Reimann Joachim, Flock Ulrika, Lepp Håkan, Honigmann Alf, Adelroth Pia
Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
Biochim Biophys Acta. 2007 May;1767(5):362-73. doi: 10.1016/j.bbabio.2007.03.006. Epub 2007 Mar 16.
Nitric oxide reductase (NOR) from P. denitrificans is a membrane-bound protein complex that catalyses the reduction of NO to N(2)O (2NO+2e(-)+2H(+)-->N(2)O+H(2)O) as part of the denitrification process. Even though NO reduction is a highly exergonic reaction, and NOR belongs to the superfamily of O(2)-reducing, proton-pumping heme-copper oxidases (HCuOs), previous measurements have indicated that the reaction catalyzed by NOR is non-electrogenic, i.e. not contributing to the proton electrochemical gradient. Since electrons are provided by donors in the periplasm, this non-electrogenicity implies that the substrate protons are also taken up from the periplasm. Here, using direct measurements in liposome-reconstituted NOR during reduction of both NO and the alternative substrate O(2), we demonstrate that protons are indeed consumed from the 'outside'. First, multiple turnover reduction of O(2) resulted in an increase in pH on the outside of the NOR-vesicles. Second, comparison of electrical potential generation in NOR-liposomes during oxidation of the reduced enzyme by either NO or O(2) shows that the proton transfer signals are very similar for the two substrates proving the usefulness of O(2) as a model substrate for these studies. Last, optical measurements during single-turnover oxidation by O(2) show electron transfer coupled to proton uptake from outside the NOR-liposomes with a tau=15 ms, similar to results obtained for net proton uptake in solubilised NOR [U. Flock, N.J. Watmough, P. Adelroth, Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen, Biochemistry 44 (2005) 10711-10719]. NOR must thus contain a proton transfer pathway leading from the periplasmic surface into the active site. Using homology modeling with the structures of HCuOs as templates, we constructed a 3D model of the NorB catalytic subunit from P. denitrificans in order to search for such a pathway. A plausible pathway, consisting of conserved protonatable residues, is suggested.
反硝化假单胞菌中的一氧化氮还原酶（NOR）是一种膜结合蛋白复合物，作为反硝化过程的一部分，它催化将NO还原为N₂O（2NO + 2e⁻ + 2H⁺ → N₂O + H₂O）。尽管NO还原是一个高度放能反应，且NOR属于还原O₂、质子泵血红素 - 铜氧化酶（HCuOs）超家族，但先前的测量表明，NOR催化的反应是不产生电的，即对质子电化学梯度没有贡献。由于电子由周质中的供体提供，这种不产生电意味着底物质子也从周质中摄取。在此，通过在脂质体重构的NOR中对NO和替代底物O₂进行还原期间的直接测量，我们证明质子确实是从“外部”消耗的。首先，O₂的多次周转还原导致NOR囊泡外部的pH升高。其次，比较NOR脂质体在被NO或O₂氧化还原酶期间产生的电势表明，两种底物的质子转移信号非常相似，证明了O₂作为这些研究的模型底物的有效性。最后，在O₂单周转氧化期间的光学测量表明，电子转移与从NOR脂质体外部摄取质子耦合，时间常数τ = 15毫秒，类似于在可溶NOR中净质子摄取获得的结果[U. Flock, N.J. Watmough, P. Adelroth, 细菌一氧化氮还原酶在氧气还原期间的电子/质子耦合，生物化学44 (2005) 10711 - 10719]。因此，NOR必须包含一条从周质表面通向活性位点的质子转移途径。使用以HCuOs结构为模板的同源建模，我们构建了反硝化假单胞菌NorB催化亚基的三维模型，以寻找这样一条途径。提出了一条由保守的可质子化残基组成的合理途径。
