Almeida M Gabriela, Silveira Célia M, Guigliarelli Bruno, Bertrand Patrick, Moura José J G, Moura Isabel, Léger Christophe
REQUIMTE, CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2829-516, Monte de Caparica, Portugal.
FEBS Lett. 2007 Jan 23;581(2):284-8. doi: 10.1016/j.febslet.2006.12.023. Epub 2006 Dec 19.
Cytochrome c nitrite reductase is a multicenter enzyme that uses a five-coordinated heme to perform the six-electron reduction of nitrite to ammonium. In the sulfate reducing bacterium Desulfovibrio desulfuricans ATCC 27774, the enzyme is purified as a NrfA2NrfH complex that houses 14 hemes. The number of closely-spaced hemes in this enzyme and the magnetic interactions between them make it very difficult to study the active site by using traditional spectroscopic approaches such as EPR or UV-Vis. Here, we use both catalytic and non-catalytic protein film voltammetry to simply and unambiguously determine the reduction potential of the catalytic heme over a wide range of pH and we demonstrate that proton transfer is coupled to electron transfer at the active site.
细胞色素c亚硝酸还原酶是一种多中心酶，它利用一个五配位血红素来将亚硝酸盐六电子还原为铵。在硫酸盐还原菌脱硫脱硫弧菌ATCC 27774中，该酶作为一种包含14个血红素的NrfA2NrfH复合物被纯化出来。这种酶中紧密排列的血红素数量以及它们之间的磁相互作用使得使用传统光谱方法（如电子顺磁共振或紫外可见光谱）研究活性位点变得非常困难。在这里，我们使用催化和非催化蛋白质膜伏安法，在很宽的pH范围内简单而明确地测定催化血红素的还原电位，并且我们证明质子转移在活性位点与电子转移相偶联。
