木质素过氧化物酶对细胞色素c的不可逆氧化作用。

Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland 97291-1000, USA.

Biochemistry. 1998 Feb 17;37(7):2029-36. doi: 10.1021/bi972198e.

Lignin peroxidase (LiP) from Phanerochaete chrysosporium catalyzes irreversible oxidative damage to ferricytochrome c (Cc3+) in the presence of H2O2 and 3,4-dimethoxybenzyl (veratryl) alcohol (VA). Atomic absorption analysis and UV/vis spectroscopy indicate that the oxidation of Cc3+ is accompanied by a loss of heme iron from the protein and probably oxidation of the porphyrin ring. At H2O2 concentrations of 7.5 microM or higher, this oxidation of Cc3+ by LiP is strictly dependent on the presence of VA. The latter is not oxidized to veratraldehyde at a significant rate in the presence of either ferrocytochrome c (Cc2+) or Cc3+, indicating it is not stimulating the reactions by specifically reducing LiP compound II. LiP is inactivated rapidly in 100 microM H2O2, and the presence of 500 microM VA protects LiP from this inactivation. Neither 20 microM Cc3+ nor 20 microM VA alone can protect LiP from inactivation; however, 20 microM each of VA and Cc3+ together protect LiP fully. This and other results strongly suggest that VA is acting as a protein-bound redox mediator in the oxidation of Cc3+. SDS-PAGE analysis of the Cc3+ oxidation products demonstrates the formation of some covalently linked dimer of Cc3+ in addition to the oxidized Cc3+ monomer. Amino acid analysis of the dimeric and monomeric products indicates the presence of oxidized Met and Tyr residues. This suggests that Tyr residues on the surface of the protein are oxidized to Tyr radicals during LiP oxidation and that some of these radicals subsequently undergo intermolecular radical coupling, resulting in dimerization of some of the Cc3+ molecules. However, most of the Cc3+ molecules appear to be irreversibly oxidized without dimerization. These results demonstrate that Cc3+ can serve as a useful polymeric model of the lignin substrate in studying the enzymatic mechanism of lignin oxidation and the role of VA in the reaction.

来自黄孢原毛平革菌的木质素过氧化物酶（LiP）在过氧化氢（H₂O₂）和3,4 - 二甲氧基苄基（藜芦醇，VA）存在的情况下，催化对高铁细胞色素c（Cc³⁺）的不可逆氧化损伤。原子吸收分析和紫外/可见光谱表明，Cc³⁺的氧化伴随着蛋白质中血红素铁的损失以及卟啉环可能的氧化。在H₂O₂浓度为7.5微摩尔或更高时，LiP对Cc³⁺的这种氧化严格依赖于VA的存在。在亚铁细胞色素c（Cc²⁺）或Cc³⁺存在下，VA不会以显著速率氧化为藜芦醛，这表明它不是通过特异性还原LiP化合物II来刺激反应。LiP在100微摩尔H₂O₂中会迅速失活，500微摩尔VA的存在可保护LiP免于这种失活。单独的20微摩尔Cc³⁺或20微摩尔VA都不能保护LiP免于失活；然而，20微摩尔的VA和Cc³⁺一起能完全保护LiP。这一结果及其他结果强烈表明，VA在Cc³⁺的氧化过程中充当蛋白质结合的氧化还原介质。对Cc³⁺氧化产物的SDS - PAGE分析表明，除了氧化的Cc³⁺单体之外，还形成了一些共价连接的Cc³⁺二聚体。对二聚体和单体产物的氨基酸分析表明存在氧化的甲硫氨酸和酪氨酸残基。这表明在LiP氧化过程中，蛋白质表面的酪氨酸残基被氧化为酪氨酸自由基，并且其中一些自由基随后发生分子间自由基偶联，导致一些Cc³⁺分子二聚化。然而，大多数Cc³⁺分子似乎在没有二聚化的情况下被不可逆地氧化。这些结果表明，在研究木质素氧化的酶促机制以及VA在反应中的作用时，Cc³⁺可作为木质素底物的有用聚合物模型。