Yue K T, Bhattacharyya A K, Zhelyaskov V R, Edmondson D E
Department of Physics, Emory University, Atlanta, Georgia 30322.
Arch Biochem Biophys. 1993 Jan;300(1):178-85. doi: 10.1006/abbi.1993.1025.
The flavoprotein monoamine oxidase B (MAO B) from bovine liver, as isolated, has an unusual additional absorption band at 412 nm, which is similar to the absorption of its anionic flavin semiquinone form, (Fl.-), and other typical (Fl.-) flavoproteins. Denaturation of the enzyme results in the elimination of this anomalous absorption. The resonance Raman (RR) spectrum of MAO B as isolated is virtually identical to that of its dithionite-reduced (Fl.-) form. Both spectra show features similar to those of the RR spectrum of the (Fl.-) form of Aspergillus niger glucose oxidase (GO) in the region between 300 and 1700 cm-1 with 406.7 nm excitation. These features are readily distinguishable from those of oxidized flavin, neutral flavin semiquinone, and hemoprotein, strongly suggesting the presence of an (Fl.-) form in MAO B as isolated, even with preparations isolated in the absence of light. There are significant differences between the RR spectra of the (Fl.-) form of MAO B and those of GO or the published RR spectra of the (Fl.-) form of D-amino acid oxidase with excess substrate analog. At least some of these differences can be attributed to the different binding of flavin in the three enzymes. No EPR signals due to (Fl.-) are observed in MAO B as isolated. The dithionite-reduced (Fl.-) form exhibits approximately 50% less EPR signal than that expected from the absorption spectrum, which suggests a possible coupling of the (Fl.-) flavin with a paramagnetic center of unknown identity in the protein. The implications of these observations on MAO B with the current view of its catalytic mechanism are discussed.
从牛肝中分离得到的黄素蛋白单胺氧化酶B（MAO B），在412 nm处有一个异常的附加吸收带，这与它的阴离子黄素半醌形式（Fl.-）以及其他典型的（Fl.-）黄素蛋白的吸收相似。该酶的变性导致这种异常吸收的消除。分离得到的MAO B的共振拉曼（RR）光谱与其连二亚硫酸盐还原的（Fl.-）形式的光谱几乎相同。在406.7 nm激发下，在300至1700 cm-1区域内，这两种光谱都显示出与黑曲霉葡萄糖氧化酶（GO）的（Fl.-）形式的RR光谱相似的特征。这些特征很容易与氧化黄素、中性黄素半醌和血红蛋白的特征区分开来，这强烈表明即使在无光条件下分离得到的制剂中，分离得到的MAO B中也存在（Fl.-）形式。MAO B的（Fl.-）形式的RR光谱与GO的（Fl.-）形式的RR光谱或已发表的过量底物类似物存在时D-氨基酸氧化酶的（Fl.-）形式的RR光谱之间存在显著差异。这些差异中至少有一些可归因于三种酶中黄素的不同结合方式。在分离得到的MAO B中未观察到由于（Fl.-）产生的电子顺磁共振（EPR）信号。连二亚硫酸盐还原的（Fl.-）形式的EPR信号比吸收光谱预期的少约50%，这表明（Fl.-）黄素可能与蛋白质中身份不明的顺磁中心发生了耦合。本文讨论了这些关于MAO B的观察结果对其当前催化机制观点的影响。
