Department of Chemistry, Hugel Science Center, Lafayette College, Easton, Pennsylvania 18042, USA.
J Phys Chem B. 2010 May 27;114(20):7121-30. doi: 10.1021/jp102275r.
Escherichia coli DNA photolyase and cryptochrome 1 isolated from Vibrio cholerae, a member of the CRY-DASH family, are directly compared using a variety of experimental methods including UV-vis and Raman spectroscopy, reduction potential measurements, and isothermal titration calorimetry. The semiquinone form of the cryptochrome has an absorption spectrum that is red-shifted from that of the photolyase, but the Raman spectrum indicates that the FAD binding pocket is similar to that of photolyase. The FADH(-)/FADH* reduction potential of the cryptochrome is significantly higher than that of the photolyase at 164 mV vs NHE, but it also increases upon substrate binding (to 195 mV vs NHE), an increase similar to what is observed in photolyase. The FADH(-)/FADH* reduction potential for both proteins was found to be insensitive to ATP binding. Isothermal titration calorimetry found that photolyase binds tighter to substrate (K(A) approximately 10(5) M(-1) for photolyase and approximately 10(4) M(-1) for cryptochrome 1), and the binding constants for both proteins were slightly sensitive to oxidation state. Based upon this work, it appears that this cryptochrome has significant spectroscopic and electrochemical similarities to CPD photolyase. The thermodynamic cycle of the enzymatic repair in the context of this work is discussed.
从霍乱弧菌中分离出的大肠杆菌 DNA 光解酶和隐色体 1 是 CRY-DASH 家族的成员，使用各种实验方法（包括紫外-可见和拉曼光谱、还原电势测量和等温热滴定法）对其进行了直接比较。隐色体的半醌形式的吸收光谱与光解酶的吸收光谱相比发生了红移，但拉曼光谱表明 FAD 结合口袋与光解酶相似。隐色体的 FADH(-)/FADH还原电势比光解酶高 164 mV（相对于 NHE），但在底物结合后也会增加（至 195 mV（相对于 NHE）），这种增加与光解酶中观察到的相似。发现两种蛋白质的 FADH(-)/FADH还原电势均不受 ATP 结合的影响。等温滴定量热法发现光解酶与底物的结合更紧密（光解酶的 K(A)约为 10(5) M(-1)，隐色体 1 的 K(A)约为 10(4) M(-1)），并且两种蛋白质的结合常数对氧化态略有敏感。基于这项工作，似乎这种隐色体与 CPD 光解酶具有显著的光谱和电化学相似性。讨论了这项工作中酶修复的热力学循环。
