Myers C R, Myers J M
Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee 53226, USA.
Biochim Biophys Acta. 1997 Jun 12;1326(2):307-18. doi: 10.1016/s0005-2736(97)00034-5.
The metal-reducing bacterium Shewanella putrefaciens MR-1 is known to localize a majority of its membrane-bound cytochromes to its outer membrane when grown under anaerobic conditions. In this study, pyridine hemochrome spectra confirmed that these outer membrane cytochromes are c-type, and electrophoretic data demonstrated the presence of four distinct outer membrane cytochromes, with apparent molecular masses of 150, 83, 65, and 53 kDa. Fourth-order derivative analysis of 77 K spectra of the outer membrane revealed four spectrally distinct c-type hemes, with peaks at 545.4, 548.0, 550.6, and 552.6 nm. Outer membrane cytochromes in the reduced state were rapidly re-oxidized by oxidized iron and manganese, which have previously been shown to serve as electron acceptors for anaerobic respiration in this bacterium. The 83-kDa outer membrane cytochrome was purified and a specific polyclonal antibody was generated against this protein. Western blot analysis demonstrated that the vast majority of this protein was localized to the outer membrane and an intermediate density membrane fraction of similar composition. Its levels, but not its subcellular distribution, were somewhat influenced by the electron acceptor used to support anaerobic growth, with levels higher in fumarate-grown cells relative to iron(III)- or trimethylamine N-oxide-grown cells. Its specific content in cells grown under aerobic conditions was only 14% of that of fumarate-grown cells, suggesting that a switch to anaerobic conditions significantly increases the de novo synthesis of this outer membrane cytochrome.
已知金属还原菌腐败希瓦氏菌MR-1在厌氧条件下生长时，其大部分膜结合细胞色素定位于外膜。在本研究中，吡啶血色素光谱证实这些外膜细胞色素为c型，电泳数据表明存在四种不同的外膜细胞色素，表观分子量分别为150、83、65和53 kDa。外膜77 K光谱的四阶导数分析显示有四种光谱不同的c型血红素，其峰值分别在545.4、548.0、550.6和552.6 nm处。还原态的外膜细胞色素会被氧化态的铁和锰迅速重新氧化，铁和锰先前已被证明可作为该细菌厌氧呼吸的电子受体。纯化了83 kDa的外膜细胞色素，并针对该蛋白制备了特异性多克隆抗体。蛋白质免疫印迹分析表明，该蛋白的绝大多数定位于外膜和组成相似的中等密度膜组分。其水平受支持厌氧生长的电子受体的一定影响，但其亚细胞分布不受影响，相对于铁（III）或三甲胺N-氧化物培养的细胞，延胡索酸培养的细胞中该蛋白水平更高。在有氧条件下生长的细胞中其特定含量仅为延胡索酸培养细胞的14%，这表明转变为厌氧条件会显著增加这种外膜细胞色素的从头合成。
