Boll M, Fuchs G
Mikrobiologie, Institut Biologie II, Universität Freiburg, Germany.
Eur J Biochem. 1998 Feb 1;251(3):946-54. doi: 10.1046/j.1432-1327.1998.2510946.x.
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 Pi. 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 63500 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 epsilon415 = 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 activity was obtained with 6 microM reduced ferredoxin, at an estimated cellular concentration of 70 microM ferredoxin. Both the low apparent Km value and the turnover number are consistent with the proposed role of ferredoxin in aromatic-ring reduction.
在缺氧条件下，大多数芳香族化合物通过苯甲酰辅酶A进行代谢，苯甲酰辅酶A被苯甲酰辅酶A还原酶（去芳香化）还原；这种酶最近在嗜芳香陶厄氏菌中被描述[博尔，M. & 富克斯，G.（1995年）《欧洲生物化学杂志》234卷，921 - 933页]。它催化反应：苯甲酰辅酶A + 2e⁻ + 2H⁺ + 2MgATP + 2H₂O → 环己 - 1,5 - 二烯 - 1 - 羧基辅酶A + 2MgADP + 2Pi。这种铁硫蛋白的天然分子量为160 - 170 kDa，由四个不同的亚基组成。此外可能存在一种黄素。确定了潜在辅基和天然电子供体的性质。纯化的苯甲酰辅酶A还原酶制剂每摩尔酶含有0.25 - 0.3摩尔FAD。用芳香族底物厌氧培养的细胞含有一种铁氧化还原蛋白，它是主要的（如果不是唯一的）铁氧化还原蛋白。从200克细胞中纯化得到，产量为60毫克，并测定了其N端氨基酸序列。通过电喷雾质谱法测定其天然分子量为9659 ± 2 Da。该蛋白每摩尔含有7.6 ± 0.6摩尔铁和7.6 ± 1摩尔酸不稳定硫。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纳米和390纳米处有最大值（氧化态）。估计的摩尔吸收系数在280纳米处为63500 M⁻¹cm⁻¹，在390纳米处为40500 M⁻¹cm⁻¹。氧化态和还原态之间的差光谱在415纳米处有最大值，Δε415 = 8200 M⁻¹cm⁻¹。每加入1摩尔连二亚硫酸盐，1摩尔铁氧化还原蛋白被还原，表明存在两个[4Fe - 4S]簇。铁硫簇的平均中点电位为 - 450毫伏。克隆并测序了铁氧化还原蛋白基因。它位于一个基因簇中，该基因簇编码参与厌氧芳香族代谢的酶。嗜芳香陶厄氏菌铁氧化还原蛋白的氨基酸序列与其他几种含有2个[4Fe - 4S]簇的铁氧化还原蛋白高度相似，例如来自梭菌属和光合细菌的铁氧化还原蛋白。与用人造电子供体还原甲基紫精获得的速率相比，还原态铁氧化还原蛋白作为苯甲酰辅酶A还原的电子供体的速率高3倍。天然电子供体的周转数为5 s⁻¹，可以解释以苯甲酸盐为底物时的细菌生长速率。在估计细胞内铁氧化还原蛋白浓度为70微摩尔时，6微摩尔还原态铁氧化还原蛋白可获得半数最大酶活性。低表观Km值和周转数都与铁氧化还原蛋白在芳香环还原中所提出的作用一致。
