Björnberg Olof, Viennet Thibault, Skjoldager Nicklas, Ćurović Aida, Nielsen Kristian Fog, Svensson Birte, Hägglund Per
Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark , Building 224, Søltofts Plads, DK-2800 Kongens Lyngby, Denmark.
Biochemistry. 2015 Mar 3;54(8):1628-37. doi: 10.1021/bi5013639. Epub 2015 Feb 20.
Thioredoxin, involved in numerous redox pathways, is maintained in the dithiol state by the nicotinamide adenine dinucleotide phosphate-dependent flavoprotein thioredoxin reductase (TrxR). Here, TrxR from Lactococcus lactis is compared with the well-characterized TrxR from Escherichia coli. The two enzymes belong to the same class of low-molecular weight thioredoxin reductases and display similar kcat values (∼25 s(-1)) with their cognate thioredoxin. Remarkably, however, the L. lactis enzyme is inactivated by visible light and furthermore reduces molecular oxygen 10 times faster than E. coli TrxR. The rate of light inactivation under standardized conditions (λmax=460 nm and 4 °C) was reduced at lowered oxygen concentrations and in the presence of iodide. Inactivation was accompanied by a distinct spectral shift of the flavin adenine dinucleotide (FAD) that remained firmly bound. High-resolution mass spectrometric analysis of heat-extracted FAD from light-damaged TrxR revealed a mass increment of 13.979 Da, relative to that of unmodified FAD, corresponding to the addition of one oxygen atom and the loss of two hydrogen atoms. Tandem mass spectrometry confined the increase in mass of the isoalloxazine ring, and the extracted modified cofactor reacted with dinitrophenyl hydrazine, indicating the presence of an aldehyde. We hypothesize that a methyl group of FAD is oxidized to a formyl group. The significance of this not previously reported oxidation and the exceptionally high rate of oxygen reduction are discussed in relation to other flavin modifications and the possible occurrence of enzymes with similar properties.
硫氧还蛋白参与众多氧化还原途径，由烟酰胺腺嘌呤二核苷酸磷酸依赖性黄素蛋白硫氧还蛋白还原酶（TrxR）维持在二硫醇状态。在此，将乳酸乳球菌的TrxR与已得到充分表征的大肠杆菌TrxR进行比较。这两种酶属于同一类低分子量硫氧还蛋白还原酶，与其同源硫氧还蛋白显示出相似的催化常数（约25 s⁻¹）。然而，值得注意的是，乳酸乳球菌的这种酶会被可见光灭活，而且还原分子氧的速度比大肠杆菌TrxR快10倍。在标准化条件下（λmax = 460 nm和4℃），光灭活速率在较低氧浓度和存在碘化物的情况下会降低。灭活过程伴随着紧密结合的黄素腺嘌呤二核苷酸（FAD）出现明显的光谱位移。对光损伤的TrxR经热提取的FAD进行高分辨率质谱分析显示，相对于未修饰的FAD，质量增加了13.979 Da，这对应于添加了一个氧原子并损失了两个氢原子。串联质谱分析确定了异咯嗪环质量的增加，并且提取的修饰辅因子与2,4 - 二硝基苯肼反应，表明存在醛基。我们推测FAD的一个甲基被氧化成了甲酰基。结合其他黄素修饰以及具有类似性质的酶可能的存在情况，讨论了这种此前未报道的氧化反应的意义以及异常高的氧还原速率。
