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
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
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DOI：
10.1046/j.1432-1327.1998.2510946.x
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发表时间：
1998-02-01
期刊：
影响因子：
--
通讯作者：
Fuchs, G
中科院分区：
文献类型：
--
作者：
Boll, M;Fuchs, G
在缺氧条件下，大多数芳香族化合物通过苯甲酰基-CoA代谢，苯甲酰基-CoA被苯甲酰基-CoA还原酶还原（脱芳香化）;这种酶最近在细菌Thauera aromatica中描述[Boll. M. & Fuchs. G.（1995）Eur. J. Biochem.234，931-933]。它催化反应苯甲酰辅酶A + 2 e（-）+ 2 H+ + 2 MgATP + 2 H2O -->环己-1，5-二烯-1-羧基辅酶A + 2 MgADP + 2 P。铁硫蛋白的天然分子量为160-170 kDa，由四个不同的亚基组成。确定了潜在辅基和天然电子供体的性质。纯化的苯甲酰-CoA还原酶制剂含有0.25-0.3 mol FAD/mol酶。用芳香底物厌氧生长的细胞含有铁氧还蛋白，其代表主要的铁氧还蛋白，如果不是唯一的话。从200 g细胞中纯化，产量为60 mg，并测定了其N-末端氨基酸序列。天然分子量为9659 ± 2 Da，通过电喷雾质谱法测定。蛋白质含有7.6 +/- 0.6 mol铁和7.6 +/- 1 mol酸不稳定硫/mol。该蛋白的紫外-可见光谱是铁氧化还原蛋白的典型光谱，在280 nm和390 nm（氧化态）处具有最大吸收，在280 nm处和390 nm处的估计摩尔吸收系数分别为63500 M-1 cm（-1）和40500 M-1 cm（-1）。氧化和还原形式之间的差异光谱在415 nm处具有最大值，Δ λ（415）= 8200 M-1 cm（-1），1 mol铁氧还蛋白变为还原/mol连二亚硫酸盐。铁硫簇的平均中点电位为-450 mV。克隆并测序了铁氧还蛋白基因，该基因位于编码厌氧芳香族代谢酶的基因簇中。芳香族铁氧还蛋白与其他几种含2个[4Fe-4S]簇的铁氧还蛋白具有很高的相似性。例如来自梭菌属和光养细菌。还原铁氧还蛋白作为电子供体苯甲酰辅酶A还原的速率比人工电子供体还原甲基紫精获得的速率高三倍。用天然电子供体的周转数为5s（-1）可以解释细菌以苯甲酸为底物的生长速率。在70 μ M铁氧还蛋白的估计细胞浓度下，用6 μ M还原铁氧还蛋白获得半最大酶活性。低的表观Km值和周转数与铁氧还蛋白在芳环还原中的作用一致。
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.