Nicolussi Andrea, Auer Markus, Weissensteiner Julia, Schütz Georg, Katz Sonja, Maresch Daniel, Hofbauer Stefan, Bellei Marzia, Battistuzzi Gianantonio, Furtmüller Paul G, Obinger Christian
Department of Chemistry, Division of Biochemistry, Vienna Institute of BioTechnology, BOKU-University of Natural Resources and Life Sciences , Muthgasse 18, A-1190 Vienna, Austria.
Department of Life Sciences, University of Modena and Reggio Emilia , via Campi 103, 41125 Modena, Italy.
Biochemistry. 2017 Aug 29;56(34):4525-4538. doi: 10.1021/acs.biochem.7b00632. Epub 2017 Aug 16.
The existence of covalent heme to protein bonds is the most striking structural feature of mammalian peroxidases, including myeloperoxidase and lactoperoxidase (LPO). These autocatalytic posttranslational modifications (PTMs) were shown to strongly influence the biophysical and biochemical properties of these oxidoreductases. Recently, we reported the occurrence of stable LPO-like counterparts with two heme to protein ester linkages in bacteria. This study focuses on the model wild-type peroxidase from the cyanobacterium Lyngbya sp. PCC 8106 (LspPOX) and the mutants D109A, E238A, and D109A/E238A that could be recombinantly produced as apoproteins in Escherichia coli, fully reconstituted to the respective heme b proteins, and posttranslationally modified by hydrogen peroxide. This for the first time allows not only a direct comparison of the catalytic properties of the heme b and PTM forms but also a study of the impact of D109 and E238 on PTM and catalysis, including Compound I formation and the two-electron reduction of Compound I by bromide, iodide, and thiocyanate. It is demonstrated that both heme to protein ester bonds can form independently and that elimination of E238, in contrast to exchange of D109, does not cause significant structural rearrangements or changes in the catalytic properties neither in heme b nor in the PTM form. The obtained findings are discussed with respect to published structural and functional data of human peroxidases.
共价血红素与蛋白质键的存在是包括髓过氧化物酶和乳过氧化物酶（LPO）在内的哺乳动物过氧化物酶最显著的结构特征。这些自催化的翻译后修饰（PTM）已被证明会强烈影响这些氧化还原酶的生物物理和生化特性。最近，我们报道了在细菌中存在具有两个血红素与蛋白质酯键的稳定LPO样对应物。本研究聚焦于来自蓝藻Lyngbya sp. PCC 8106的模型野生型过氧化物酶（LspPOX）以及突变体D109A、E238A和D109A/E238A，它们可以在大肠杆菌中作为脱辅基蛋白进行重组生产，完全重组成各自的血红素b蛋白，并通过过氧化氢进行翻译后修饰。这首次不仅允许直接比较血红素b和PTM形式的催化特性，还能研究D109和E238对PTM和催化的影响，包括化合物I的形成以及溴离子、碘离子和硫氰酸盐对化合物I的双电子还原。结果表明，两个血红素与蛋白质酯键都可以独立形成，并且与D109的交换相比，E238的消除在血红素b形式和PTM形式中均不会引起显著的结构重排或催化特性变化。我们结合已发表的人类过氧化物酶的结构和功能数据对所得结果进行了讨论。
