Eaves D J, Grove J, Staudenmann W, James P, Poole R K, White S A, Griffiths I, Cole J A
School of Biochemistry, University of Birmingham, UK.
Mol Microbiol. 1998 Apr;28(1):205-16. doi: 10.1046/j.1365-2958.1998.00792.x.
Cytochrome c552 is the terminal component of the formate-dependent nitrite reduction pathway of Escherichia coli. In addition to four 'typical' haem-binding motifs, CXXCH-, characteristic of c-type cytochromes, the N-terminal region of NrfA includes a motif, CWSCK. Peptides generated by digesting the cytochrome from wild-type bacteria with cyanogen bromide followed by trypsin were analysed by on-line HPLC MS/MS in parent scanning mode. A strong signal at mass 619, corresponding to haem, was generated by fragmentation of a peptide of mass 1312 that included the sequence CWSCK. Neither this signal nor the haem-containing peptide of mass 1312 was detected in parallel experiments with cytochrome that had been purified from a transformant unable to synthesize NrfE, NrfF and NrfG: this is consistent with our previous report that NrfE and NrfG (but not NrfF) are essential for formate-dependent nitrite reduction. Redox titrations clearly revealed the presence of high and low mid-point potential redox centres. The best fit to the experimental data is for three n=1 components with mid-point redox potentials (pH 7.0) of +45 mV (21% of the total absorbance change), -90 mV (36% of the total) and -210mV (43% of the total). Plasmids in which the lysine codon of the cysteine-lysine motif, AAA, was changed to the histidine codon CAT (to create a fifth 'typical' haem c-binding motif), or to the isoleucine and leucine codons, ATT and CTT, were unable to transform a Nrf deletion mutant to Nrf+ or to restore formate-dependent nitrite reduction to the transformants. The presence of a 50 kDa periplasmic c-type cytochrome was confirmed by staining proteins separated by SDS-PAGE for covalently bound haem, but the methyl-viologen-dependent nitrite reductase activities associated with the mutated proteins, although still detectable, were far lower than that of the native protein. The combined data establish not only that there is a haem group bound covalently to the cysteine-lysine motif of cytochrome c552 but also that one or more products of the last three genes of the nrf operon are essential for the haem ligation to this motif.
细胞色素c552是大肠杆菌中甲酸依赖型亚硝酸盐还原途径的末端组分。除了四个“典型的”血红素结合基序CXXCH-（c型细胞色素的特征基序）外，NrfA的N端区域还包含一个基序CWSCK。用溴化氰消化野生型细菌中的细胞色素，然后用胰蛋白酶消化产生的肽段，通过在线HPLC MS/MS在母离子扫描模式下进行分析。质量为1312且包含序列CWSCK的肽段断裂产生了一个质量为619的强信号，对应于血红素。在从无法合成NrfE、NrfF和NrfG的转化体中纯化的细胞色素的平行实验中，未检测到该信号或质量为1312的含血红素肽段：这与我们之前的报告一致，即NrfE和NrfG（但不是NrfF）对于甲酸依赖型亚硝酸盐还原是必需的。氧化还原滴定清楚地揭示了存在高和低中点电位的氧化还原中心。对实验数据的最佳拟合是三个n = 1组分，其在pH 7.0时的中点氧化还原电位分别为+45 mV（占总吸光度变化的21%）、-90 mV（占总变化的36%）和-210 mV（占总变化的43%）。将半胱氨酸-赖氨酸基序的赖氨酸密码子AAA分别改变为组氨酸密码子CAT（以产生第五个“典型的”血红素c结合基序）、异亮氨酸密码子ATT和亮氨酸密码子CTT的质粒，无法将Nrf缺失突变体转化为Nrf+，也无法恢复转化体中甲酸依赖型亚硝酸盐还原。通过对SDS-PAGE分离的蛋白质进行共价结合血红素染色，证实了50 kDa周质c型细胞色素的存在，但与突变蛋白相关的甲基紫精依赖性亚硝酸盐还原酶活性，虽然仍可检测到，但远低于天然蛋白。综合数据不仅证实了有一个血红素基团共价结合到细胞色素c552的半胱氨酸-赖氨酸基序上，还证实了nrf操纵子最后三个基因的一个或多个产物对于该基序的血红素连接是必需的。
