Létoffé S, Deniau C, Wolff N, Dassa E, Delepelaire P, Lecroisey A, Wandersman C
Unité des Membranes Bactériennes, Institut Pasteur (CNRS URA2172), 25 rue du Dr Roux, 75724 Paris Cedex 15, France.
Mol Microbiol. 2001 Jul;41(2):439-50. doi: 10.1046/j.1365-2958.2001.02530.x.
Bacterial extracellular haemophores also named HasA for haem acquisition system form an independent family of haemoproteins that take up haem from host haeme carriers and shuttle it to specific receptors (HasR). Haemophore receptors are required for the haemophore-dependent haem acquisition pathway and alone allow free or haemoglobin-bound haem uptake, but the synergy between the haemophore and its receptor greatly facilitates this uptake. The three-dimensional structure of the Serratia marcescens holo-haemophore (HasASM) has been determined previously and revealed that the haem iron atom is ligated by tyrosine 75 and histidine 32. The phenolate of tyrosine 75 is also tightly hydrogen bonded to the Ndelta atom of histidine 83. Alanine mutagenesis of these three HasASM residues was performed, and haem-binding constants of the wild-type protein, the three single mutant proteins, the three double mutant proteins and the triple mutant protein were compared by absorption spectrometry to probe the roles of H32, Y75 and H83 in haem binding. We show that one axial iron ligand is sufficient to ligate haem efficiently and that H83 may become an alternative iron ligand in the absence of Y75 or both H32 and Y75. All the single mutant proteins retained the ability to stimulate haemophore-dependent haem uptake in vivo. Thus, the residues H32, Y75 and H83 are not individually necessary for haem delivery to the receptor. The binding of haem-free and haem-loaded HasASM proteins to HasRSM-producing strains was studied. Both proteins bind to HasRSM with similar apparent Kd. The double mutant H32A-Y75A competitively inhibits binding to the receptor of both holo-HasASM and apo-HasASM, showing that there is a unique or overlapping site on HasRSM for the apo- and holo-haemophores. Thus, we propose a new mechanism for haem uptake, in which haem is exchanged between haem-loaded haemophores and unloaded haemophores bound to the receptor without swapping of haemophores on the receptor.
细菌细胞外血红蛋白载体也被称为HasA（用于血红素获取系统），它构成了一个独立的血红蛋白家族，能从宿主血红素载体中摄取血红素，并将其转运至特定受体（HasR）。血红素载体受体是血红素载体依赖性血红素获取途径所必需的，单独即可实现游离或与血红蛋白结合的血红素摄取，但血红素载体与其受体之间的协同作用极大地促进了这种摄取。此前已确定了粘质沙雷氏菌全血红素载体（HasASM）的三维结构，结果显示血红素铁原子由酪氨酸75和组氨酸32配位。酪氨酸75的酚盐也与组氨酸83的Nδ原子紧密形成氢键。对这三个HasASM残基进行了丙氨酸诱变，并通过吸收光谱法比较了野生型蛋白、三个单突变蛋白、三个双突变蛋白和三突变蛋白的血红素结合常数，以探究H32、Y75和H83在血红素结合中的作用。我们发现一个轴向铁配体足以有效地配位血红素，并且在没有Y75或同时没有H32和Y75的情况下，H83可能成为替代铁配体。所有单突变蛋白在体内均保留了刺激血红素载体依赖性血红素摄取的能力。因此，H32、Y75和H83残基并非将血红素递送至受体所必需的单个残基。研究了无血红素和负载血红素的HasASM蛋白与产生HasRSM的菌株的结合情况。两种蛋白与HasRSM的结合表观Kd相似。双突变体H32A - Y75A竞争性抑制全HasASM和脱辅基HasASM与受体的结合，表明在HasRSM上存在一个脱辅基和全血红素载体的独特或重叠位点。因此，我们提出了一种新的血红素摄取机制，即负载血红素的血红素载体与结合在受体上的未负载血红素载体之间进行血红素交换，而无需受体上的血红素载体进行交换。
