Biochemical and genetic characterization of PpcA, a periplasmic c-type cytochrome in Geobacter sulfurreducens
Biochemical and genetic characterization of PpcA, a periplasmic c-type cytochrome in Geobacter sulfurreducens
复制标题
DOI：
10.1042/bj20020597
复制
发表时间：
2003-01-01
影响因子：
4.1
通讯作者：
Lovley, DR
中科院分区：
文献类型：
--
作者：
Lloyd, JR;Leang, C;Lovley, DR
一个9.6 kDa的周质C-型细胞色素，指定PpcA，从铁（III）-还原细菌Geetriumsulfur-reducens纯化和表征。纯化的蛋白质是碱性的（pI 9.5），含有三个血红素，并且具有与先前描述的Geelophylla metallirereducens和Desulfuromonas acetoxidans的三血红素c（7）细胞色素的那些密切相关的N-末端氨基酸序列。从G.硫还原菌（sulfurreducens）基因组的N端序列，编码71个氨基酸的蛋白质，分子量为9.58 kDa，与D.乙酰氧烷。为了确定PpcA的生理作用，用单步重组方法制备敲除突变体。乙酸盐依赖的Fe（III）还原显着抑制在两个增长的文化和细胞悬浮液的突变体。当ppcA以反式表达时，用乙酸盐还原Fe（III）的全部能力被恢复。电子从乙酸转移到蒽醌2，6-二磺酸盐（AQDS;腐殖酸类似物）和U（VI）也受到损害的突变体，但乙酸依赖的还原延胡索酸没有改变。还原率的Fe（III），AQDS，U（VI）和富马酸盐也是相同的野生型和ppcA突变体时，提供氢作为电子供体。结合前人对G.这些结果表明，PpcA作为一个中间电子载体从乙酸到终端铁（III）还原酶的外膜，也参与了电子从乙酸转移到U（VI）和腐殖酸。
A 9.6 kDa periplasmic c-type cytochrome, designated PpcA, was purified from the Fe(III)-reducing bacterium Geobacter sulfur-reducens and characterized. The purified protein is basic (pI 9.5), contains three haems and has an N-terminal amino acid sequence closely related to those of the previously described trihaem c(7) cytochromes of Geobacter metallireducens and Desulfuromonas acetoxidans. The gene encoding PpcA was identified from the G. sulfurreducens genome using the N-terminal sequence, and encodes a protein of 71 amino acids (molecular mass 9.58 kDa) with 49% identity to the c(7) cytochrome of D. acetoxidans. In order to determine the physiological role of PpcA, a knockout mutant was prepared with a single-step recombination method. Acetate-dependent Fe(III) reduction was significantly inhibited in both growing cultures and cell suspensions of the mutant. When ppcA was expressed in trans, the full capacity for Fe(III) reduction with acetate was restored. The transfer of electrons from acetate to anthraquinone 2,6-disulphonate (AQDS; a humic acid analogue) and to U(VI) was also compromised in the mutant, but acetate-dependent reduction of fumarate was not altered. The rates of reduction of Fe(III), AQDS, U(VI) and fumarate were also the same in the wild type and ppcA mutant when hydrogen was supplied as the electron donor. When taken together with previous studies on other electron transport proteins in G. sulfurreducens, these results suggest that PpcA serves as an intermediary electron carrier from acetate to terminal Fe(III) reductases in the outer membrane, and is also involved in the transfer of electrons from acetate to U(VI) and humics.