, the cylindrical gel was removed from the glass tube and placed in the slot on top of a discontinuous slab gel system similar to that of Laemmli (13). It consisted of a 376 polyacrylamide stacking gel on a 10°C polyacrylamide separating gel (10 x 15 X 0.1 cm). Agarose (1%) containing 62.5 mM Tris/HCl buffer, pH 6.8, 0.1% SDS, 10% 2-mercaptoethanol, and 10% glycerol at 80 "C was added on top of the stacking gel to cover the cylindrical gel. Amino Arid Ana/.vsi.s-Amino acid analysis was performed on a Hitachi KLA-5 automatic amino acid analywr. Samples were hydrolyzed in (i N HCI for 22.48. and 72 h at 110 "C in a vacuum (14). Halfcystine was determined as cvsteic acid after performic acid oxidation (15). Tryptophan was determined spectrophotometrically according to the method of Coodwin and Morton (16).

NH,-terminn/Ancl/.vsis-ldentification of the NH2-terminal amino
acids of benzoate-l;~-dioxvgenase suhunits was made by reaction of the enzyme protein with dansvl chloride followed by acid hydrolysis and two-dimensional thin laver chromatography on polyamide sheets according to the procedure of Gray (17).
Isoelecfric Focusing-Anal\fticaI isoelectric focusing was performed on Sci polyacrylamide gels containing 'L'r Ampholine (mixture of 4 parts of pH :1.5-5.0 Ampholine and 1 part of pH 3.5-10 Ampholine) and 6 M urea, essentially according to the procedure described by Wrigley (18). Electrofocusing was carried out at 200 V for 4 h at 4 "C. Gels were sliced into 2-nun sections and placed into 0.5 ml of water. After standing overnight at 4 "C, the pH of water extract of each piece was measured. In a parallel experiment, gels were stained with Coomassie brilliant blue (G-250) (19).
Determination ofbon-The iron content was determined by using the o-phenanthroline method described bv Massev (20). Ofher Determinations-The concentration of benzoate-1.2-dioxygenase was estimated by measuring the absorbance at 279 nm, taking 39.900 as the molar extinction coefficient ( 7 ) . The protein concentrations of subunits were estimated from the absorbance at 280 nm based on their contents of tryptophan and tyrosine residues (24). All spectrophotometric measurements were carried out with a Shimadzu UV200 recording spectrophotometer.

