Institute for Integrative Biology of the Cell (I2BC), CNRS CEA Univ. Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France.
Synchrotron SOLEIL, L'Orme des Merisiers, Gif-sur-Yvette, France.
FEBS J. 2020 Jan;287(2):295-309. doi: 10.1111/febs.15004. Epub 2019 Jul 26.
Pseudomonas aeruginosa secretes pyoverdine, a major siderophore to get access to iron, an essential nutrient. Pyoverdine scavenges ferric iron in the bacterial environment with the resulting complex internalized by bacteria. Releasing of iron from pyoverdine in the periplasm involves an iron reduction by an inner membrane reductase and two solute-binding proteins (SBPs) FpvC and FpvF in association with their ABC transporter. FpvC and FpvF belong to two different subgroups of SBPs within the structural cluster A: FpvC and FpvF were proposed to be a metal-binding protein and a ferrisiderophore-binding protein respectively. Here, we report the redox state and the binding mode of iron to FpvC. We first solved the crystal structure of FpvC bound to a fortuitous Ni by single anomalous dispersion method. Using a different protein purification strategy, we determined the structure of FpvC with manganese and iron, which binds to FpvC in a ferrous state as demonstrated by electron paramagnetic resonance. FpvC is the first example of a hexahistidine metal site among SBPs in which the Fe redox state is stabilized under aerobic conditions. Using biophysics methods, we showed that FpvC reversibly bind to a broad range of divalent ions. The structure of a mutant mimicking the apo FpvC reveals a protein in an open state with large conformational changes when compared with the metal-bound FpvC. These results highlight that the canonical metal site in FpvC is distinct from those yet described in SBPs and they provide new insights into the mechanism of PVD-Fe dissociation in P. aeruginosa.
铜绿假单胞菌分泌绿脓菌素，这是一种主要的铁载体，用于获取铁这种必需的营养物质。绿脓菌素在细菌环境中抢夺三价铁，形成的复合物被细菌内化。周质中绿脓菌素释放铁涉及内膜还原酶和两个溶质结合蛋白（SBPs）FpvC 和 FpvF 与它们的 ABC 转运蛋白一起将铁还原。FpvC 和 FpvF 属于结构簇 A 中两个不同的 SBPs 亚群：FpvC 和 FpvF 分别被认为是金属结合蛋白和亚铁载体结合蛋白。在这里，我们报告了 FpvC 中铁的氧化还原状态和结合模式。我们首先通过单异常分散法解决了 FpvC 与偶然 Ni 结合的晶体结构。使用不同的蛋白质纯化策略，我们确定了 FpvC 与锰和铁结合的结构，电子顺磁共振证明铁以亚铁状态结合到 FpvC 上。FpvC 是 SBPs 中第一个六组氨酸金属位点的例子，其中 Fe 氧化还原状态在有氧条件下得到稳定。通过生物物理方法，我们表明 FpvC 可逆地结合到广泛的二价离子。模拟 apo FpvC 的突变体的结构揭示了与金属结合的 FpvC 相比，蛋白处于开放状态并发生较大的构象变化。这些结果表明，FpvC 中的典型金属位点与 SBPs 中描述的那些不同，并为铜绿假单胞菌中 PVD-Fe 解离的机制提供了新的见解。
