Analysis of cellular function of HBP23 on redox regulation

HBP 23（血红素结合蛋白，M.W.：23 kDa），一种对血红素具有高结合亲和力的大鼠胞质蛋白，是过氧化物氧还蛋白家族的成员，其表现出硫氧还蛋白依赖性过氧化物酶活性。突变体Cys 52 Ser、Cys 173 Ser和Cys 52 - 173 Ser均未表现出活性，这与突变体Cys 83 Ser的活性不同。氧化形式的HBP 23（Cys 83 Ser）的2.6A-可溶性晶体结构揭示了独特的二聚体结构，其中Cys-52通过C-末端尾部交换与来自另一个亚基的Cys-173形成二硫键。内部二硫键被两个精氨酸残基Arg-123和Arg-151包围。在这些位置的突变显示出降低的活性。因此，半胱氨酸残基参与过氧化催化并形成二硫键作为中间体。Arg-123和Arg-151残基可能通过与Cys-52相互作用而降低pK水平，从而显示出增强的反应性。HBP 23以血红素/蛋白质的1：1摩尔比与血红素相互作用。血红素部分抑制HBP 23上硫氧还蛋白依赖的过氧化物酶活性。共振拉曼光谱分析表明血红素与HBP 23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分析表明，HBP 23在大鼠肝细胞溶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.