Identification and characterization of the natural electron donor ferredoxin and of FAD as a possible prosthetic group of benzoyl‐CoA reductase (dearomatizing), a key enzyme of anaerobic aromatic metabolism

Abstract

Under anoxic conditions most aromatic compounds are metabolized via benzoyl‐CoA which becomes reduced by benzoyl‐CoA reductase (dearomatizing); this enzyme was recently described in the bacterium Thauera aromatica [Boll, M. & Fuchs, G. (1995) Eur. J. Biochem. 234, 921−933]. It catalyzes the reaction benzoyl‐CoA + 2 e⁻ + 2 H⁺ + 2 MgATP + 2 H₂O ← cyclohexa‐1,5‐diene‐1‐carboxyl‐CoA + 2 MgADP + 2 P_i. The iron‐sulfur protein has a native molecular mass of 160−170 kDa and consists of four different subunits. In addition a flavin may be present. The nature of the potential prosthetic group and the natural electron donor were determined. Purified benzoyl‐CoA reductase preparations contained 0.25−0.3 mol FAD/mol enzyme. Cells grown anaerobically with aromatic substrates contained a ferredoxin which represented the main, if not the only ferredoxin present. It was purified from 200 g cells with a yield of 60 mg and its N‐terminal amino acid sequence was determined. The native molecular mass was 9659 ± 2 Da as determined by electrospray mass spectrometry. The protein contained 7.6 ± 0.6 mol iron and 7.6 ± 1 mol acid‐labile sulfur/mol. The ultraviolet−visible spectrum of the protein was typical for ferredoxins with maxima at 280 nm and 390 nm (in the oxidized state). The estimated molar absorption coefficients were 63 500 M⁻¹ cm⁻¹ at 280 nm and 40 500 M⁻¹ cm⁻¹ at 390 nm. The difference spectrum between the oxidized and the reduced form had a maximum at 415 nm with Δε₄₁₅= 8200 M⁻¹ cm⁻¹. 1 mol ferredoxin became reduced/mol dithionite added, suggesting the presence of two [4Fe‐4S] clusters. The average midpoint potential of the iron‐sulfur clusters was −450 mV. The ferredoxin gene was cloned and sequenced. It was located in a gene cluster coding for enzymes involved in anaerobic aromatic metabolism. The amino acid sequence of the T. aromatica ferredoxin showed high similarities to several other ferredoxins containing 2[4Fe‐4S] clusters, e.g. from Clostridia and phototrophic bacteria. The reduced ferredoxin served as electron donor for benzoyl‐CoA reduction at a three times higher rate compared with the rate obtained with the artificial electron donor reduced methyl viologen. The turnover number with the natural electron donor of 5 s⁻¹ can explain the bacterial growth rate with benzoate as substrate. Half‐maximal enzyme acitivity was obtained with 6 μM reduced ferredoxin, at an estimated cellular concentration of 70 μM ferredoxin. Both the low apparent K_m value and the turnover number are consistent with the proposed role of ferredoxin in aromatic‐ring reduction.