基于甲烷氧化菌素修饰电极的颗粒性甲烷单加氧酶催化甲烷氧化研究

Particulate methane monooxygenase (PMMO) is a membrane-bound protein that catalyzes the oxidation of methane to methanol in methanotrophs. Understanding the function mechanism of pMMO has important implications for the catalytic conversion of methane and for the development of new, environmentally friendly catalysts. Methanobactin (Mb) is a copper-binding small peptide that is associated with pMMO and appears to be involved in oxidation of methane by pMMO. Mb has been shown to participate the electron transfer to the activity centers of pMMO. Although pMMO metal centers have been characterized spectroscopically, and the crystal structure has been determined, most questions regarding its metal center function, natural electron donor and active site composition remain unknown and controversial; the potential function mechanism of Mb on pMMO is also not elucidated. It is one of the trends of chemistry to use electrocehemical methods in studying biologieal phenomena. This project will undertake direct electrochemistry of pMMO using a Mb-functionalized gold nanoparticles modified gold electrode. Mb will be used as an efficient electron mediator to achieve a communication between the electrode and the redox-active moiety of pMMO. Mb-functionalized gold nanoparticles modified gold electrode will also provide a more congenial surface to mimic the physiological situation and microenvironment. Electrochemical technique such as Cyclic voltammogram(CV), Differential-pulse voltammogram（DPV）, electrochemical impedance spectroscopy ( EIS), optical spectroscopy and in situ spectroelectrochemistry will be employed to probe and characterize the redox potentials of the pMMO metal active centers, the intermolecular electron transfer between Mb and pMMO and the function of Mb. Electrochemically driven cofactor-independent methane oxygenation catalyzed by pMMO in aqueous and nonaqueous system will be investigated. The results obtained from these works will be used in the determination of mechanism of pMMO catalyzed methane oxidization and in the realization of electrochemically driven cofactor-independent methane oxygenation.

颗粒性甲烷单加氧酶（pMMO）是甲烷氧化菌产生的催化甲烷氧化为甲醇的膜蛋白，阐明其作用机理对实现温和条件下甲烷的催化转化和开发新型环境友好催化剂意义重大。甲烷氧化菌素（Mb）是pMMO复合物上的铜结合小肽，参与了pMMO催化甲烷氧化过程的电子传递。有关pMMO的金属中心光谱信息和晶体结构数据虽已获得，但在催化甲烷氧化过程中金属中心功能、生理电子供体、活性位点等方面还存在广泛争论；Mb作用于pMMO的功能机理也未阐明。电化学是研究生命现象的重要手段。本项目使用Mb功能化纳米金修饰电极进行pMMO直接电化学研究。利用Mb在电极和pMMO氧化还原中心之间实现有效电子传递，为pMMO模拟天然内膜系统的生理状态和微环境。通过电化学、谱学和现场光谱电化学表征，水及非水介质生物电催化甲烷氧化研究，获得pMMO金属中心功能信息，阐明催化甲烷氧化的电子传递路径和Mb功能机理，实现无辅酶依赖生物电催化甲烷氧化

颗粒性甲烷单加氧酶（pMMO）是甲烷氧化菌产生的催化甲烷氧化为甲醇的膜蛋白，阐明其作用机理对实现温和条件下甲烷的催化转化和开发新型环境友好催化剂意义重大。甲烷氧化菌素（Mb）是pMMO复合物上的铜结合小肽，参与了pMMO催化甲烷氧化过程的电子传递。本项目利用分离纯化到的pMMO和Mb进行细胞外研究，发现在人工电子供体对苯二酚存在下，Mb在细胞外可以减轻pMMO催化过程中产生的活性氧物种引起的自身失活。Mb存在下pMMO可使用对苯二酚作为人工电子供体驱动甲烷单加氧化反应。Mb/Cu比例变化时，可以形成具有不同的活性氧清除拟酶活性的3种配合物（Mb4-Cu、Mb2-Cu和Mb-Cu）。被功能化到纳米金上后，Mb/Cu配合物拟酶活性提高了近一个数量级。铜离子介导Mb功能化纳米金的配位组装可以进一步提高拟酶活性，推测在二维和三维纳米结构中构建的Mb2-Cu催化中心是其拟酶活性提高的主要原因。多催化中心的纳米团簇组装结构表现出更高的拟酶活性。在上述研究基础上，利用Mb功能化纳米金铜配位组装并对金电极进行修饰实现了Mb-Cu和金电极之间的电子传递，Mb功能化纳米金修饰电极表现出了良好过氧化物酶（POD）拟酶活性、超氧化物歧化酶（SOD）拟酶活性和生物电催化活性氧清除能力。Mb功能化纳米金可以通过Au-S键等结合pMMO。利用Mb功能化纳米金修饰，在电极和pMMO氧化还原中心之间实现了有效电子传递。使用Mb功能化纳米金修饰电极进行pMMO直接电化学研究，分析了pMMO生物电催化动力学行为和Mb对pMMO作用。当测试环境中只含甲烷或氧气时pMMO直接电化学过程为单电子传递，而同时含有甲烷和氧气时为双电子传递。氧物种含量对pMMO直接电化学行为影响显著。pMMO直接电化学结果同样推测出Mb不仅起到接收和向pMMO传递电子作用，也起到稳定pMMO结构和维持特定氧化还原状态的作用。进行了电化学现场谱学表征指认pMMO金属活性中心研究。将pMMO溶解在电解液中，DPV扫描发现无论氧物种含量如何，始终有两个电位峰存在。电化学现场谱学研究pMMO直接电化学的对应谱学信息在335~360 nm波长范围内的变化规律，推测pMMO催化中心为双铜金属中心。基于pMMO直接生物电催化行为，对Mb功能化纳米金修饰电极生物电催化甲烷氧化和TCE环氧化反应的动力学参数进行了测定。