Yu Lu, Liu Aokun, Kuang Jian, Wei Ruotong, Wang Zhiwen, Tian Changlin
High Magnetic Field Laboratory, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, China.
Department of Endocrinology, Institute of Endocrine and Metabolic Disease, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Hefei National Research Center for Physical Sciences at the Microscale, Joint Center for Biological Analytical Chemistry, Anhui Engineering Laboratory of Peptide Drug, Anhui Laboratory of Advanced Photonic Science and Technology, University of Science and Technology of China, Hefei, 230026, China.
Magn Reson Lett. 2024 Mar 28;4(3):200116. doi: 10.1016/j.mrl.2024.200116. eCollection 2024 Aug.
Bacterial small laccases (SLAC) are promising industrial biocatalysts due to their ability to oxidize a broad range of substrates with exceptional thermostability and tolerance for alkaline pH. Electron transfer between substrate, copper centers, and O is one of the key steps in the catalytic turnover of SLAC. However, limited research has been conducted on the electron transfer pathway of SLAC and SLAC-catalyzed reactions, hindering further engineering of SLAC to produce tunable biocatalysts for novel applications. Herein, the combinational use of electron paramagnetic resonance (EPR) and ultraviolet-visible (UV-vis) spectroscopic methods coupled with redox titration were employed to monitor the electron transfer processes and obtain further insights into the electron transfer pathway in SLAC. The reduction potentials for type 1 copper (T1Cu), type 2 copper (T2Cu) and type 3 copper (T3Cu) were determined to be 367 ± 2 mV, 378 ± 5 mV and 403 ± 2 mV, respectively. Moreover, the reduction potential of a selected substrate of SLAC, hydroquinone (HQ), was determined to be 288 mV using cyclic voltammetry (CV). In this way, an electron transfer pathway was identified based on the reduction potentials. Specifically, electrons are transferred from HQ to T1Cu, then to T2Cu and T3Cu, and finally to O. Furthermore, superhyperfine splitting observed via EPR during redox titration indicated a modification in the covalency of T2Cu upon electron uptake, suggesting a conformational alteration in the protein environment surrounding the copper sites, which could potentially influence the reduction potential of the copper sites during catalytic processes. The results presented here not only provide a comprehensive method for analyzing the electron transfer pathway in metalloenzymes through reduction potential measurements, but also offer valuable insights for further engineering and directed evolution studies of SLAC in the aim for biotechnological and industrial applications.
细菌小漆酶（SLAC）是很有前景的工业生物催化剂，因为它们能够氧化多种底物，具有出色的热稳定性和对碱性pH的耐受性。底物、铜中心和氧之间的电子转移是SLAC催化周转的关键步骤之一。然而，关于SLAC的电子转移途径和SLAC催化反应的研究有限，这阻碍了对SLAC进行进一步工程改造以生产用于新型应用的可调生物催化剂。在此，采用电子顺磁共振（EPR）和紫外可见（UV-vis）光谱方法结合氧化还原滴定的组合使用来监测电子转移过程，并进一步深入了解SLAC中的电子转移途径。确定1型铜（T1Cu）、2型铜（T2Cu）和3型铜（T3Cu）的还原电位分别为367±2 mV、378±5 mV和403±2 mV。此外，使用循环伏安法（CV）测定SLAC的选定底物对苯二酚（HQ）的还原电位为288 mV。通过这种方式，基于还原电位确定了电子转移途径。具体而言，电子从HQ转移到T1Cu，然后转移到T2Cu和T3Cu，最后转移到O。此外，在氧化还原滴定过程中通过EPR观察到的超超精细分裂表明，T2Cu在摄取电子时共价性发生了改变，这表明铜位点周围蛋白质环境的构象发生了变化，这可能会在催化过程中影响铜位点的还原电位。这里展示的结果不仅提供了一种通过还原电位测量分析金属酶中电子转移途径的综合方法，还为旨在用于生物技术和工业应用的SLAC的进一步工程改造和定向进化研究提供了有价值的见解。
