We report that the metal-sequestering human host-defense protein calprotectin (CP, S100A8/S100A9 oligomer) affects the redox speciation of iron (Fe) in bacterial growth media and buffered aqueous solution. Under aerobic conditions and in the absence of an exogenous reducing agent, CP-Ser (S100A8(C42S)/S100A9(C3S) oligomer) depletes Fe from three different bacterial growth media preparations over a 48 h timeframe (T = 30 °C). The presence of the reducing agent β-mercaptoethanol accelerates this process and allows CP-Ser to deplete Fe over a ≈1 h timeframe. Fe-depletion assays performed with metal-binding-site variants of CP-Ser show that the hexahistidine (His₆) site, which coordinates Fe(II) with high affinity, is required for Fe depletion. An analysis of Fe redox speciation in buffer containing Fe(III) citrate performed under aerobic conditions demonstrates that CP-Ser causes a time-dependent increase in the [Fe(II)]/[Fe(III)] ratio. Taken together, these results indicate that the hexahistidine site of CP stabilizes Fe(II) and thereby shifts the redox equilibrium of Fe to the reduced ferrous state under aerobic conditions. We also report that the presence of bacterial metabolites affects the Fe-depleting activity of CP-Ser. Supplementation of bacterial growth media with an Fe(III)-scavenging siderophore (enterobactin, staphyloferrin B, or desferrioxamine B) attenuates the Fe-depleting activity of CP-Ser. This result indicates that formation of Fe(III)–siderophore complexes blocks CP-mediated reduction of Fe(III) and hence the ability of CP to coordinate Fe(II). In contrast, the presence of pyocyanin (PYO), a redox-cycling phenazine produced by Pseudomonas aeruginosa that reduces Fe(III) to Fe(II), accelerates Fe depletion by CP-Ser under aerobic conditions. These findings indicate that the presence of microbial metabolites that contribute to metal homeostasis at the host/pathogen interface can affect the metal-sequestering function of CP.

中文翻译：

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人钙卫蛋白会影响铁的氧化还原形态†

我们报告说，金属螯合人宿主防御蛋白钙卫蛋白（CP，S100A8 / S100A9寡聚物）影响细菌生长培养基和缓冲水溶液中铁（Fe）的氧化还原形态。在有氧条件下并且在没有外源还原剂的情况下，CP-Ser（S100A8（C42S）/ S100A9（C3S）低聚物）会在48小时内（T = 30°C）消耗三种不同细菌生长培养基制剂中的铁。还原剂β-巯基乙醇的存在加速了该过程，并使CP-Ser在约1小时的时间内耗尽了Fe。用CP-Ser的金属结合位点变体进行的铁耗竭试验表明，六组氨酸（His ₆）位点与Fe（IIFe耗尽需要具有高亲和力的）。在有氧条件下对含有柠檬酸铁（Ⅲ）的缓冲液中的铁氧化还原形态的分析表明，CP-Ser引起[铁（Ⅱ）] / [铁（Ⅲ）]比率随时间的增加。综上所述，这些结果表明CP的六组氨酸位点稳定了Fe（II），从而在有氧条件下将Fe的氧化还原平衡转变为还原的亚铁态。我们还报告说，细菌代谢产物的存在会影响CP-Ser的铁消耗活性。用Fe（III）补充细菌生长培养基）清除铁载体（肠杆菌肽，葡萄铁蛋白B或去铁胺B）会减弱CP-Ser的铁消耗活性。该结果表明Fe（III）-铁载体配合物的形成阻止了CP介导的Fe（III）的还原，从而阻止了CP协调Fe（II）的能力。相反，绿脓杆菌（Pseudomonas aeruginosa）产生的氧化还原循环吩嗪（Pyocyanin（PYO））的存在将Fe（III）还原为Fe（II），在有氧条件下通过CP-Ser加速了Fe的消耗。这些发现表明，在宿主/病原体界面处有助于金属稳态的微生物代谢物的存在会影响CP的金属螯合功能。