Ethylbenzene Dehydrogenase, a Novel Hydrocarbon-oxidizing Molybdenum/Iron-Sulfur/Heme Enzyme

The initial enzyme of ethylbenzene metabolism in denitrifying Azoarcus strain EbN1, ethylbenzene dehydrogenase, was purified and characterized. The soluble periplasmic enzyme is the first known enzyme oxidizing a nonactivated hydrocarbon without molecular oxygen as cosubstrate. It is a novel molybdenum/iron-sulfur/heme protein of 155 kDa, which consists of three subunits (96, 43, and 23 kDa) in an αβγ structure. The N-terminal amino acid sequence of the α subunit is similar to that of other molybdenum proteins such as selenate reductase from the related speciesThauera selenatis. Ethylbenzene dehydrogenase is unique in that it oxidizes the hydrocarbon ethylbenzene, a compound without functional groups, to (S)-1-phenylethanol. Formation of the product was evident by coupling to an enantiomer-specific (S)-1-phenylethanol dehydrogenase from the same organism. The apparent K m of the enzyme for ethylbenzene is very low at <2 μm. Oxygen does not affect ethylbenzene dehydrogenase activity in extracts but inactivates the purified enzyme, if the heme b cofactor is in the reduced state. A variant of ethylbenzene dehydrogenase exhibiting significant activity also with the homolog n-propylbenzene was detected in a relatedAzoarcus strain (PbN1). The initial enzyme of ethylbenzene metabolism in denitrifying Azoarcus strain EbN1, ethylbenzene dehydrogenase, was purified and characterized. The soluble periplasmic enzyme is the first known enzyme oxidizing a nonactivated hydrocarbon without molecular oxygen as cosubstrate. It is a novel molybdenum/iron-sulfur/heme protein of 155 kDa, which consists of three subunits (96, 43, and 23 kDa) in an αβγ structure. The N-terminal amino acid sequence of the α subunit is similar to that of other molybdenum proteins such as selenate reductase from the related speciesThauera selenatis. Ethylbenzene dehydrogenase is unique in that it oxidizes the hydrocarbon ethylbenzene, a compound without functional groups, to (S)-1-phenylethanol. Formation of the product was evident by coupling to an enantiomer-specific (S)-1-phenylethanol dehydrogenase from the same organism. The apparent K m of the enzyme for ethylbenzene is very low at <2 μm. Oxygen does not affect ethylbenzene dehydrogenase activity in extracts but inactivates the purified enzyme, if the heme b cofactor is in the reduced state. A variant of ethylbenzene dehydrogenase exhibiting significant activity also with the homolog n-propylbenzene was detected in a relatedAzoarcus strain (PbN1). polyacrylamide gel electrophoresis inductively coupled plasma optical emission spectroscopy Three bacterial species capable of anaerobic degradation of the aromatic hydrocarbon ethylbenzene are known to date. All of these are denitrifying bacteria that belong to the genus Azoarcus of the β-proteobacteria. For one of these strains, Azoarcussp. EB-1, ethylbenzene is the only known hydrocarbon utilized as growth substrate (1Ball H.A. Johnson H.A. Reinhard M. Spormann A.M. J. Bacteriol. 1996; 178: 5755-5761Crossref PubMed Google Scholar). The other two strains utilize either ethylbenzene or an alternative hydrocarbon compound, namely toluene (strain EbN1) orn-propylbenzene (strain PbN1) (2Rabus R. Widdel F. Arch. Microbiol. 1995; 163: 96-103Crossref PubMed Scopus (312) Google Scholar). The proposed pathway of anaerobic degradation of ethylbenzene by these bacteria is shown in Fig. 1. It is initiated by a novel biochemical reaction, namely an oxygen-independent oxidation of ethylbenzene to (S)-1-phenylethanol. This intermediate is then oxidized further to acetophenone by an alcohol dehydrogenase (1Ball H.A. Johnson H.A. Reinhard M. Spormann A.M. J. Bacteriol. 1996; 178: 5755-5761Crossref PubMed Google Scholar, 2Rabus R. Widdel F. Arch. Microbiol. 