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 metabolism

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, 931-933]. 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. 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 40500 M-1 cm(-1) at 390 nm. The difference spectrum between the oxidized and the reduced form had a maximum at 415 nm with Delta epsilon(415) = 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. aromatico 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 far 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 activity was obtained with 6 mu M reduced ferredoxin at an estimated cellular concentration of 70 mu M ferredoxin. Both the low apparent K-m value and the turnover number are consistent with the proposed role of ferredoxin in aromatic-ring reduction.