Hori H, Tsubaki M, Mogi T, Anraku Y
Department of Biophysical Engineering, Faculty of Engineering Science, Osaka University, Toyonaka, Osaka, Japan.
J Biol Chem. 1996 Apr 19;271(16):9254-8. doi: 10.1074/jbc.271.16.9254.
The heme axial ligands of bd-type ubiquinol oxidase of Escherichia coli were studied by EPR and optical spectroscopies using nitric oxide (NO) as a monitoring probe. We found that NO bound to ferrous heme d of the air-oxidized and fully reduced enzymes with very high affinity and to ferrous heme b595 of the fully reduced enzyme with low affinity. EPR spectrum of the 14NO complex of the reduced enzyme exhibited an axially symmetric signal with g-values at g = 2.041 and g = 1.993 and a clear triplet of triplet (or a triplet of doublet for the 15NO complex) superhyperfine structure originating from a nitrogenous proximal ligand trans to NO was observed. This EPR species was assigned to the ferrous heme d-NO complex. This suggests that the proximal axial ligand of heme d is a histidine residue in an anomalous condition or other nitrogenous amino acid residue. Furthermore, the EPR line shape of the ferrous heme d-NO was slightly influenced by the oxidation state of the heme b595. This indicates that heme d exists in close proximity to heme b595 forming a binuclear center. Another axially symmetric EPR signal with g-values at g(parallel) = 2.108 and g(perpendicular) = 2.020 appeared after prolonged incubation of the reduced enzyme with NO and was attributed to the ferrous heme b595-NO complex.
利用一氧化氮（NO）作为监测探针，通过电子顺磁共振（EPR）和光谱学方法研究了大肠杆菌bd型泛醇氧化酶的血红素轴向配体。我们发现，NO与空气氧化的完全还原酶中的亚铁血红素d具有非常高的亲和力结合，与完全还原酶中的亚铁血红素b595具有低亲和力结合。还原酶的14NO复合物的EPR谱显示出一个轴向对称信号，g值为g = 2.041和g = 1.993，并且观察到一个清晰的三重态三重态（或15NO复合物的双重态三重态）超精细结构，其源于与NO反位的含氮近端配体。该EPR物种被归为亚铁血红素d-NO复合物。这表明血红素d的近端轴向配体是处于异常状态的组氨酸残基或其他含氮氨基酸残基。此外，亚铁血红素d-NO的EPR线形略微受到血红素b595氧化态的影响。这表明血红素d与血红素b595紧密相邻存在，形成一个双核中心。在还原酶与NO长时间孵育后，出现了另一个轴向对称的EPR信号，g值为g（平行）= 2.108和g（垂直）= 2.020，该信号归因于亚铁血红素b595-NO复合物。
