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首页> 外文期刊>Biochemistry >Mapping the Iron Binding Site(s) on the Small Tetraheme Cytochrome of Shewanella oneidensis MR-1
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Mapping the Iron Binding Site(s) on the Small Tetraheme Cytochrome of Shewanella oneidensis MR-1

机译：绘制沙瓦氏假单胞菌MR-1的小四氢血红素细胞色素上的铁结合位点

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摘要

In the model microbe Shewanella oneidensis, multi-heme proteins are utilized for respiratory metabolism where metals serve as the terminal electron acceptor. Among those is the periplasm-localized small tetraheme cytochrome (STC). STC has been extensively characterized structurally and electrochemically to which electron flow in and out of the protein has been modeled. However, until the present work, no kinetic studies have been performed to probe the route of electron flow or to determine the iron-binding site on STC. Using iron chelated by EDTA, NTA, or citrate, we have used chemical modification, site-directed mutagenesis along with isothermal titration calorimetry (ITC), and stopped-flow measurements to identify the iron binding site of STC. Chemical modifications of STC revealed that carboxyl groups on STC are involved in binding of EDTA-Fe~(3+). Scanning mutagenesis was performed on Asp and Glu to probe the putative iron-binding site on STC. Two STC mutants (D21N; D80N) showed -70% decrease in observed electron transfer rate constant with EDTA-Fe~(3+) from transient-state kinetic measurements. The impaired reactivity of STC (D80N/D21N) with EDTA-Fe~(3+) was further confirmed by a significant decrease (>10-fold) in iron binding affinity.
机译：在模型微生物希瓦氏菌中，多血红素蛋白被用于呼吸代谢，其中金属充当末端电子受体。其中包括周质定位的小四血红素细胞色素（STC）。 STC在结构和电化学方面已得到广泛表征，已经模拟了电子流入和流出蛋白质的过程。但是，直到目前的工作，还没有进行动力学研究来探测电子流的路径或确定STC上的铁结合位点。使用经EDTA，NTA或柠檬酸盐螯合的铁，我们已使用化学修饰，定点诱变以及等温滴定热量法（ITC）和定流测量来确定STC的铁结合位点。对STC的化学修饰表明，STC上的羧基与EDTA-Fe〜（3+）的结合有关。在Asp和Glu上进行诱变扫描以探测STC上假定的铁结合位点。两种STC突变体（D21N; D80N）的瞬态动力学测量结果表明，用EDTA-Fe〜（3+）观察到的电子传递速率常数降低了-70％。铁结合亲和力的显着降低（> 10倍）进一步证实了STC（D80N / D21N）与EDTA-Fe〜（3+）的反应性受损。

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