Nishina Y, Sato K, Miura R, Matsui K, Shiga K
Department of Physiology, Kumamoto University School of Medicine, Kumamoto, 860-0811, Japan.
J Biochem. 1998 Jul;124(1):200-8. doi: 10.1093/oxfordjournals.jbchem.a022081.
4-Carbonyl-18O]-enriched lumiflavin, riboflavin, and FMN were prepared by incubating each corresponding non-labeled flavin in 1 M Na18OH (H218O) at 25 degrees C. [4-Carbonyl-18O]FAD was prepared from the corresponding riboflavin by using FAD synthetase. Isotope effects by [4-carbonyl-18O]-labeling confirmed that the 1,709-cm-1 band in the IR spectrum of lumiflavin and the 1,711-cm-1 band in the Raman spectrum of FAD are mainly derived from C(4)=O stretching vibrational mode. The 1,605-cm-1 Raman band of the anionic reduced flavin in the purple intermediate of D-amino acid oxidase (DAO) with D-proline or D-alanine does not shift in DAO reconstituted with [4-carbonyl-18O]FAD, although it shifts with [4,10a-13C2]- or [4a-13C]FAD. Thus the band is mainly due to the C(4a)=C(10a) stretching vibrational mode and includes no contribution from C(4)=O stretching vibration. The band frequencies cover a fairly wide range (1,602-1,620 cm-1) depending on the enzymes. The frequencies of the reduced flavin in the purple intermediates of the dehydrogenases (medium-chain acyl-CoA, short-chain acyl-CoA, and isovaleryl-CoA dehydrogenases) are higher than those of the oxidases (DAO and L-phenylalanine oxidase). This indicates that the C(4a)=C(10a) bond order of reduced flavin in the dehydrogenases with the low reactivity for molecular oxygen is stronger than that in the oxidases with high reactivity. Therefore, the band frequency of C(4a)=C(10a) stretching may serve as an indicator of the reactivity of flavoprotein with molecular oxygen. Furthermore, strong hydrogen bonding of flavin at the N(1) moiety with the hydroxyl group of Thr136 in MCAD is probably responsible for the strong bond of the C(4a)=C(10a) of reduced flavin in the dehydrogenase.
通过在25℃下将每种相应的未标记黄素在1M Na¹⁸OH（H₂¹⁸O）中孵育，制备了富含[4-羰基-¹⁸O]的黄素单核苷酸、核黄素和黄素腺嘌呤二核苷酸。通过使用黄素腺嘌呤二核苷酸合成酶，从相应的核黄素制备了[4-羰基-¹⁸O]黄素腺嘌呤二核苷酸。[4-羰基-¹⁸O]标记的同位素效应证实，黄素单核苷酸红外光谱中的1709cm⁻¹谱带和黄素腺嘌呤二核苷酸拉曼光谱中的1711cm⁻¹谱带主要源自C(4)=O伸缩振动模式。在用D-脯氨酸或D-丙氨酸处理的D-氨基酸氧化酶（DAO）的紫色中间体中，阴离子还原黄素的1605cm⁻¹拉曼谱带在用[4-羰基-¹⁸O]黄素腺嘌呤二核苷酸重构的DAO中不发生位移，尽管它会随着[4,10a-¹³C₂]-或[4a-¹³C]黄素腺嘌呤二核苷酸发生位移。因此，该谱带主要归因于C(4a)=C(10a)伸缩振动模式，不包括C(4)=O伸缩振动的贡献。根据酶的不同，谱带频率覆盖相当宽的范围（1602 - 1620cm⁻¹）。脱氢酶（中链酰基辅酶A、短链酰基辅酶A和异戊酰辅酶A脱氢酶）紫色中间体中还原黄素的频率高于氧化酶（DAO和L-苯丙氨酸氧化酶）。这表明对分子氧反应性低的脱氢酶中还原黄素的C(4a)=C(10a)键级比反应性高的氧化酶中的更强。因此，C(4a)=C(10a)伸缩的谱带频率可作为黄素蛋白与分子氧反应性的指标。此外，黄素在N(1)部分与中链酰基辅酶A脱氢酶中苏氨酸136的羟基形成的强氢键可能是脱氢酶中还原黄素的C(4a)=C(10a)强键的原因。
