Morris C J, Black A C, Pealing S L, Manson F D, Chapman S K, Reid G A, Gibson D M, Ward F B
Institute of Cell and Molecular Biology, University of Edinburgh, U.K.
Biochem J. 1994 Sep 1;302 ( Pt 2)(Pt 2):587-93. doi: 10.1042/bj3020587.
The major soluble cytochrome isolated from microaerobically grown cells of Shewanella putrefaciens has been shown to be a novel type of flavocytochrome with fumarate reductase activity. This flavocytochrome, located in the periplasmic fraction of cell extracts, has been purified to homogeneity and shown to contain 4 mol of haem c and 1 mol of non-covalently bound FAD per mol of protein. An M(r) value of 63,800 is estimated from sequence analysis assuming 4 mol of haem/mol of protein. In the presence of the artificial electron donor, reduced methyl viologen, the flavocytochrome catalysed the reduction of fumarate but not that of nitrite, dimethylsulphoxide, trimethylamine-N-oxide or sulphite. The pH optimum was 7.4 with calculated pKa values of 6.8 and 8.0 for contributing catalytic groups. The Km and kcat. values for fumarate reduction were 21 microM and 250 s-1 respectively, whereas the corresponding values for succinate oxidation with 2,6-dichlorophenol-indophenol as electron carriers were 200 microM and 0.07 s-1 respectively. Mesaconic acid was a competitive inhibitor of fumarate reduction with a Ki of 2 microM. Zymogram staining of polyacrylamide gels with purified protein showed a band of fumarate reductase activity. Polyclonal antibodies, raised to the purified flavocytochrome, were shown to titrate out fumarate reductase activity. We conclude that the physiological role of this enzyme is as a fumarate reductase. Optical absorption spectra of the flavocytochrome indicated that all the haems were of the c-type and gave alpha, beta and gamma peaks at 552.3, 523 and 418 nm in the reduced spectrum with epsilon values of 30.2, 15.9 and 188.2 mM-1.cm-1 respectively. Oxidized spectra showed no 695 nm band that would be indicative of His-Met coordination. Two redox potentials were resolved at -220 mV and -320 mV. The cytochrome was reduced by formate in the presence of particulate cell fractions. The relationship of this cytochrome to other low-potential flavocytochromes c is discussed.
从腐败希瓦氏菌微需氧生长细胞中分离出的主要可溶性细胞色素已被证明是一种具有延胡索酸还原酶活性的新型黄素细胞色素。这种黄素细胞色素位于细胞提取物的周质部分，已被纯化至同质，并显示每摩尔蛋白质含有4摩尔血红素c和1摩尔非共价结合的FAD。根据序列分析，假设每摩尔蛋白质含有4摩尔血红素，估计其分子量（M(r)）值为63,800。在人工电子供体还原型甲基紫精存在下，黄素细胞色素催化延胡索酸的还原，但不催化亚硝酸盐、二甲基亚砜、三甲胺-N-氧化物或亚硫酸盐的还原。最适pH为7.4，对催化基团贡献的计算pKa值为6.8和8.0。延胡索酸还原的Km和kcat值分别为21μM和250 s-1，而以2,6-二氯酚靛酚作为电子载体时琥珀酸氧化的相应值分别为200μM和0.07 s-1。中康酸是延胡索酸还原的竞争性抑制剂，Ki为2μM。用纯化的蛋白质对聚丙烯酰胺凝胶进行酶谱染色，显示出一条延胡索酸还原酶活性带。针对纯化的黄素细胞色素产生的多克隆抗体被证明能滴定出延胡索酸还原酶活性。我们得出结论，这种酶的生理作用是作为延胡索酸还原酶。黄素细胞色素的光吸收光谱表明，所有血红素均为c型，在还原光谱中分别在552.3、523和418 nm处给出α、β和γ峰，ε值分别为30.2、15.9和188.2 mM-1·cm-1。氧化光谱未显示出指示His-Met配位的695 nm带。在-220 mV和-320 mV处解析出两个氧化还原电位。在存在颗粒细胞组分的情况下，细胞色素被甲酸还原。讨论了这种细胞色素与其他低电位黄素细胞色素c的关系。
