Lipman R S, Jorns M S
Department of Biochemistry, Medical College of Pennsylvania, Philadelphia, USA.
Biochemistry. 1996 Jun 18;35(24):7968-73. doi: 10.1021/bi9605548.
The folate chromophore in native Escherichia coli DNA photolyase ([6R]-5,10-CH+-H4Pte(Glu)n=3-6) serves as an antenna, transferring light energy to the fully reduced flavin (FADH2) reaction center at high efficiency (EET = 0.92). Apophotolyase reconstituted after an overnight incubation with [6R,S]-5,10-CH+-H4folate (a monoglutamate analogue of the native cofactor) contains equimolar amounts of the [6R]- and [6S]-isomers, suggesting similar binding affinities. A rapid, biphasic increase in fluorescence (approximately 100-fold) is observed upon binding of 5,10-CH+-H4folate to apophotolyase at 5 degrees C; the [6S]-isomer binds about 25-fold faster than the [6R]-isomer. Although identical absorption and fluorescence emission maxima are observed for enzyme reconstituted with [6S]-, [6R]-, or [6R,S]-5,10-CH+-H4folate, folate fluorescence quantum yield values vary depending on the stereochemical configuration at the 6 position (theta = 0.18, 0.82, or 0.46, respectively, at 5 degrees C), a feature not seen with free folate. The fluorescence of enzyme-bound folate is quenched upon flavin binding; the efficiency of quenching by flavin radical (EQ = 0.96) or FADH2 (EQ = 0.89) is the same for both folate isomers. In contrast, energy transfer from folate to FADH2 is sensitive to the stereochemical configuration at the 6 position. The efficiency of energy transfer observed for enzyme containing FADH2 and [6S]-, [6R]-, or [6R,S]-5,10-CH+-H4folate (theta = 0.26, 0.66, or 0.44, respectively) is directly proportional to the fluorescence quantum yield observed for folate in the absence of FADH2, as expected for Förster-type energy transfer. Although less efficient, the unnatural [6S]-isomer is catalytically functional, a feature not previously observed with other folate-dependent enzymes. Fluorescence quantum yield studies at 77 K with free (theta = 0.67) and enzyme-bound (theta = 1.0) folate suggest that differences in solvent exposure may contribute to the fluorescence efficiency differences observed with the enzyme-bound folate isomers at 5 degrees C.
天然大肠杆菌DNA光解酶中的叶酸发色团（[6R]-5,10-CH⁺-H₄Pte(Glu)ₙ，n = 3 - 6）充当天线，将光能高效地转移到完全还原的黄素（FADH₂）反应中心（能量转移效率EET = 0.92）。脱辅基光解酶与[6R,S]-5,10-CH⁺-H₄叶酸（天然辅因子的单谷氨酸类似物）孵育过夜后重构，含有等摩尔量的[6R]-和[6S]-异构体，表明它们具有相似的结合亲和力。在5℃下，5,10-CH⁺-H₄叶酸与脱辅基光解酶结合时，观察到荧光快速双相增加（约100倍）；[6S]-异构体的结合速度比[6R]-异构体快约25倍。尽管用[6S]-、[6R]-或[6R,S]-5,10-CH⁺-H₄叶酸重构的酶观察到相同的吸收和荧光发射最大值，但叶酸荧光量子产率值因6位的立体化学构型而异（在5℃下分别为θ = 0.18、0.82或0.46），这是游离叶酸所没有的特征。黄素结合后，酶结合叶酸的荧光被淬灭；黄素自由基（淬灭效率EQ = 0.96）或FADH₂（淬灭效率EQ = 0.89）对两种叶酸异构体的淬灭效率相同。相比之下，从叶酸到FADH₂的能量转移对6位的立体化学构型敏感。对于含有FADH₂和[6S]-、[6R]-或[6R,S]-5,10-CH⁺-H₄叶酸的酶，观察到的能量转移效率（分别为θ = 0.26、0.66或0.44）与在没有FADH₂时观察到的叶酸荧光量子产率成正比，这符合Förster型能量转移的预期。尽管效率较低，但非天然的[6S]-异构体具有催化功能，这是其他叶酸依赖性酶以前未观察到的特征。在77K下对游离叶酸（θ = 0.67）和酶结合叶酸（θ = 1.0）进行的荧光量子产率研究表明，溶剂暴露的差异可能导致在5℃下观察到的酶结合叶酸异构体的荧光效率差异。
