de Poulpiquet Anne, Kjaergaard Christian H, Rouhana Jad, Mazurenko Ievgen, Infossi Pascale, Gounel Sébastien, Gadiou Roger, Giudici-Orticoni Marie Thérèse, Solomon Edward I, Mano Nicolas, Lojou Elisabeth
Aix Marseille Univ, CNRS, BIP, 31 chemin Aiguier, 13402 Marseille, France.
Department of Chemistry, Stanford University, Stanford, California 94305, United States.
ACS Catal. 2017 Jun 2;7(6):3916-3923. doi: 10.1021/acscatal.7b01286. Epub 2017 Apr 27.
Bilirubin oxidases (BODs) belong to the multi-copper oxidase (MCO) family and efficiently reduce O at neutral pH and in physiological conditions where chloride concentrations are over 100 mM. BODs were consequently considered to be Cl resistant contrary to laccases. However, there has not been a detailed study on the related effect of chloride and pH on the redox state of immobilized BODs. Here, we investigate by electrochemistry the catalytic mechanism of O reduction by the thermostable BOD immobilized on carbon nanofibers in the presence of NaCl. The addition of chloride results in the formation of a redox state of the enzyme, previously observed for different BODs and laccases, which is only active after a reductive step. This behavior has not been previously investigated. We show for the first time that the kinetics of formation of this state is strongly dependent on pH, temperature, Cl concentration and on the applied redox potential. UV-visible spectroscopy allows us to correlate the inhibition process by chloride with the formation of the alternative resting form of the enzyme. We demonstrate that O is not required for its formation and show that the application of an oxidative potential is sufficient. In addition, our results suggest that the reactivation may proceed thought the T3 β.
胆红素氧化酶（BODs）属于多铜氧化酶（MCO）家族，在中性pH值和氯化物浓度超过100 mM的生理条件下能有效还原氧气。因此，与漆酶相反，BODs被认为对氯离子具有抗性。然而，关于氯离子和pH值对固定化BODs氧化还原状态的相关影响尚未有详细研究。在此，我们通过电化学方法研究了在NaCl存在下固定在碳纳米纤维上的热稳定BOD还原氧气的催化机制。添加氯离子会导致形成一种酶的氧化还原状态，这种状态之前在不同的BODs和漆酶中观察到，且只有在经过还原步骤后才具有活性。这种行为此前尚未被研究过。我们首次表明，这种状态的形成动力学强烈依赖于pH值、温度、氯离子浓度以及所施加的氧化还原电位。紫外可见光谱使我们能够将氯离子的抑制过程与酶的另一种静止形式的形成联系起来。我们证明其形成不需要氧气，并且表明施加氧化电位就足够了。此外，我们的结果表明再活化可能通过T3β进行。