1995; 163: 96-103Crossref PubMed Scopus (312) Google Scholar, 3Rabus R. Heider J. Arch. Microbiol. 1998; 170: 377-384Crossref Scopus (125) Google Scholar, 4Johnson H.A. Spormann A.M. J. Bacteriol. 1999; 181: 5662-5668Crossref PubMed Google Scholar). Activities of an ethylbenzene-oxidizing enzyme and an enantio-specific (S)-1-phenylethanol dehydrogenase have been reported in cell extracts of strain EB-1 (4Johnson H.A. Spormann A.M. J. Bacteriol. 1999; 181: 5662-5668Crossref PubMed Google Scholar), and a substrate-specific (S)-1-phenylethanol dehydrogenase has been purified and characterized from strain EbN1. 1O. Kniemeyer and J. Heider, submitted for publication. The intermediate acetophenone is apparently degraded further by carboxylation to benzoylacetate to yield benzoyl-CoA and acetyl-CoA eventually (Fig. 1; for review, see Ref. 5Heider J. Spormann A.M. Beller H.R. Widdel F. FEMS Microbiol. Rev. 1999; 22: 459-473Crossref Google Scholar). The catabolic pathway ofn-propylbenzene in strain PbN1 is supposed to be analogous to that of ethylbenzene, yielding benzoyl-CoA and propionyl-CoA as intermediates (2Rabus R. Widdel F. Arch. Microbiol. 1995; 163: 96-103Crossref PubMed Scopus (312) Google Scholar). Toluene degradation in strain EbN1 proceeds via a completely different pathway and involves the formation of benzylsuccinate from toluene and fumarate as initial reaction (3Rabus R. Heider J. Arch. Microbiol. 1998; 170: 377-384Crossref Scopus (125) Google Scholar, 6Biegert T. Fuchs G. Heider J. Eur. J. Biochem. 1996; 238: 661-668Crossref PubMed Scopus (220) Google Scholar,7Beller H.R. Spormann A.M. J. Bacteriol. 1997; 179: 670-676Crossref PubMed Google Scholar). In this report, we analyze the biochemical properties of the first enzyme of anaerobic ethylbenzene metabolism, ethylbenzene dehydrogenase. The enzyme was purified and shown to be a new periplasmic molybdenum/iron-sulfur/heme protein that oxidizes ethylbenzene stereospecifically to (S)-1-phenylethanol. We also provide evidence that the same enzyme catalyzes anaerobic oxidation of ethylbenzene and n-propylbenzene. Strain EbN1 was isolated previously from an enrichment culture on ethylbenzene by Rabus and Widdel (2Rabus R. Widdel F. Arch. Microbiol. 1995; 163: 96-103Crossref PubMed Scopus (312) Google Scholar). Growth of the bacteria in 1–2-liter scale cultures was performed as described previously (2Rabus R. Widdel F. Arch. Microbiol. 1995; 163: 96-103Crossref PubMed Scopus (312) Google Scholar, 3Rabus R. Heider J. Arch. Microbiol. 1998; 170: 377-384Crossref Scopus (125) Google Scholar). Cells were grown by subsequent transfer for at least 30 generations on the same substrate prior to harvesting for the described experiments. Harvesting was performed anoxically while the cultures were in the exponential growth phase. Fermenter cultures (200 liters) were set up as described previously (3Rabus R. Heider J. Arch. Microbiol. 1998; 170: 377-384Crossref Scopus (125) Google Scholar) and run in fed-batch mode with a growth-limiting and exponentially increasing feeding rate of nitrate and discontinuous supply of ethylbenzene. Growth rates of 0.015–0.025 h−1 and cell yields of 200–300 g (wet mass)/fermenter were usually obtained. Extract preparation was usually performed aerobically. Cells (10 g, wet mass) were suspended in 10 ml of water and passed through a French pressure cell at 137 megapascals. Cell debris and membranes were removed by ultracentrifugation (1 h at 100,000 × g). Washed membrane fractions were prepared from the supernatant of a 20,000 × g centrifugation step, which was centrifuged at 100,000 × g for 1 h. The pellet was washed and resuspended in the same volume of basal buffer (10 mm Tris-Cl, 1 mm MgCl2, 10% glycerol, pH 7.5). For anaerobic extract preparation, all solutions were degassed and stored under nitrogen, and all handling steps were performed in an anaerobic glove box as described earlier (3Rabus R. Heider J. Arch. Microbiol. 