Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX, U.S.A.
Department of Anesthesiology, University of Nebraska Medical Center, Omaha, NE 68198, U.S.A.
Biochem J. 2021 May 14;478(9):1795-1808. doi: 10.1042/BCJ20210091.
To inculcate biocatalytic activity in the oxygen-storage protein myoglobin (Mb), a genetically engineered myoglobin mutant H64DOPA (DOPA = L-3,4-dihydroxyphenylalanine) has been created. Incorporation of unnatural amino acids has already demonstrated their ability to accomplish many non-natural functions in proteins efficiently. Herein, the presence of redox-active DOPA residue in the active site of mutant Mb presumably stabilizes the compound I in the catalytic oxidation process by participating in an additional hydrogen bonding (H-bonding) as compared to the WT Mb. Specifically, a general acid-base catalytic pathway was achieved due to the availability of the hydroxyl moieties of DOPA. The reduction potential values of WT (E° = -260 mV) and mutant Mb (E° = -300 mV), w.r.t. Ag/AgCl reference electrode, in the presence of hydrogen peroxide, indicated an additional H-bonding in the mutant protein, which is responsible for the peroxidase activity of the mutant Mb. We observed that in the presence of 5 mM H2O2, H64DOPA Mb oxidizes thioanisole and benzaldehyde with a 10 and 54 folds higher rate, respectively, as opposed to WT Mb. Based on spectroscopic, kinetic, and electrochemical studies, we deduce that DOPA residue, when present within the distal pocket of mutant Mb, alone serves the role of His/Arg-pair of peroxidases.
为了在储氧蛋白肌红蛋白（Mb）中引入生物催化活性，已经构建了一种经过基因工程改造的肌红蛋白突变体 H64DOPA（DOPA = L-3,4-二羟基苯丙氨酸）。非天然氨基酸的掺入已经证明了它们在蛋白质中高效完成许多非天然功能的能力。在此，突变体 Mb 活性位点中存在氧化还原活性的 DOPA 残基，通过参与额外的氢键（H 键），与 WT Mb 相比，可能在催化氧化过程中稳定化合物 I。具体而言，由于 DOPA 的羟基部分的可用性，实现了一般的酸碱催化途径。WT（E° = -260 mV）和突变体 Mb（E° = -300 mV）的还原电位值，相对于 Ag/AgCl 参比电极，在存在过氧化氢的情况下，表明突变体蛋白中存在额外的 H 键，这是突变体 Mb 过氧化物酶活性的原因。我们观察到，在存在 5 mM H2O2 的情况下，H64DOPA Mb 分别以 10 倍和 54 倍的更高速率氧化硫代茴香醚和苯甲醛，而 WT Mb 则没有。基于光谱、动力学和电化学研究，我们推断当 DOPA 残基存在于突变体 Mb 的远端口袋中时，它独自充当过氧化物酶的 His/Arg 对的作用。
