Hemp James, Christian Caroline, Barquera Blanca, Gennis Robert B, Martínez Todd J
Department of Chemistry, University of Illinois, Urbana, Illinois 61801, USA.
Biochemistry. 2005 Aug 16;44(32):10766-75. doi: 10.1021/bi050464f.
In the respiratory chains of mitochondria and many aerobic prokaryotes, heme-copper oxidases are the terminal enzymes that couple the reduction of molecular oxygen to proton pumping, contributing to the protonmotive force. The cbb(3) oxidases belong to the superfamily of enzymes that includes all of the heme-copper oxidases. Sequence analysis indicates that the cbb(3) oxidases are missing an active-site tyrosine residue that is absolutely conserved in all other known heme-copper oxidases. In the other heme-copper oxidases, this tyrosine is known to be subject to an unusual post-translational modification and to play a critical role in the catalytic mechanism. The absence of this tyrosine in the cbb(3) oxidases raises the possibility that the cbb(3) oxidases utilize a different catalytic mechanism from that of the other members of the superfamily. Using homology modeling, quantum chemistry, and molecular dynamics, a model of the structure of subunit I of a cbb(3) oxidase (Vibrio cholerae) was constructed. The model predicts that a tyrosine residue structurally analogous to the active-site tyrosine in other oxidases is present in the cbb(3) oxidases but that the tyrosine originates from a different transmembrane helix within the protein. The predicted active-site tyrosine is conserved in the sequences of all of the known cbb(3) oxidases. Mutagenesis of the tyrosine to phenylalanine in the V. cholerae oxidase resulted in a fully assembled enzyme with nativelike structure but lacking catalytic activity. These findings strongly suggest that all of the heme-copper oxidases utilize the same catalytic mechanism and provide an unusual example in which a critical active-site residue originates from different places within the primary sequence for different members of the same superfamily.
在线粒体和许多需氧原核生物的呼吸链中，血红素 - 铜氧化酶是将分子氧的还原与质子泵浦偶联的终端酶，有助于质子动力势的形成。cbb(3)氧化酶属于包括所有血红素 - 铜氧化酶的酶超家族。序列分析表明，cbb(3)氧化酶缺少一个在所有其他已知血红素 - 铜氧化酶中绝对保守的活性位点酪氨酸残基。在其他血红素 - 铜氧化酶中，已知该酪氨酸会经历一种不寻常的翻译后修饰，并在催化机制中起关键作用。cbb(3)氧化酶中缺少这种酪氨酸增加了cbb(3)氧化酶利用与超家族其他成员不同的催化机制的可能性。利用同源建模、量子化学和分子动力学，构建了一种cbb(3)氧化酶（霍乱弧菌）亚基I的结构模型。该模型预测，在cbb(3)氧化酶中存在一个在结构上类似于其他氧化酶活性位点酪氨酸的酪氨酸残基，但该酪氨酸来自蛋白质内不同的跨膜螺旋。预测的活性位点酪氨酸在所有已知cbb(3)氧化酶的序列中都是保守的。将霍乱弧菌氧化酶中的酪氨酸突变为苯丙氨酸导致形成一种具有天然结构但缺乏催化活性的完全组装酶。这些发现强烈表明，所有血红素 - 铜氧化酶都利用相同的催化机制，并提供了一个不同寻常的例子，即对于同一超家族的不同成员，一个关键的活性位点残基来自一级序列中的不同位置。