1998; 170: 377-384Crossref Scopus (125) Google Scholar). Strain PbN1 (2Rabus R. Widdel F. Arch. Microbiol. 1995; 163: 96-103Crossref PubMed Scopus (312) Google Scholar) was grown in 2-liter bottles under the same conditions as described for strain EbN1. The hydrocarbon substrates were added to the cultures in an inert carrier phase (2,2,4,4,6,8,8-heptamethylnonane) containing 2% (v/v) ethylbenzene or 4% (v/v) n-propylbenzene. Shortest doubling times of 10.5 h on ethylbenzene and 12 h on n-propylbenzene were recorded. Ethylbenzene dehydrogenase was routinely assayed in 100 mm Tris-Cl buffer (pH 7.5) containing 0.2 mm ferricenium hexafluorophosphate as electron acceptor. Enzyme solution was added, and the reactions were started by adding ethylbenzene or n-propylbenzene (final concentration, 100 μm) from saturated aqueous solutions, which contained 2 mm ethylbenzene (8Dean J.A. Lange's Handbook of Chemistry. 14th Ed. McGraw-Hill Inc., New York1992: 1.202Google Scholar) or 1 mm n-propylbenzene (9Mackay D. Shiu W.Y. J. Phys. Chem. Ref. Data. 1981; 10: 1175-1199Crossref Scopus (685) Google Scholar). Decrease of absorption of the ferricenium ion was followed at 290 nm (Δε = 9,000m−1cm−1). The tests were routinely performed under aerobic conditions because identical activities were observed in control tests under anaerobic conditions. To assess the pH optimum of ethylbenzene dehydrogenase, the enzyme assay was also performed in sodium phosphate buffers within a pH range of 6.0–8.0. Alternative assays for ethylbenzene oxidation were set up with 0.1 mmdichlorophenol indophenol as electron acceptor in the presence and absence of the redox mediator phenazine methosulfate (0.05 mm). These tests were performed under anaerobic conditions as described above and were monitored for dichlorophenol indophenol reduction at 546 nm. Reversibility of the ethylbenzene dehydrogenase reaction was tested under strictly anaerobic conditions in 100 mm Tris-Cl buffer (pH 7.5), containing 1 mmmethyl viologen and 0.5 mm dithionite. Oxidation of reduced methyl viologen was followed at 710 nm (ε = 2,400m−1cm−1). After adding the enzyme, the reaction was started by adding 1 mm (S)-1-phenylethanol. The same buffer was also used to assess the purified enzyme for possible selenate reductase or nitrate reductase activities. In these cases, the reaction was started by the addition of 1 mmrespective electron acceptor. (S)-1-Phenylethanol dehydrogenase activity was assayed in 100 mm Tris-Cl buffer (pH 7.5) containing 2 mm MgCl2, 0.5 mm NAD, and 1 mm (S)-1-phenylethanol and enzyme. Malate dehydrogenase activity was measured in 100 mm potassium phosphate buffer (100 mm, pH 7) containing 0.25 mm NADH, 0.2 mm oxaloacetate, and cell extract. Reduction of NAD+ or oxidation of NADH was followed photometrically at 365 nm (ε = 3.4 mm−1cm−1). All column chromatography steps were performed in an anaerobic glove box with an FPLC System (Amersham Pharmacia Biotech). Extract of ethylbenzene-grown cells of strain EbN1 (20 ml of a 100,000 × g supernatant) was applied to a DEAE-Sepharose column (Amersham Pharmacia Biotech; 2.2-cm diameter, 50-ml volume), which had been equilibrated with basal buffer (2 mm Tris acetate buffer, pH 8.0, and 10% w/v glycerol). The column was washed at a flow rate of 5 ml min−1for 2 column volumes and eluted with a gradient from 0 to 50 mm KCl in basal buffer over 500 ml. Fractions of 7 ml were collected. Ethylbenzene dehydrogenase activity eluted in a volume of 80 ml between 40 and 50 mm KCl. A yield of 77% and an enrichment factor of 20 were obtained after this step (see Table III). The active fractions were applied on a ceramic hydroxyapatite column (10 ml; Bio-Rad, Hercules, CA), which had been