Structure and function complex flavo-proteins which produce free radicals

哺乳动物黄嘌呤氧化还原酶是以脱氢酶的形式合成的，但可以通过蛋白分解或半胱氨酸残基的修饰而很容易地转化为氧化物型(XO)。牛乳XDH和XO的晶体结构已有报道。我们确定了一个独特的氨基酸簇，它通过形成XDH/XO转变的继电系统的核心和通过选通通向FAD环的溶剂通道来发挥双重作用。更详细的结构比较和定点突变分析实验表明，Phe549、Arg335、Trp336和Arg427位于将连接肽的修饰传递到活性位点环(Gln423-Lys433)的中继系统的中心。进一步解决了黄嘌呤氧化还原酶在慢底物上羟化反应的关键中间体的晶体结构，在该中间体中形成了产物的碳氧键，但产物仍与钼络合。该中间体在640 nm附近有一个稳定的、宽的电荷转移带。络合物的晶体结构表明，催化不稳定的Mo-OH氧已经与底物的碳原子形成了键。通常见于酶活性部位的水分子不存在于目前的结构中，这可能解释了该中间体对氢氧化物取代配体的稳定性。

Mammalian xanthine oxidoreductase is synthesized as a dehydrogenase (XDH) but can be readily converted to its oxidase form (XO), either by proteolysis or modification of cysteine residues. The crystal structures of bovine milk XDH and XO demonstrated previously. We identify a unique cluster of amino acids, which plays a dual role by forming the core of a relay system for the XDH/XO transition and by gating a solvent channel leading toward the FAD ring. A more detailed structural comparison and site-directed mutagenesis analysis experiments showed that Phe549, Arg335, Trp336 and Arg427 sit at the center of a relay system that transmits modifications of the linker peptide to the active site loop (Gln423-Lys433). Further, we solved the crystal structure of the key intermediate in the hydroxylation reaction of xanthine oxidoreductase with a slow substrate, in which the carbon-oxygen bond of the product is formed, yet the product remains complexed to the molybdenum. This intermediate displays a stable, broad charge-transfer band at around 640 nm. The crystal structure of the complex indicates that the catalytically labile Mo-OH oxygen has formed a bond with a carbon atom of the substrate. A water molecule usually seen in the active site of the enzyme is absent in the present structure, which probably accounts for the stability of this intermediate toward ligand displacement by hydroxide.