Analysis of cellular function of HBP23 on redox regulation

HBP23（血红素结合蛋白，M.W.: 23kDa）是一种与血红素具有高结合亲和力的大鼠细胞质蛋白，是过氧化物还蛋白家族的成员，具有硫氧还蛋白依赖性过氧化物酶活性。突变体Cys52Ser、Cys173Ser和Cys52-173Ser没有表现出与突变体Cys83Ser不同的活性。HBP23 （Cys83Ser）在氧化形态下的2.6A分辨率晶体结构揭示了一种独特的二聚体结构，其中Cys-52与另一个亚基的Cys-173通过c端尾部交换形成二硫键。内部二硫键由两个精氨酸残基Arg-123和Arg-151包围。这些位点的突变表现出活性降低。因此，半胱氨酸残基参与过氧化催化并作为中间体形成二硫键。残基Arg-123和Arg-151可能通过降低pK水平与Cys-52相互作用而表现出增强的反应性。HBP23与血红素/蛋白的摩尔比为1:1。血红素部分抑制HBP23上的硫氧嘧啶依赖性过氧化物酶活性。共振拉曼光谱分析表明，血红素与HBP23的氨基酸残基结合。虽然血红素的作用目前还不清楚，但它很有趣，因为这种蛋白质也是由血红素诱导的。Western-blot分析显示HBP23在大鼠肝细胞质组分中是二聚体和十聚体的混合物。

HBP23 (Heme Binding Protein, M.W. : 23kDa), a rat cytosolic protein with high binding affinity for heme, is a member of the peroxiredoxin family, which exhibits thioredoxin-dependent peroxidase activity. The mutants, Cys52Ser, Cys173Ser and Cys52-173Ser, did not show the activity, which was different from that of the mutant (Cys83Ser). A 2.6A-resolutin crystal structure of HBP23 (Cys83Ser) in oxidative form revealed a unique dimer structure in which Cys-52 forms a disulfide bond with Cys-173 from another subunit by C-terminal tail swapping. The internal disulfide bond was surrounding by the two arginine residues, Arg-123 and Arg-151. The mutations at the positions showed reduced activity. Thus, the cysteine residues are involved in the peroxidation catalysis and form a disulfide bond as an intermediate. The residues, Arg-123 and Arg-151 may display enhanced reactivity by interacting with Cys-52 due to decrease the level of pK. HBP23 interacts with heme in a 1 : 1 molar ratio of heme/protein. Thioredoxn-dependent peroxidase activity on HBP23 was inhibited in partially by heme. It was suggested that heme bound to the amino acid residue(s) of HBP23 by analysis of Resonance Raman spectra. Although the role of heme is not clear at the moment, it is of interest, because this protein is also induced by heme. The Western-blot analysis showed that HBP23 is a mixture of dimer and decamer in the rat liver cytosolic fraction.