equilibrated with basal buffer. The column was washed with 2 volumes of basal buffer. A gradient over 100 ml was then applied from 0 to 300 mmpotassium phosphate, and fractions of 5 ml were collected. Enzyme activity eluted in a volume of 40 ml when 160–250 mmpotassium phosphate was applied. Active fractions were pooled, and ethylbenzene dehydrogenase was concentrated by ammonium under anaerobic conditions of ammonium of ethylbenzene is in a new A is Cells of strain EbN1 were grown and as described were by a of J. Microbiol. Scopus Google Scholar, J. Bacteriol. PubMed Google Scholar). cells g, wet mass) were resuspended in ml of buffer mm Tris-Cl, pH mm and × were added, and the was on for to proteins were prepared by centrifugation of the for 30 at × of the periplasmic proteins were in the the pellet contained the These were washed in buffer, suspended in ml of buffer (20 mm Tris-Cl, 10 mm MgCl2, 10% glycerol, pH containing 10 and by one through a French pressure The membrane and soluble fractions of the cell were by centrifugation at 100,000 × were to in and Inc., New Scholar) or by the in and Inc., New Scholar) with as and discontinuous was performed in polyacrylamide to in and Inc., New Scholar). were dehydrogenase, and were by the (Amersham Pharmacia Biotech). were with a in fractions was and as described Scopus Google Scholar). The molecular of ethylbenzene dehydrogenase was by gel on a column (Amersham Pharmacia and by of purified enzyme on polyacrylamide containing different polyacrylamide between and were and and the and of were used as for a in and Inc., New Scholar). of Chem. Scopus Google Scholar), J. Chem. Scholar), and Biochem. PubMed Scopus Google Scholar) was performed by a of in purified enzyme was performed by inductively coupled plasma optical emission spectroscopy a at the of of For protein cell extract or purified enzyme was by and on a membrane a as described in J. Chem. PubMed Google Scholar). on membrane were by The proteins were to degradation with yields of hexafluorophosphate was a Biochem. PubMed Scopus Google all other were from or and were of the A enzyme assay was for the first enzyme of anaerobic ethylbenzene degradation of strain EbN1, ethylbenzene dehydrogenase. The electron dichlorophenol indophenol or phenazine methosulfate were tested without for coupling to ethylbenzene significant activity of an ethylbenzene dehydrogenase was detected in extracts of ethylbenzene-grown cells with the ferricenium as electron acceptor. The assay was on the of and a pH optimum of was activities were obtained under and strictly that molecular oxygen is not for ethylbenzene in activity was when extracts were to pH or to pH prior to the enzyme Ethylbenzene dehydrogenase activity was detected in the soluble after 100,000 activity was in washed membrane cell an of was that ethylbenzene is oxidized to acetophenone in these a of the ethylbenzene oxidation rate in 100,000 × g was of This is to a substrate degradation rate of of in cells at the of harvesting low ethylbenzene dehydrogenase activity was in extracts of cells grown on or or on and in cells activity was also observed when ethylbenzene was by n-propylbenzene in the assays with ethylbenzene-grown only at of the activity measured with ethylbenzene dehydrogenase and n-propylbenzene dehydrogenase activities in 100,000 × g extracts of grown cells of Azoarcus strains EbN1 and activity activity was measured in washed membrane The are of at least two were not in a new activity was measured in washed membrane The are of at least two were not Cells of strain which were grown in 2-liter cultures on ethylbenzene or were also tested for dehydrogenase activities for ethylbenzene cells of strain PbN1 contained two times activities of ethylbenzene dehydrogenase observed in strain EbN1 In extracts of strain n-propylbenzene was oxidized