DsrJ, an Essential Part of the DsrMKJOP Transmembrane Complex in the Purple Sulfur Bacterium Allochromatium vinosum, Is an Unusual Triheme Cytochrome c
DsrJ, an Essential Part of the DsrMKJOP Transmembrane Complex in the Purple Sulfur Bacterium Allochromatium vinosum, Is an Unusual Triheme Cytochrome c
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DOI：
10.1021/bi1007673
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发表时间：
2010-09-28
期刊：
影响因子：
2.9
通讯作者：
Dahl, Christiane
中科院分区：
文献类型：
--
作者：
Grein, Fabian;Venceslau, Sofia S.;Dahl, Christiane
DsrMKJOP 跨膜复合物在异化硫代谢中具有最重要的功能，不仅在许多硫氧化生物体中，而且在硫酸盐还原原核生物中。在这里，我们重点关注该复合物的一个单独成分，即来自紫硫细菌 Allochromatium vinosum 的三血红素细胞色素 c DsrJ。在 A. vinosum 中，DsrJ 的信号肽不会被切除，而是充当膜锚。序列分析表明存在三种具有双His、His/Met 的血红素c 物种，并且可能存在非常不寻常的His/Cys 连接。在大肠杆菌中作为重组蛋白产生的 A. vinosum DsrJ 确实含有三种血红素，电子顺磁共振 (EPR) 光谱提供了其中一种血红素可能但仅部分的 His/Cys 血红素连接的证据。该血红素显示出异质配位，Met 是另一个候选配体。使用定点诱变将半胱氨酸 46 替换为丝氨酸，突变蛋白由于 His/Cys 协调而显示 EPR 信号强度小幅下降，但 UV-vis 和 RR 光谱相同。野生型蛋白中血红素的氧化还原电位被测定为-20、-200和-220 mV，并且发现在突变蛋白中几乎相同。然而，在体内，相同的配体交换导致表型显着改变，凸显了 Cys46 的重要性。我们的结果表明 Cys46 可能参与催化硫化学而不是电子转移。其他体内实验表明，A. vinosum 中的 DsrJ 可以被来自硫酸盐还原剂 Desulfovibrio vulgaris 的同源蛋白功能性取代。
The DsrMKJOP transmembrane complex has a most important function in dissimilatory sulfur metabolism, not only in many sulfur-oxidizing organisms but also in sulfate-reducing prokaryotes. Here, we focused on an individual component of this complex, the triheme cytochrome c DsrJ from the purple sulfur bacterium Allochromatium vinosum. In A. vinosum, the signal peptide of DsrJ is not cleaved off but serves as a membrane anchor. Sequence analysis suggested the presence of three heme c species with bis-His, His/Met, and possibly a very unusual His/Cys ligation. A. vinosum DsrJ produced as a recombinant protein in Escherichia coli indeed contained three hemes, and electron paramagnetic resonance (EPR) spectroscopy provided evidence of possible, but only partial, His/Cys heme ligation in one of the hemes. This heme shows heterogeneous coordination, with Met being another candidate ligand. Cysteine 46 was replaced with serine using site-directed mutagenesis, with the mutant protein showing a small decrease in the magnitude of the EPR signal attributed to His/Cys coordination, but identical UV-vis and RR spectra. The redox potentials of the hemes in the wild-type protein were determined to be -20, -200, and -220 mV and were found to be virtually identical in the mutant protein. However, in vivo the same ligand exchange led to a dramatically altered phenotype, highlighting the importance of Cys46. Our results suggest that Cys46 may be involved in catalytic sulfur chemistry rather than electron transfer. Additional in vivo experiments showed that DsrJ can be functionally replaced in A. vinosum by the homologous protein from the sulfate reducer Desulfovibrio vulgaris.