Alberta J A, Andersson L A, Dawson J H
Department of Chemistry, University of South Carolina, Columbia 29208.
J Biol Chem. 1989 Dec 5;264(34):20467-73.
Secondary amine mono-oxygenase from Pseudomonas aminovorans catalyzes the NAD(P)H- and dioxygen-dependent N-dealkylation of secondary amines to yield a primary amine and an aldehyde. Heme iron, flavin, and non-heme iron prosthetic groups are known to be present in the oligomeric enzyme. The N-dealkylation reaction is also catalyzed by the only other heme-containing mono-oxygenase, cytochrome P-450. In order to identify the heme iron axial ligands of secondary amine mono-oxygenase so as to better define the structural requirements for oxygen activation by heme enzymes, we have investigated the spectroscopic properties of the enzyme. The application of three different spectroscopic techniques, UV-visible absorption, magnetic circular dichroism and electron paramagnetic resonance, to study eight separate enzyme derivatives has provided extensive and convincing evidence for the presence of a proximal histidine ligand. This conclusion is based primarily on comparisons of the spectral properties of the enzyme with those of parallel derivatives of myoglobin (histidine proximal ligand) and P-450 (cysteinate proximal ligand). Spectral studies of ferric secondary amine mono-oxygenase as a function of pH have led to the proposal that the distal ligand is water. Deprotonation of the distal water ligand occurs upon either raising the pH to 9.0 or substrate (dimethylamine) binding. In contrast, the deoxyferrous enzyme appears to have a weakly bound nitrogen donor distal ligand. Initial spectroscopic studies of the iron-sulfur units in the enzyme are interpreted in terms of a pair of Fe2S2 clusters. Secondary amine mono-oxygenase is unique in its ability to function as cytochrome P-450 in activating molecular oxygen but to do so with a myoglobin-like active site. As such, it provides an important system with which to probe structure-function relations in heme-containing oxygenases.
食氨假单胞菌的仲胺单加氧酶催化依赖于NAD(P)H和双加氧的仲胺N-脱烷基反应，生成伯胺和醛。已知寡聚酶中存在血红素铁、黄素和非血红素铁辅基。N-脱烷基反应也由唯一另一种含血红素的单加氧酶——细胞色素P-450催化。为了确定仲胺单加氧酶的血红素铁轴向配体，以便更好地定义血红素酶激活氧的结构要求，我们研究了该酶的光谱性质。应用三种不同的光谱技术，紫外-可见吸收光谱、磁圆二色光谱和电子顺磁共振光谱，对八种不同的酶衍生物进行研究，为存在近端组氨酸配体提供了广泛且有说服力的证据。这一结论主要基于该酶与肌红蛋白（近端配体为组氨酸）和P-450（近端配体为半胱氨酸）的平行衍生物的光谱性质比较。对高铁仲胺单加氧酶随pH变化的光谱研究表明，远端配体是水。当pH升高到9.0或底物（二甲胺）结合时，远端水配体发生去质子化。相比之下，脱氧亚铁酶似乎有一个弱结合的氮供体远端配体。对该酶中铁硫单元的初步光谱研究解释为一对Fe2S2簇。仲胺单加氧酶在激活分子氧方面具有独特的能力，能够像细胞色素P-450一样发挥作用，但具有类似肌红蛋白的活性位点。因此，它为研究含血红素加氧酶的结构-功能关系提供了一个重要的体系。