Molecular Weights-Purified
benzoate-1,2-dioxygenase, which was homogeneous on polyacrylamide disc gel electrophoresis (7), showed two protein bands on gel electrophoresis in 0.1% SDS as shown in Fig. 1A. The molecular weights of the two protein bands were determined to be 50,000 (designated a subunit) and 20,000 (p subunit) according to the method of Weber and Osborn (12) as shown in Fig. 2. When the two protein bands on SDS gel were stained with Amido black 10B or Coomassie brilliant blue (R-250) and scanned with a densitometer at 580 nm to determine the relative amount of protein in each band, a ratio of protein staining of 5:2 for the bands of a subunit and p subunit was obtained, indicating a molar ratio of 1:l for a subunit and p subunit based on their molecular weights of 50,000 and 20,000.
The molecular weight of purified benzoate-1,2-dioxygenase, as determined by low speed sedimentation equilibrium, was reported to be 273,000 (7). However, the purified enzyme was found to have a tendency to aggregate as shown in Fig. 3. When the purified enzyme was stored in a frozen state at -20 "C for 1 month at a concentration of 54 mg/ml, approximately 20% of the enzyme appeared to become aggregated forms. Since the elution position of the aggregates of the enzyme preceded that of /3-galactosidase, the molecular weight of the aggregates appeared to be higher than 520,000. In order to obtain an accurate value of the molecular weight of the enzyme, high speed sedimentation equilibrium analysis (10) of the enzyme was performed immediately after removal of the aggregates by gel filtration. The value, 201,200 k 11,500 (Table I), was considerably lower than that reported previ-Cross-linking Studies-When benzoate-1,2-dioxygenase was treated with DTBP and analyzed by SDS-polyacrylamide gel electrophoresis, the patterns of protein-staining bands, as shown in Fig. 4, were obtained. The molecular weight of each ously. band was estimated to be 43,000, 70,000, 98,000, 120,000, 145,000, 170,000, and 190,000, using cross-linked bovine serum albumin as a standard marker. These values of the molecular weights tentatively suggested that each of the bands might correspond to p2, cup, a2, a$, a3, a$, and a:&, respectively.
When the tube gel which had been electrophoresed for separation of the enzyme cross-linked with DTBP as described above was subjected to electrophoresis of the second dimension as described under "Experimental Procedures," the patterns of protein bands as shown in Fig. 5 were obtained. All which was stored at -20 "C for 1 month at the protein concentration of 54 mg/ml in 50 mM Tris/HCI buffer, pH 6.8, containing 5% dimethyl sulfoxide, 0.1 M NaCI, and 1 mM dithiothreitol. was applied to a column (1.9 X 50 cm) of Sephadex C-200 equilibrated with the same buffer. The column was eluted in fractions of 1 ml, and the absorbance a t 280 nm of each fraction was measured. For calibration of the column the following standard proteins were used: The species assumed to be ap and a2/3 in the first dimension produced protein bands in both positions corresponding to a and / 3 in the second dimension. The two slowest migrating bands in the fust dimension produced protein-staining bands in a position corresponding to p in the second dimension, suggesting that these species might be a:$ and but not ad. These results, taken together with the densitometric observation described above, suggested that the subunit structure of benzoate-1,2-dioxygenase might be a:&. Separation of Suhunits--Benzoate-l,2-dioxygenase was separated into a and p subunits by gel filtration chromatog-  Two-dimensional polyacrylamide gel electrophoresis of benzoate-1.2-dioxygenase cross-linked with DTBP. Benzoate-1.2-dioxvgenase (2 mg/ml) was cross-linked with IITBI' (5 mg/ ml) in 0.2 M triethanolamine/HCI buffer, pH 8.5, at room temperature for 2 h. Two samples of the cross-linked enzyme, each containing 20 p g of protein, were subjected to SIX-polyacrylamide disc gel electrophoresis, and one of the two gels was stained with Coomassie brilliant blue (H-250) ( A ) . The unstained disc gel was placed over a slab gel, and electrophoresis was carried out as described under "Experimental Procedures.'' After electrophoresis of the second dimension. the gel was stained with Coomassie brilliant blue (H-250) (B). raphy on Ultrogel AcA44 in 50 mM Tris/HCl buffer, pH 6.8, containing 2 mM dithiothreitol, 6 M urea, and 5% dimethyl sulfoxide as shown in Fig. 6. Each subunit, a and /I, was eluted in fractions of 38 to 46 and 48 to 58, respectively, and each Subunit Structure of Benzoate-1,2-dioxygenase subunit thus obtained was shown to be free from the other on SDS-polyacrylamide gel electrophoresis (Fig. 1, B and C). Absorbance at 415 nm, presumably due to iron-sulfur cluster, was eluted at a region corresponding to a subunit on Ultrogel AcA44 gel filtration. Iron determination of each fraction of the gel filtration revealed that about 70% of,iron was eluted a t a region corresponding to a subunit, and the remainder was eluted in the column volume. These results, taken together, indicated that an iron-sulfur cluster might lie on a subunit of benzoate-1,2-dioxygenase. Both subunit preparations thus obtained were dialyzed against 50 mM Tris/HCl buffer, pH 6.8, containing 5% dimethyl sulfoxide and 1 mM dithiothreitol, and then used for the studies described below.
Amino Acid Compositions of Subunits-The results of amino acid analyses of a and p subunits are summarized in Table 11. The numbers of a and p subunits in benzoate-1,2-  dioxygenase, m and n, were calculated from the results of their amino acid analyses according to the method of least squares by the following formula.

P = [ N , -(ma + n/3JI2
where Ni is the number of each amino acid residue in the native enzyme, a& is that of a subunit, and pz is that of p subunit. The following two equations must .be satisfied in order to minimize P.

,=I
The values of m and n were calculated to be 2.9 and 2.8, respectively, from the two equations, supporting the contention that the subunit structure of benzoate-1,2-dioxygenase is a&.