at a rate of of that measured with ethylbenzene. strain PbN1 was grown on the activity of the enzyme was but the of the rates or ethylbenzene as substrates was the same as with ethylbenzene-grown cells These that ethylbenzene and n-propylbenzene are oxidized by a enzyme. activities for substrates were to the observed growth rates of strain PbN1 on either cells of strain EbN1 contained of which were in cells grown on the intermediate of the The in cell extracts by are shown in Fig. other were also observed in cells grown on or acetophenone with cells (Fig. of the catabolic of anaerobic ethylbenzene of cells of strain PbN1 in of identical were observed in cells grown on either hydrocarbon substrate with cells (Fig. in extracts of ethylbenzene-grown cells strain EbN1 were by chromatography on DEAE-Sepharose in 10 mm Tris-Cl buffer (pH 7.5). these of ethylbenzene dehydrogenase activity was in the and (S)-1-Phenylethanol from the cell extract were on the of the first two of ethylbenzene the we the oxidation of ethylbenzene to as detected in cell is by ethylbenzene dehydrogenase or it on the subsequent (S)-1-phenylethanol dehydrogenase Enzyme assays with ethylbenzene dehydrogenase containing which are of (S)-1-phenylethanol dehydrogenase, an of The to when the tests were with NAD+ and purified (S)-1-phenylethanol and at when the same was performed in the absence of ethylbenzene dehydrogenase catalyzes a oxidation of ethylbenzene, and stereospecifically which is oxidized to acetophenone by (S)-1-phenylethanol dehydrogenase in cell extract. The NADH by the alcohol dehydrogenase is apparently by an with ferricenium as electron acceptor. After from cells of ethylbenzene-grown strain EbN1 by in buffer, of the ethylbenzene dehydrogenase was the The periplasmic protein was also in these as from of the enzyme dehydrogenase was in the of (S)-1-phenylethanol dehydrogenase activities in the fractions that of this enzyme was and was in the This that ethylbenzene oxidation to (S)-1-phenylethanol in the further oxidation of (S)-1-phenylethanol to acetophenone in the This is by the of ethylbenzene oxidation ferricenium reduction in the different In an of was coupling of the ethylbenzene dehydrogenase activity with (S)-1-phenylethanol dehydrogenase, the periplasmic fractions an of of ethylbenzene dehydrogenase in Azoarcus strain were grown in the presence of nitrate and ethylbenzene. activities are in of 1 of at least two are were in a new Cells were grown in the presence of nitrate and ethylbenzene. activities are in of 1 of at least two are were ethylbenzene dehydrogenase not to DEAE-Sepharose when 10 mm Tris-Cl buffer (pH 7.5) was the buffer was to a 2 mm Tris acetate buffer (pH 7.5). these the enzyme to the and was then eluted from the column by a KCl enzyme activity in extracts was not by enzyme from the first column activity under conditions. of the enzyme was performed under which in yields of and an enrichment factor of 20 after the first column on ceramic hydroxyapatite was performed as The enzyme eluted from the DEAE-Sepharose column was applied on this column and eluted by a potassium phosphate gradient (pH 7.5). A enrichment factor of at a yield of was obtained after this The enzyme was after the as shown by A of the is in Ethylbenzene dehydrogenase consists of three subunits of 43, and 23 kDa, as by of the purified enzyme (Fig. The apparent molecular of the enzyme was as 155 by gel and of polyacrylamide These are with an αβγ of the enzyme. The N-terminal amino acid of the three subunits were from enzyme that had been by The of the α subunit of purified ethylbenzene dehydrogenase was of the and subunits were obtained. a sequence of the α subunit was obtained when the was from cell extracts and used for The N-terminal amino acid sequence of the α subunit was similar to that of other such as