Gabricević Mario, Anderson Damon S, Mietzner Timothy A, Crumbliss Alvin L
Department of Chemistry, Duke University, Box 90346, Durham, North Carolina 27708-0346, USA.
Biochemistry. 2004 May 18;43(19):5811-9. doi: 10.1021/bi036217y.
Iron transport across the periplasmic space to the cytoplasmic membrane of certain Gram-negative bacteria is mediated by a ferric binding protein (Fbp). This requires Fe(3+) loading of Fbp at the inner leaflet of the outer membrane. A synergistic anion is required for tight Fe(3+) sequestration by Fbp. Although phosphate fills this role in the protein isolated from bacterial cell lysates, nitrilotriacetate anion (NTA) can also satisfy this requirement in vitro. Here, we report the kinetics and mechanism of Fe(3+) loading of Fbp from Fe(NTA)(aq) in the presence of phosphate at pH 6.5. The reaction proceeds in four kinetically distinguishable steps to produce Fe(3+)Fbp(PO(4)) as a final product. The first three steps exhibit half-lives ranging from ca. 20 ms to 0.5 min, depending on the concentrations, and produce Fe(3+)Fbp(NTA) as an intermediate product of significant stability. The rate for the first step is accelerated with an increasing phosphate concentration, while that of the third step is retarded by phosphate. Conversion of Fe(3+)Fbp(NTA) to Fe(3+)Fbp(PO(4)) in the fourth step is a slow process (half-life approximately 2 h) and is facilitated by free phosphate. A mechanism for the Fe(3+)-loading process is proposed in which the synergistic anions, phosphate and NTA, play key roles. These data suggest that not only is a synergistic anion required for tight Fe(3+) sequestration by Fbp, but also the synergistic anion plays a critical role in the process of inserting Fe(3+) into the Fbp binding site.
铁通过某些革兰氏阴性菌的周质空间转运至细胞质膜是由铁结合蛋白（Fbp）介导的。这需要在外膜内小叶处将Fe(3+)加载到Fbp上。Fbp紧密螯合Fe(3+)需要一种协同阴离子。尽管磷酸盐在从细菌细胞裂解物中分离出的蛋白质中发挥此作用，但次氮基三乙酸阴离子（NTA）在体外也能满足这一要求。在此，我们报告了在pH 6.5且存在磷酸盐的情况下，Fbp从Fe(NTA)(aq)加载Fe(3+)的动力学和机制。该反应按四个动力学上可区分的步骤进行，最终产物为Fe(3+)Fbp(PO(4))。前三个步骤的半衰期约为20毫秒至0.5分钟，具体取决于浓度，并产生Fe(3+)Fbp(NTA)作为具有显著稳定性的中间产物。第一步的速率随磷酸盐浓度增加而加快，而第三步的速率则受磷酸盐抑制。第四步中Fe(3+)Fbp(NTA)转化为Fe(3+)Fbp(PO(4))是一个缓慢的过程（半衰期约2小时），且游离磷酸盐可促进此过程。我们提出了一个Fe(3+)加载过程的机制，其中协同阴离子磷酸盐和NTA发挥关键作用。这些数据表明，Fbp紧密螯合Fe(3+)不仅需要协同阴离子，而且协同阴离子在将Fe(3+)插入Fbp结合位点的过程中也起着关键作用。
