Oxidation of heme to β- and δ-biliverdin by Pseudomonas aeruginosa heme oxygenase as a consequence of an unusual seating of the heme
Oxidation of heme to β- and δ-biliverdin by Pseudomonas aeruginosa heme oxygenase as a consequence of an unusual seating of the heme
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DOI：
10.1021/ja0274960
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发表时间：
2002-12-18
影响因子：
15
通讯作者：
Rivera, M
中科院分区：
文献类型：
--
作者：
Caignan, GA;Deshmukh, R;Rivera, M
本文用H-1 NMR、C-13 NMR和共振拉曼光谱研究了铜绿假单胞菌血红素加氧酶（pa-HO）对血红素氧化的特殊区域选择性，即血红素氧化为δ-胆绿素（70%）和β-胆绿素（30%）的起源。而共振拉曼表明，血红素铁连接在pa-HO是同源的，在以前研究的α-羟基化血红素加氧酶中观察到的，NMR光谱研究表明，在这种酶中的血红素是坐在一种方式，这是不同于观察到的所有其他α-羟基化血红素加氧酶的结构是已知的。在pa-HO中，血红素在平面内旋转110度，因此主要取向异构体的δ-内消旋碳位于HO折叠内，在α-羟基化酶通常放置α-内消旋碳的地方。由pa-HO显示的不寻常的血红素座位放置血红素丙酸酯，使得这些基团指向溶剂暴露的血红素边缘的方向，并且似乎在很大程度上源自多肽和血红素丙酸酯之间不存在稳定的相互作用，这通常在α-羟基化血红素加氧酶中发现。这些相互作用通常涉及脑膜炎奈瑟氏球菌HO中的Lys-16和Tyr-112，以及人和大鼠HO-1中的Lys-16和Tyr-134。pa-HO中相应的残基分别是Asn-19和Phe-117。我们发现pa-HO的Asn-19 Lys/Phe-117 Tyr双突变体以两种不同血红素位置的分子混合物形式存在;一个座位与野生型pa-HO所显示的座位相同，而替代的座位与α-羟基化血红素加氧酶的典型座位非常相似，并且与野生型座位的相关性类似于110度，血红素的平面旋转。此外，在pa-HO双突变体中的这些血红素座位中的每一个产生两个血红素异构体取向的子集，所述两个血红素异构体取向通过围绕α-γ-中轴的180度旋转而彼此相关。这些分子在溶液中的共存，在H-1 NMR光谱中的峰下的相应面积所建议的比例，解释了与双突变体，我们发现产生α-（55%），δ-（35%）和β-胆绿素（10%）观察到的血红素氧化的不寻常的区域选择性。α-胆绿素是通过类似于α-羟基化酶的血红素的氧化而获得的，而β-和δ-胆绿素是由野生型pa-HO中血红素的氧化而形成的。
The origin of the unusual regioselectivity of heme oxygenation, i.e. the oxidation of heme to delta-biliverdin (70%) and beta-biliverdin (30%), that is exhibited by heme oxygenase from Pseudomonas aeruginosa (pa-HO) has been studied by H-1 NMR, C-13 NMR, and resonance Raman spectroscopies. Whereas resonance Raman indicates that the heme-iron ligation in pa-HO is homologous to that observed in previously studied a-hydroxylating heme oxygenases, the NMR spectroscopic studies suggest that the heme in this enzyme is seated in a manner that is distinct from that observed for all other a-hydroxylating heme oxygenase enzymes for which a structure is known. In pa-HO, the heme is rotated in-plane similar to110degrees, so the delta-meso-carbon of the major orientational isomer is located within the HO-fold in the place where the a-hydroxylating enzymes typically place the alpha-meso-carbon. The unusual heme seating displayed by pa-HO places the heme propionates so that these groups point in the direction of the solvent-exposed heme edge and appears to originate in large part from the absence of stabilizing interactions between the polypeptide and the heme propionates, which are typically found in alpha-hydroxylating heme oxygenase enzymes. These interactions typically involve Lys-16 and Tyr-112, in Neisseriae meningitidis HO, and Lys-16 and Tyr-134, in human and rat HO-1. The corresponding residues in pa-HO are Asn-19 and Phe-117, respectively. In agreement with this hypothesis, we found that the Asn-19 Lys/Phe-117 Tyr double mutant of pa-HO exists as a mixture of molecules exhibiting two distinct heme seatings; one seating is identical to that exhibited by wild-type pa-HO, whereas the alternative seating is very similar to that typical of alpha-hydroxylating heme oxygenase enzymes and is related to the wild-type seating by similar to110degrees in-plane rotation of the heme. Furthermore, each of these heme seatings in the pa-HO double mutant gives rise to a subset of two heme isomeric orientations that are related to each other by 180degrees rotation about the alpha-gamma-meso-axis. The coexistence of these molecules in solution, in the proportions suggested by the corresponding area under the peaks in the H-1 NMR spectrum, explains the unusual regioselectivity of heme oxygenation observed with the double mutant, which we found produces alpha- (55%), delta- (35%), and beta-biliverdin (10%). a-Biliverdin is obtained by oxidation of the heme seated similar to that of a-hydroxylating enzymes, whereas beta- and delta-biliverdin are formed from the oxidation of heme seated as in wild-type pa-HO.