Pearson Arwen R, Wilmot Carrie M
Department of Biochemistry, Molecular Biology and Biophysics, The University of Minnesota, 6-155 Jackson Hall, 321 Church St., Minneapolis, MN 55455, USA.
Biochim Biophys Acta. 2003 Apr 11;1647(1-2):381-9. doi: 10.1016/s1570-9639(03)00099-2.
Methylamine dehydrogenase (MADH) is produced by a range of gram-negative methylotrophic and autotrophic bacteria, and allows the organisms to utilise methylamine as the sole source of carbon. The enzyme catalyses the oxidation of methylamine to formaldehyde and ammonia, leaving it in a two-electron reduced state. To complete the catalytic cycle, MADH is reoxidised via an electron transfer (ET) chain. The redox center in the enzyme is the organic cofactor tryptophan tryptophylquinone (TTQ) derived from the posttranslational modification of two Trp residues in the protein. This cofactor has spectral features in the visible region, which change during catalytic turnover, defining spectrally distinct reaction intermediates that reflect the electronic state of the TTQ. In the case of the Paracoccus denitrificans enzyme the physiologic ET chain involves the protein redox partner amicyanin (a blue copper protein). A stable binary (MADH/amicyanin) complex can be formed, and its crystal structure has been solved to 2.5 A resolution by Chen et al. [Biochemistry 21 (1992) 4959]. These crystals were shown to be competent for catalysis and ET by Merli et al. [J. Biol. Chem. 271 (1996) 9177] using single crystal polarised absorption spectroscopy. Through a novel combination of single crystal visible microspectrophotometry, X-ray crystallography and freeze-trapping, we have trapped reaction intermediates of the enzyme in complex with its physiological redox partner amicyanin in the crystalline state. We will present data confirming that catalysis and ET in the binary complex crystals can be tracked by single crystal visible microspectrophotometry. We will also show that the reaction pathway is unperturbed by the presence of cryoprotectant solution, enabling direct freeze-trapping of reaction intermediates within the crystal. We will present new data demonstrating that the binary complex crystals are also capable of exhibiting UV light-dependent oxidase activity, as observed in solution [Biochim. Biophys. Acta 1364 (1998) 297].
甲胺脱氢酶（MADH）由多种革兰氏阴性甲基营养菌和自养菌产生，使这些生物体能够利用甲胺作为唯一碳源。该酶催化甲胺氧化为甲醛和氨，使其处于双电子还原状态。为了完成催化循环，MADH通过电子传递（ET）链进行再氧化。酶中的氧化还原中心是有机辅因子色氨酸色氨酰醌（TTQ），它源自蛋白质中两个Trp残基的翻译后修饰。该辅因子在可见光区域具有光谱特征，在催化周转过程中会发生变化，定义了反映TTQ电子状态的光谱上不同的反应中间体。就反硝化副球菌的酶而言，生理ET链涉及蛋白质氧化还原伙伴蓝铜蛋白（一种蓝色铜蛋白）。可以形成稳定的二元（MADH/蓝铜蛋白）复合物，Chen等人[《生物化学》21（1992）4959]已将其晶体结构解析至2.5 Å分辨率。Merli等人[《生物化学杂志》271（1996）9177]使用单晶偏振吸收光谱法表明这些晶体具有催化和ET能力。通过将单晶可见显微分光光度法、X射线晶体学和冷冻捕获进行新颖的结合，我们已在结晶状态下捕获了该酶与其生理氧化还原伙伴蓝铜蛋白复合物的反应中间体。我们将展示数据，证实二元复合物晶体中的催化和ET可以通过单晶可见显微分光光度法进行追踪。我们还将表明，反应途径不受冷冻保护剂溶液的存在干扰，从而能够直接在晶体内冷冻捕获反应中间体。我们将展示新数据，证明二元复合物晶体也能够表现出溶液中观察到的紫外光依赖性氧化酶活性[《生物化学与生物物理学报》1364（1998）297]。
