Vibrio vulnificus, a pathogenic bacterium that causes serious infections in humans, requires iron for growth. Clinical isolate, V. vulnificus M2799, secretes a catecholate siderophore, namely, vulnibactin, to capture iron (III) from the environment. Growth experiments using a deletion mutant indicated that VuuB, a member of the FAD-containing siderophore-interacting protein family, plays a crucial role in Fe³⁺-vulnibactin reduction. IutB, a member of the ferric-siderophore reductase family, stands a substitute for VuuB in its absence. It remained unclear why V. vulnificus M2799 has two proteins with relevant functions. Here we biochemically characterized VuuB and IutB using purified recombinant proteins. Purified VuuB, a flavoprotein, catalyzed the reduction of Fe³⁺-nitrilotriacetic acid as its electron acceptor, in the presence of NADH as its electron donor and FAD as its cofactor. IutB catalyzed the reduction of Fe³⁺-nitrilotriacetic acid, in the presence of NADH, NADPH, or reduced glutathione as its electron donor. The optimal pH values and temperatures of VuuB and IutB were 7.0 and 37 °C, and 8.5 and 45 °C, respectively. On analyzing their ferric-chelate reductase activities, both VuuB and IutB were found to catalyze the reduction of Fe³⁺-aerobactin, Fe³⁺-vibriobactin, and Fe³⁺-vulnibactin. When the biologically relevant substrate, Fe³⁺-vulnibactin, was used, the levels of ferric-chelate reductase activities were similar between VuuB and IutB. Finally, the mRNA levels were quantified by qRT-PCR in M2799 cells cultivated under low-iron conditions. The number of vuuB mRNA was 8.5 times greater than that of iutB. The expression ratio correlated with the growth of their mutants in the presence of vulnibactin.

创伤弧菌是一种导致人类严重感染的病原菌，需要铁才能生长。临床分离株V. vulnificus M2799 分泌儿茶酚酸铁载体，即 vulnibactin，以从环境中捕获铁 (III)。使用缺失突变体的生长实验表明，VuuB（含 FAD 的铁载体相互作用蛋白家族的成员）在 Fe ³⁺ -vulnibactin 还原中起关键作用。IutB 是三价铁-铁载体还原酶家族的成员，在没有 VuuB 时可以替代它。尚不清楚为什么V. vulnificusM2799 有两种具有相关功能的蛋白质。在这里，我们使用纯化的重组蛋白对 VuuB 和 IutB 进行了生化表征。纯化的 VuuB 是一种黄素蛋白，在 NADH 作为其电子供体和 FAD 作为其辅助因子的情况下，催化 Fe ^{3+ -}次氮基三乙酸作为其电子受体的还原。在 NADH、NADPH 或还原型谷胱甘肽作为其电子供体存在的情况下，IutB 催化 Fe ^{3+ -}次氮基三乙酸的还原。VuuB 和 IutB 的最佳 pH 值和温度分别为 7.0 和 37°C，以及 8.5 和 45°C。通过分析它们的铁螯合还原酶活性，发现 VuuB 和 IutB 都可以催化 Fe ³⁺ -aerobactin、Fe ³⁺ -vibriobactin 和 Fe ³⁺的还原-vulnibactin。当使用生物学相关底物 Fe ³⁺ -vulnibactin 时，VuuB 和 IutB 之间的铁螯合还原酶活性水平相似。最后，在低铁条件下培养的 M2799 细胞中，通过 qRT-PCR 量化 mRNA 水平。数vuuB表达比更大的8.5倍iutB。表达比率与它们的突变体在 vulnibactin 存在下的生长相关。