Kavakli I Halil, Sancar Aziz
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA.
Biochemistry. 2004 Dec 7;43(48):15103-10. doi: 10.1021/bi0478796.
Escherichia coli DNA photolyase contains FADH(-) as the catalytic cofactor. The cofactor becomes oxidized to the FADH() blue neutral radical during purification. The E-FADH() form of the enzyme is catalytically inert but can be converted to the active E-FADH(-) form by a photoreduction reaction that involves intraprotein electron transfer from Trp306. It is thought that the E-FADH() form is also transiently generated during pyrimidine dimer repair by photoinduced electron transfer, and it has been suggested that the FADH() that is generated after each round of catalysis must be photoreduced before the enzyme can engage in subsequent rounds of repair. In this study, we introduced the Trp306Phe mutation into the chromosomal gene and tested the non-photoreducible W306F mutant for photorepair in vivo. We find that both wild-type and W306F mutant photolyases carry out at least 25 rounds of photorepair at the same rate. We conclude that photoreduction by intraprotein electron transfer is not part of the photolyase photocycle under physiological conditions.
大肠杆菌DNA光解酶含有FADH(-)作为催化辅因子。在纯化过程中，该辅因子会被氧化为FADH()蓝色中性自由基。酶的E-FADH()形式无催化活性，但可通过涉及从Trp306进行蛋白内电子转移的光还原反应转化为活性E-FADH(-)形式。据认为，在嘧啶二聚体修复过程中，通过光诱导电子转移也会短暂生成E-FADH()形式，并且有人提出，每一轮催化后产生的FADH()必须在酶能够进行后续轮次的修复之前进行光还原。在本研究中，我们将Trp306Phe突变引入染色体基因，并测试了不可光还原的W306F突变体在体内的光修复能力。我们发现野生型和W306F突变体光解酶都以相同的速率进行至少25轮的光修复。我们得出结论，在生理条件下，通过蛋白内电子转移进行的光还原不是光解酶光循环的一部分。