NHt-terminal Amino Acid Residues of Subunits-The
NHn-terminal amino acids of both subunits of benzoate-1,2dioxygenase were determined according to the dansylation method as described under "Experimental Procedures." In each case, a single major dansyl-serine was identified after acid hydrolysis, indicating that the NHz termini of both a and p subunits might be serine.
Isoelectric Points of Subunits-Isoelectric focusing of each subunit of benzoate-l,2-dioxygenase on polyacrylamide gel in the presence of 6 M urea revealed a single protein band with an isoelectric point of pH 5.6 for a subunit and pH 4.8 for p subunit. An isoelectric point for native enzyme was reported to be pH 4.5 (7).
Absorption Spectra of Subunits- Fig. 7 shows the visible absorption spectra of benzoate-1,2-dioxygenase and its a subunit. Both measurements were performed at the same concentration of a subunit. The native enzyme exhibited a broad absorption spectrum with maxima at about 325 and 464 nm and with a shoulder at about 560 nm; a subunit also exhibited a broad absorption spectrum with maxima at about 325,415, and 450 nm, presumably due to iron-sulfur cluster of [2Fe-2S] type. Thus, both absorption spectra of the native enzyme and Absorption spectra of benzoate-1,2-dioxygenase and its a subunit. The concentration of benzoate-1,2-dioxygenase (-) was 4.2 p~ and that of a subunit (. . . .) was 12.5 PM in 50 mM Tris/HCl buffer, pH 6.8, containing 5% dimethyl sulfoxide and 1 mM dithiothreitol. subunit appeared to resemble each other both in shape and intensity, suggesting that the visible absorption of benzoate-1,2-dioxygenase might be primarily derived from iron-sulfur cluster on a subunit. In contrast to a subunit, / 3 subunit showed no significant absorption in the visible range.
Iron and Labile Sulfide Contents of Subunits-Iron and labile sulfide contents of benzoate-1,2-dioxygenase and its a and , / 3 subunits are summarized in Table 111. The iron and labile sulfide contents of the native enzyme were calculated to be 8.2 and 5.9 mol/mol of enzyme, based on a molecular weight of 201,000 of the enzyme. The labile sulfide content of a subunit, 1.9 mol/mol, accounted for the total labile sulfide content of the enzyme, based on the finding that the subunit structure of the enzyme is a,&. The value of the iron content of subunit, 1.8 mol/mol, corresponded to approximately 708 of the total iron content of the enzyme. These results provided the evidence for the contention that an iron-sulfur cluster of [2Fe-2S] type might be on each a subunit of benzoate-1,2dioxygenase and, furthermore, suggested that additional iron atoms might be contained in the enzyme. The preparation of , 8 subunit had no significant amounts of both iron and labile sulfide.

DISCUSSION
The benzoate-1,2-dioxygenase system which catalyzes the double hydroxylation of benzoate consists of two protein components, benzoate-1,2-dioxygenase reductase and benzoate-1,2-dioxygenase (3-7). The former is an iron-sulfur flavoprotein containing one FAD and one iron-sulfur cluster of [2Fe-2S] type (5, 6), and the latter is an iron-sulfur protein with iron-sulfur clusters of [2Fe-2S] type (7). In the present study, this iron-sulfur protein, benzoate-1,2-dioxygenase, was shown to be composed of nonidentical subunits which include a larger iron-sulfur cluster-containing polypeptide ( a ) with a molecular weight of 50,000 and a smaller polypeptide (p) with a molecular weight of 20,000.
The molecular weight estimation (Table I), SDS-polyacrylamide gel electrophoresis (Fig. 2), cross-linking studies (Figs.  4 and 5 ) , and amino acid analyses (Table 11), taken together, strongly suggested that the subunit structure of benzoate-1,2dioxygenase might be a:&. Since the majority of proteins consisting of nonidentical subunits are known to be the dimers and tetramers (25), benzoate-1,2-dioxygenase appears to be a rare case. Toluene dioxygenase which catalyzes the double hydroxylation of toluene has been reported to be composed of two nonidentical subunits, one with a molecular weight of 52,500 and the other with a molecular weight of 20,800 (26). Although the subunit structure of toluene dioxygenase has not been reported, this enzyme appears to exist as an a& form, taking into account its molecular weight of 151,000 (26).
Each subunit of benzoate-1,2-dioxygenase was obtained without contamination of the other by gel filtration in the presence of 6 M urea (Fig. 6). The preparation of CY subunit thus obtained appeared to still retain iron-sulfur cluster, judging from the contents of iron and labile sulfide (Table 111) and the visible absorption spectrum (Fig. 7). The absorption spectrum of a subunit was very similar to those of spinach ferredoxin (27) and adrenodoxin (28), indicating that the absorption might be derived from iron-sulfur cluster of [2Fe-2S] type bound to a subunit. Thus, an iron-sulfur cluster of [2Fe-2S] type appeared to be attached to each cy subunit as a prosthetic group of benzoate-l,2-dioxygenase.
The finding that benzoate-1,2-dioxygenase contained 8.2 mol of iron and 5.9 mol of labile sulfide/mol of enzyme suggested the presence of additional iron atoms besides ironsulfur clusters. The amounts of the additional iron atoms, 2.3 mol/mol of enzyme, corresponded to approximately 30% of the total iron atoms of the enzyme. Gel filtration of benzoate-1,2-dioxygenase in the presence of 6 M urea produced , 6' subunit, a subunit which still retained about 2 mol each of iron and labile sulfide (Table III), and unbound iron, the amounts of which corresponded to about 30% of total iron of the enzyme (Fig. 6). These, together with the subunit structure of a&, suggested that the enzyme might contain three iron atoms in addition to three iron-sulfur clusters as prosthetic groups. The terminal oxygenase of 4-methoxybenzoate-Odemethylase system has been reported to have iron-sulfur clusters of [2Fe-2S] type and high spin ferric ions as active cofactors (29,30). It is, therefore, reasonably assumed that benzoate-1,2-dioxygenase may be composed of three active units, each consisting of one iron atom, one [2Fe-2S]-a subunit, and one , B subunit.