selenate reductase of or nitrate with nitrate reductase subunit 1 of The obtained for the other subunits and not significant with known of and were in purified ethylbenzene dehydrogenase. in ethylbenzene dehydrogenase was as 0.1 of by and as 0.1 of by was at a of 2 of with 1 of of by of 12 of These are with the presence of one and one heme in ethylbenzene dehydrogenase, as reported previously for selenate reductase of T. T. J. Chem. 1997; PubMed Scopus Google Scholar, T. F. 10: PubMed Scopus Google Scholar). detected by in significant were and but further or were in purified enzyme. of purified ethylbenzene dehydrogenase a The of the purified enzyme a nm and absorption at and which the presence of a reduced heme b After anaerobic oxidation of the enzyme by of ferricenium the α and of the heme at and nm and the at nm was to nm The of reduced and oxidized enzyme was of the presence of a heme b cofactor (Fig. of of ethylbenzene to enzyme in of the of reduced enzyme (Fig. The of the enzyme and the enzyme obtained from the column were that ethylbenzene dehydrogenase was purified in the completely reduced of the reduced enzyme with 0.2 mm not in further reduction of the heme cofactor but in further of the absorption between and 500 nm (Fig. This is of the presence of in ethylbenzene dehydrogenase. These are not completely reduced by the substrate and a such as for The heme of the enzyme was as from enzyme, an of the α Eur. J. Biochem. 1996; PubMed Scopus Google Scholar). ethylbenzene dehydrogenase the oxidation of ethylbenzene to (S)-1-phenylethanol. The activity for purified enzyme to a very low of a similar to that for benzoyl-CoA reductase and M. Fuchs G. Eur. J. Biochem. 1995; PubMed Scopus Google Scholar, Fuchs G. Eur. J. Biochem. 1999; PubMed Scopus Google Scholar). Ethylbenzene dehydrogenase activity was saturated at low ethylbenzene which the of m for ethylbenzene. of <2 for the K m was from the substrate under conditions see Fig. of the activity with ethylbenzene was obtained was used as The of activities with the two substrates not between cell extract and purified enzyme. aromatic such as or were not oxidized by ethylbenzene dehydrogenase. The of ethylbenzene dehydrogenase to the reaction was tested by an anaerobic enzyme assay with reduced methyl viologen as electron and (S)-1-phenylethanol as reduction of (S)-1-phenylethanol to ethylbenzene was detected by this that the reaction of ethylbenzene dehydrogenase is under conditions. Ethylbenzene dehydrogenase also not methyl reduction of selenate or the of the N-terminal of the α Ethylbenzene dehydrogenase was not in assays containing sodium or sodium (1 mm of to the assay buffer a reaction, by from the ferricenium a but enzyme activities were after with ethylbenzene. ethylbenzene dehydrogenase activity in cell extracts was not by aerobic extract preparation and in for up to 12 purified enzyme, which was apparently in the reduced (see was by in with a of 7 This was by addition of the electron acceptor ferricenium hexafluorophosphate (1 to the enzyme these of the enzyme activity was after 2 h and after a in ferricenium has been shown to the heme cofactor of ethylbenzene dehydrogenase to the oxidized it be that the reduced heme is for by The enzyme is oxidized in cell extracts by electron and oxygen when these electron are removed The pathway of anaerobic ethylbenzene metabolism is initiated by two oxidation steps of ethylbenzene to (S)-1-phenylethanol and further to acetophenone (1Ball H.A. Johnson H.A. Reinhard M. Spormann A.M. J. Bacteriol. 1996; 178: 5755-5761Crossref PubMed Google Scholar, 2Rabus R. Widdel F. Arch. Microbiol. 1995; 163: 96-103Crossref PubMed Scopus (312) Google Scholar, 3Rabus R. Heider J. Arch. Microbiol. 