CYTOCHROME-BO FROM ESCHERICHIA-COLI - IDENTIFICATION OF HEME LIGANDS AND REACTION OF THE REDUCED ENZYME WITH CARBON-MONOXIDE
CYTOCHROME-BO FROM ESCHERICHIA-COLI - IDENTIFICATION OF HEME LIGANDS AND REACTION OF THE REDUCED ENZYME WITH CARBON-MONOXIDE
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DOI：
10.1042/bj2890709
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发表时间：
1993-02-01
影响因子：
4.1
通讯作者：
THOMSON, AJ
中科院分区：
文献类型：
--
作者：
CHEESMAN, MR;WATMOUGH, NJ;THOMSON, AJ
内膜由大肠杆菌菌株RG 145制备，其缺乏细胞色素bd，但过表达细胞色素bo [Au和Gennis（1987）J. Bacteriol. 169，3237-3242]。通过用辛基β-D-吡喃葡萄糖苷提取膜，然后在DEAE-Sepharose上层析，将后者纯化7倍，得到150 mg蛋白质/150 g湿重的细胞。光学电子病历和低温m.c.d.用磁性圆二色谱研究了大肠杆菌细胞色素bo中两个血红素的蛋白质配体的性质。杆菌低自旋铁血红素B，菱形电脉冲的来源。具有g = 2.98、2.26和1.50的光谱在近红外中产生电荷转移带。m.c.d.光谱在1622 nm处。因此得出结论，血红素B由两个组氨酸残基协调。低温m.c.d.连二亚硫酸盐还原的细胞色素B 0的光谱包括由于低自旋亚铁血红素B和高自旋亚铁血红素O的谱带。血红素o的条带与m.c. d的条带直接对应。五配位组氨酸连接的亚铁血红素如肌红蛋白的光谱，这意味着连接血红素的蛋白残基也是组氨酸。通过测量e.p.r.证实了这一分配。完全还原的细胞色素bo的一氧化氮衍生物的光谱。这显示了g = 2.098、2.008和1.987的菱形光谱，并且核超精细分裂与亚铁血红素通过NO和组氨酸的配位一致。观察到的NO配体N-14的超精细分裂为1.95 +/- 0.05 mT，近端组氨酸N-14的超精细分裂为0.75 +/- 0.05 mT。紧急情况报告。在低于15 K的温度下，氧化细胞色素bo的一些样品的光谱显示在g = 7.6、3.6和2.8处的宽信号，并且在甘油存在下的其它制备物在g = 10.8、3.2和2.6处产生信号。这些信号，这是取消了添加氰化物，被分配到双核中心，细胞色素o-Cu（B），表明双核网站可能显示异质性。一氧化碳与还原的酶以1：1的化学计量反应，该反应的解离常数测定为1.7 x 10（-6）M。测定了该反应的二级速率常数，结果表明与牛细胞色素aa 3的二级速率常数相似[吉布森和格林伍德（1963）生物化学杂志86，541-554]。
Inner membranes were prepared from Escherichia coli strain RG 145, which is deficient in cytochrome bd, but overexpresses cytochrome bo [Au and Gennis (1987) J. Bacteriol. 169, 3237-3242]. The latter was purified 7-fold by extracting the membranes with octyl beta-D-glucopyranoside, followed by chromatography on DEAE-Sepharose, yielding 150 mg of protein/150 g wet weight of cells. Optical e.p.r. and low-temperature m.c.d. (magnetic circular dichroism) spectroscopies were used to investigate the nature of the protein ligands to the two haems in cytochrome bo from E. coli. Low-spin ferric haem b, the origin of a rhombic e.p.r. spectrum with g = 2.98, 2.26 and 1.50, gives rise to a charge-transfer band in the near-i.r. m.c.d. spectrum at 1622 nm. It is therefore concluded that haem b is co-ordinated by two histidine residues. The low-temperature m.c.d. spectrum of dithionite-reduced cytochrome bo comprises bands due both to low-spin ferrous haem b and to high-spin ferrous haem o. The bands arising from haem o show a direct correspondence with those in the m.c.d. spectrum of five-co-ordinate histidine-ligated ferrous haems such as myoglobin, implying that the protein residue liganding haem o is also histidine. This assignment was confirmed by measuring the e.p.r. spectrum of the nitric oxide derivative of fully reduced cytochrome bo. This showed a rhombic spectrum with g = 2.098, 2.008 and 1.987, and nuclear hyperfine splitting consistent with the co-ordination of ferrous haem by NO and histidine. The hyperfine splittings observed were 1.95 +/- 0.05 mT for the N-14 of the NO ligand and 0.75 +/- 0.05 mT for the N-14 of the proximal histidine. The e.p.r. spectrum of some samples of oxidized cytochrome bo show, at temperatures below 15 K, broad signals at g = 7.6, 3.6 and 2.8, and other preparations in the presence of glycerol yield signals at g = 10.8, 3.2 and 2.6. These signals, which are abolished by the addition of cyanide, are assigned to the binuclear centre, cytochrome o-Cu(B), suggesting that the binuclear site may display heterogeneity. Carbon monoxide reacts with the reduced enzyme with a stoichiometry of 1:1 , and the dissociation constant for this reaction was determined to be 1.7 x 10(-6) M. The second-order rate constants for this reaction were measured and shown to be similar to those determined for bovine cytochrome aa3 [Gibson and Greenwood (1963) Biochem. J. 86, 541-554].