Duarte Américo G, Cordas Cristina M, Moura José J G, Moura Isabel
Requimte, Centro de Química Fina e Biotecnologia, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2829-516 Monte de Caparica, Portugal.
Biochim Biophys Acta. 2014 Mar;1837(3):375-84. doi: 10.1016/j.bbabio.2014.01.001. Epub 2014 Jan 9.
Nitric oxide reductase (NOR) from denitrifying bacteria is an integral membrane protein that catalyses the two electron reduction of NO to N2O, as part of the denitrification process, being responsible for an exclusive reaction, the NN bond formation, the key step of this metabolic pathway. Additionally, this class of enzymes also presents residual oxidoreductase activity, reducing O2 to H2O in a four electron/proton reaction. In this work we report, for the first time, steady-state kinetics with the Pseudomonas nautica NOR, either in the presence of its physiological electron donor (cyt. c552) or immobilised on a graphite electrode surface, in the presence of its known substrates, namely NO or O2. The obtained results show that the enzyme has high affinity for its natural substrate, NO, and different kinetic profiles according to the electron donor used. The kinetic data, as shown by the pH dependence, is modelled by ionisable amino acid residues nearby the di-nuclear catalytic site. The catalytic mechanism is revised and a mononitrosyl-non-heme Fe complex (FeB(II)-NO) species is favoured as the first catalytic intermediate involved on the NO reduction.
反硝化细菌中的一氧化氮还原酶（NOR）是一种整合膜蛋白，作为反硝化过程的一部分，它催化将NO双电子还原为N2O，负责唯一的反应，即NN键的形成，这是该代谢途径的关键步骤。此外，这类酶还具有残留的氧化还原酶活性，在四电子/质子反应中将O2还原为H2O。在这项工作中，我们首次报道了海生假单胞菌NOR的稳态动力学，该酶在存在其生理电子供体（细胞色素c552）时，或固定在石墨电极表面，存在其已知底物（即NO或O2）时的情况。所得结果表明，该酶对其天然底物NO具有高亲和力，并且根据所使用的电子供体具有不同的动力学特征。如pH依赖性所示的动力学数据，由双核催化位点附近的可电离氨基酸残基进行建模。对催化机制进行了修正，单亚硝酰-非血红素铁配合物（FeB(II)-NO）物种被认为是参与NO还原的第一个催化中间体。
