Isolation, characterization and gene sequence analysis of a membrane-associated 89 kDa Fe(III) reducing cytochrome c from Geobacter sulfurreducens
Isolation, characterization and gene sequence analysis of a membrane-associated 89 kDa Fe(III) reducing cytochrome c from Geobacter sulfurreducens
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DOI：
10.1042/0264-6021:3590147
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发表时间：
2001-10-01
影响因子：
4.1
通讯作者：
Lovley, DR
中科院分区：
文献类型：
--
作者：
Magnuson, TS;Isoyama, N;Lovley, DR
硫还原锗酸菌能够通过与大分子量细胞色素c相关的膜结合Fe（III）还原酶活性以Fe（III）作为末端电子受体进行无氧呼吸。该细胞色素通过膜部分的去污剂提取来纯化。Q-琼脂糖离子交换色谱法。制备电泳和MonoQ离子交换色谱。纯化细胞色素的分光光度分析显示c型血红素，没有血红素a的证据。血红素B或西罗血红素。变性聚丙烯酰胺凝胶电泳测得该细胞色素的M-r为89000。并具有通过分析等电聚焦测定的5.2的等电点。连二亚硫酸盐还原的细胞色素可以给Fe（III）-次氮基三乙酸和合成的水铁矿提供电子，从而证明细胞色素具有还原Fe（III）所需的氧化还原和热力学性质。使用循环伏安法的分析证实，还原的细胞色素可以催化电子转移到水铁矿，进一步证明了它的能力，是一个电子传递介体在厌氧Fe（III）呼吸。克隆的染色体DNA片段的序列分析揭示了一个2307 bp的开放阅读框（ferA），其编码768个氨基酸的蛋白质，对应于89 kDa的细胞色素。从开放阅读框架翻译的推导的氨基酸序列（FerA）包含12个推定的血红素结合基序。以及疏水性N-末端膜锚序列，脂质附着位点和ATP/GTP结合位点。FerA与其他已知的细胞色素的氨基酸序列显示20%或更少的同一性，尽管它确实与特征性的多血红素细胞色素c共享一些特征。
Geobacter sulfurreducens is capable of anaerobic respiration with Fe(III) as a terminal electron acceptor via a membrane-bound Fe(III) reductase activity associated with a large molecular mass cytochrome c. This cytochrome was purified by detergent extraction of the membrane fraction. Q-Sepharose ion-exchange chromatography. preparative electrophoresis, and MonoQ ion-exchange chromatography. Spectrophotometric analysis of the purified cytochrome reveals a c-type haem, with no evidence of haem a. haem b or sirohaem. The cytochrome has an M-r, of 89 000 as determined by denaturing PAGE. and has an isoelectric point of 5.2 as determined by analytical isoelectric focusing. Dithionite-reduced cytochrome can donate electrons to Fe(III)-nitrilotriacetic acid and synthetic ferrihydrite, thus demonstrating that the cytochrome has redox and thermodynamic properties required for reduction of Fe(III). Analysis using cyclic voltammetry confirmed that the reduced cytochrome can catalytically transfer electrons to ferrihydrite, further demonstrating its ability to be an electron transport mediator in anaerobic Fe(III) respiration. Sequence analysis of a cloned chromosomal DNA fragment revealed a 2307 bp open reading frame (ferA) encoding a 768 amino acid protein corresponding to the 89 kDa cytochrome. The deduced amino acid sequence (FerA) translated from the open reading frame contained 12 putative haem-binding motifs., as well as a hydrophobic N-terminal membrane anchor sequence., a lipid-attachment site and an ATP/GTP-binding site. FerA displayed 20% or less identity with amino acid sequences of other known cytochromes, although it does share some features with characterized polyhaem cytochromes c.