Butland G, Spiro S, Watmough N J, Richardson D J
Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom.
J Bacteriol. 2001 Jan;183(1):189-99. doi: 10.1128/JB.183.1.189-199.2001.
The bacterial nitric oxide reductase (NOR) is a divergent member of the family of respiratory heme-copper oxidases. It differs from other family members in that it contains an Fe(B)-heme-Fe dinuclear catalytic center rather than a Cu(B)-heme-Fe center and in that it does not pump protons. Several glutamate residues are conserved in NORs but are absent in other heme-copper oxidases. To facilitate mutagenesis-based studies of these residues in Paracoccus denitrificans NOR, we developed two expression systems that enable inactive or poorly active NOR to be expressed, characterized in vivo, and purified. These are (i) a homologous system utilizing the cycA promoter to drive aerobic expression of NOR in P. denitrificans and (ii) a heterologous system which provides the first example of the expression of an integral-membrane cytochrome bc complex in Escherichia coli. Alanine substitutions for three of the conserved glutamate residues (E125, E198, and E202) were introduced into NOR, and the proteins were expressed in P. denitrificans and E. coli. Characterization in intact cells and membranes has demonstrated that two of the glutamates are essential for normal levels of NOR activity: E125, which is predicted to be on the periplasmic surface close to helix IV, and E198, which is predicted to lie in the middle of transmembrane helix VI. The subsequent purification and spectroscopic characterization of these enzymes established that they are stable and have a wild-type cofactor composition. Possible roles for these glutamates in proton uptake and the chemistry of NO reduction at the active site are discussed.
细菌一氧化氮还原酶（NOR）是呼吸性血红素-铜氧化酶家族中的一个不同成员。它与该家族的其他成员不同，在于它含有一个Fe（B）-血红素-Fe双核催化中心而非Cu（B）-血红素-Fe中心，并且它不泵出质子。几个谷氨酸残基在NOR中保守，但在其他血红素-铜氧化酶中不存在。为了便于对反硝化副球菌NOR中的这些残基进行基于诱变的研究，我们开发了两种表达系统，能够表达无活性或活性较差的NOR，在体内进行表征并纯化。这两种系统分别是：（i）利用cycA启动子驱动NOR在反硝化副球菌中进行有氧表达的同源系统，以及（ii）一种异源系统，它提供了在大肠杆菌中表达完整膜细胞色素bc复合物的首个实例。将三个保守谷氨酸残基（E125、E198和E202）替换为丙氨酸引入NOR中，并在反硝化副球菌和大肠杆菌中表达这些蛋白质。在完整细胞和膜中的表征表明，其中两个谷氨酸对于NOR的正常活性水平至关重要：E125预计位于靠近螺旋IV的周质表面，E198预计位于跨膜螺旋VI的中部。随后对这些酶进行纯化和光谱表征，确定它们是稳定的且具有野生型辅因子组成。讨论了这些谷氨酸在质子摄取以及活性位点处NO还原化学过程中的可能作用。