1998; 170: 377-384Crossref Scopus (125) Google Scholar). In this we the first enzyme of the ethylbenzene dehydrogenase. To ethylbenzene dehydrogenase is the first described enzyme that catalyzes oxygen-independent of a of an compound without functional of ethylbenzene dehydrogenase was by coupling the reaction to that of purified (S)-1-phenylethanol dehydrogenase. This the product reported previously for a strain (4Johnson H.A. Spormann A.M. J. Bacteriol. 1999; 181: 5662-5668Crossref PubMed Google Scholar). Ethylbenzene dehydrogenase was in cells grown on ethylbenzene, and only very low activities were measured in cells grown on or Three of the described in this molecular identical to of the subunits of ethylbenzene dehydrogenase (96, and 23 Cells of the strain EbN1 contained an enzyme exhibiting n-propylbenzene dehydrogenase activity at of the activity measured with ethylbenzene. In cells of the ethylbenzene and strain PbN1 contained an enzyme exhibiting activity with either ethylbenzene or n-propylbenzene. of activity with the two in cells grown on either substrate that the same enzyme is used for metabolism of ethylbenzene This is by the apparent of the of ethylbenzene grown Ethylbenzene dehydrogenase activity with toluene as which is with that strain EbN1 toluene via a completely different the addition of the methyl to fumarate J. Spormann A.M. Beller H.R. Widdel F. FEMS Microbiol. Rev. 1999; 22: 459-473Crossref Google Scholar, 6Biegert T. Fuchs G. Heider J. Eur. J. Biochem. 1996; 238: 661-668Crossref PubMed Scopus (220) Google Scholar, H.R. Spormann A.M. J. Bacteriol. 1997; 179: 670-676Crossref PubMed Google Scholar). The that ethylbenzene oxidation in the the is oxidized further in the as evident from the of NAD+ as electron acceptor. It is and is the A of as a compound via the membrane The of a periplasmic of ethylbenzene dehydrogenase for the is that of the ethylbenzene is of the The low K m μm) of ethylbenzene dehydrogenase the that the of the enzyme for substrate is an factor in the metabolism of this the redox of the is to from of other ethylbenzene oxidation was only observed in this with an electron acceptor of redox 0 = that redox in the enzyme as as the electron acceptor have redox to oxidation rates with the inert hydrocarbon of in the enzyme a possible for the observed of ethylbenzene in tests with electron methyl 0 = ethylbenzene dehydrogenase is a periplasmic enzyme, a possible acceptor in the Azoarcus strains be The properties of ethylbenzene dehydrogenase from strain EbN1 are in to the reported of ethylbenzene oxidation in strain rates in this strain were reported and the enzyme was and not in ethylbenzene-grown cells (4Johnson H.A. Spormann A.M. J. Bacteriol. 1999; 181: 5662-5668Crossref PubMed Google Scholar). are different of ethylbenzene in different strains of Ethylbenzene dehydrogenase is a new molybdenum/iron-sulfur/heme enzyme, which is of three In to other known it be that the α subunit the molybdenum the subunit the and the the heme The enzyme, which is similar to ethylbenzene dehydrogenase with to subunit cofactor and is the characterized selenate reductase from a related T. J. Chem. 1997; PubMed Scopus Google Scholar, T. F. 10: PubMed Scopus Google Scholar). ethylbenzene dehydrogenase not reduction of selenate or characterized from T. catalyzes a similar reaction with a aromatic but this enzyme is of a heme and apparently catalyzes the oxidation of to with coupling to and without of intermediates Fuchs G. Eur. J. Biochem. 1999; PubMed Scopus Google Scholar). A is the between the of ethylbenzene dehydrogenase under in cell extracts and the of the purified enzyme by We that the enzyme to by anaerobic oxidation of the heme b that the reduced heme in the enzyme oxygen In cell ethylbenzene dehydrogenase is oxidized by with electron acceptor and only reduced when the electron acceptor is removed We G. Fuchs and F. Widdel for and We also of for and and for N-terminal