Properties and reactivity of myoglobin reconstituted with chemically modified protohemin complexes
Properties and reactivity of myoglobin reconstituted with chemically modified protohemin complexes
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DOI：
10.1021/bi000784t
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发表时间：
2000-08-08
期刊：
影响因子：
2.9
通讯作者：
Chillemi, F
中科院分区：
文献类型：
--
作者：
Monzani, E;Alzuet, G;Chillemi, F
合成复合物原血红素-6(7)-L-精酰基-L-丙氨酸(HM-RA)和原血红素-6(7)L-组氨酸甲酯(HM-H)是通过适当保护的Arg-Ala或His残基与原血红素IX缩合制备的。 HM-RA 和 HM-H 用于重建来自马心脏的脱辅基肌红蛋白，分别产生该蛋白质的 Mb-RA 和 Mb-H 衍生物。 Mb-RA 和 Mb-H 的光谱、结合和催化特性与 Mb 显着不同。如 MM 和 MD 计算所示，这些差异是由血红素周围的一些局部结构变化决定的，这些变化是由关键肽段 (Phe43-Lys47) 的迁移率增加而产生的，该肽段含有天然 Mb 中与卟啉羧酸基团之一相互作用的残基 (Lys45)。在重建的 Mb 中，该羧酸根基团与 Arg-Ala 或 His 残基结合，不再可与 Lys45 进行静电相互作用。活性位点附近肽段的移动性使得远端组氨酸能够与血红素更紧密地接触，事实上，Mb-RA 和 Mb-H 作为高自旋形式和含有双咪唑连接血红素的主要低自旋六配位形式之间的平衡存在。这两种形式在 NMR 谱中清晰可辨，这也表明它们均由辅因子重构产生的两种最稳定异构体的混合物组成，正如 MM 和 MD 计算所预期的那样。外源配体如氰化物、叠氮化物或过氧化氢可以取代结合的远端组氨酸，但它们的亲和力会降低。另一方面，相对于天然 Mb，重组 Mb 中血红素周围肽链的动员增加了血红素外围大供体分子的可及性，其中刚性主链限制了对远端口袋的访问。 Mb-RA 和 Mb-H 活性位点可及性的增加促进了在过氧化氢存在下重构蛋白催化的过氧化物酶型氧化中酚类底物的结合和电子转移。
The synthetic complexes protohemin-6(7)-L-arginyl-L-alanine (HM-RA) and protohemin-6(7)L-histidine methyl ester (HM-H) were prepared by condensation of suitably protected Arg-Ala or His residues with protohemin IX. HM-RA and HM-H were used for reconstitution of apomyoglobin from horse heart, yielding the Mb-RA and Mb-H derivatives, respectively, of the protein. The spectral, binding and catalytic properties of Mb-RA and Mb-H are significantly different from those of Mb. As shown by MM and MD calculations, these differences are determined by some local structural changes around the heme which are generated by increased mobility of a key peptide segment (Phe43-Lys47), containing the residue (Lys45) that in native Mb interacts with one of the porphyrin carboxylate groups. In the reconstituted Mbs this carboxylate group is bound to the Arg-Ala or His residue and is no longer available for electrostatic interaction with Lys45. The mobility of the peptide segment near the active site allows the distal histidine to come to a closer contact with the heme, and in fact Mb-RA and Mb-H exist as an equilibrium between a high-spin form and a major low-spin, six-coordinated form containing a bis-imidazole ligated heme. The two forms are clearly distinguishable in the NMR spectra, that also show that each of them consists of a mixture of the two most stable isomers resulting from cofactor reconstitution, as also anticipated by MM and MD calculations. Exogenous ligands such as cyanide, azide, or hydrogen peroxide can displace the bound distal histidine, but their affinity is reduced. On the other hand, mobilization of the peptide chain around the heme in the reconstituted Mbs increases the accessibility of large donor molecules at the heme periphery, with respect to native Mb, where a rigid backbone limits access to the distal pocket. The increased active site accessibility of Mb-RA and Mb-H facilitates the binding and electron transfer of phenolic substrates in peroxidase-type oxidations catalyzed by the reconstituted proteins in the presence of hydrogen peroxide.