Raibekas A A, Ramsey A J, Jorns M S
Department of Biological Chemistry, Hahnemann University School of Medicine, Philadelphia, Pennsylvania 19102.
Biochemistry. 1993 Apr 27;32(16):4420-9. doi: 10.1021/bi00067a035.
The reduction potential of flavin bearing a methylsulfonyl moiety (MeSO2) in place of a methyl group at position 8 is increased by more than 150 mV as compared with normal flavin. This substitution is accompanied by a substantial increase in reactivity with various reductants, including NADH, and greatly (10(3)-fold) enhanced susceptibility toward nucleophilic attack by sulfite at N(5). 1,5-Dihydro-8-(methylsulfonyl)riboflavin exhibits two intense, well-resolved absorption bands (lambda max = 310, 362 nm) in a region where most other reduced flavins exhibit weak, characterless absorption. This unusual spectrum is attributable to a shift of pi-electron density from the N(5) atom into the benzene ring. It is observed only with reduced flavins bearing a strongly electronegative substituent (MeSO2, CN) at the 8-position. The effect is abolished by replacing the hydrogen at N(5) with a bulky group, like sulfite, which interferes with sp2 hybridization at N(5). Reaction of 8-MeSO2-substituted flavins with thiols results in nucleophilic displacement of MeSO2- in a reaction that is about 10(3)-fold faster than an analogous nucleophilic displacement reaction observed with 8-halo-substituted flavins. The flavin ring acts as a redox switch in controlling electrophilicity at the 8-position, as judged by the fact that the displacement reactions are observed only with the oxidized flavins. Initial studies to evaluate 8-MeSO2-substituted flavins as active site probes were conducted with flavodoxin from Clostridium beijerinckii MP. 8-MeSO2FMN is rapidly bound to apoflavodoxin, accompanied by absorbance and fluorescence changes similar to those observed for FMN binding. 1,5-Dihydro-8-MeSO2FMN flavodoxin exhibits spectral properties (lambda max = 323, 382 nm) similar to those of the corresponding free flavin, except for a bathochromic shift due to a change in the polarity of the flavin environment. As judged by peak resolution and intensity, the spectral properties of 1,5-dihydro-FMN flavodoxin (lambda max = 311, 362 nm) appear to lie about midway between those observed for the free 1,5-dihydro forms of FMN versus 8-MeSO2FMN. This suggests that the protein environment may favor enhanced resonance delocalization of pi-electron density into the benzene ring of bound 1,5-dihydro-FMN, as compared with the free flavin. This hypothesis is consistent with previous NMR studies and with a proposal that electron transfer from reduced flavodoxin to other redox proteins occurs through this region of the ring. 8-MeSO2FMN bound to flavodoxin reacts readily with exogenous thiols but does not react with sulfite.(ABSTRACT TRUNCATED AT 400 WORDS)
与正常黄素相比，在第8位带有甲磺酰基（MeSO2）而非甲基的黄素的还原电位增加了超过150 mV。这种取代伴随着与各种还原剂（包括NADH）反应性的大幅增加，以及在N(5)处对亚硫酸盐亲核攻击的敏感性极大增强（10³倍）。1,5 - 二氢 - 8 - （甲磺酰基）核黄素在大多数其他还原型黄素表现出微弱、无特征吸收的区域呈现出两个强烈、分辨率良好的吸收带（λmax = 310、362 nm）。这种不寻常的光谱归因于π电子密度从N(5)原子转移到苯环。仅在第8位带有强电负性取代基（MeSO2、CN）的还原型黄素中观察到这种效应。通过用像亚硫酸盐这样的大体积基团取代N(5)上的氢来消除这种效应，亚硫酸盐会干扰N(5)处的sp²杂化。8 - MeSO2取代的黄素与硫醇的反应导致MeSO2⁻的亲核取代，该反应比用8 - 卤代取代的黄素观察到的类似亲核取代反应快约10³倍。黄素环在控制第8位的亲电性方面起到氧化还原开关的作用，这可以从仅在氧化型黄素中观察到取代反应这一事实判断出来。最初用拜氏梭菌MP的黄素氧还蛋白评估8 - MeSO2取代的黄素作为活性位点探针的研究。8 - MeSO2FMN迅速与脱辅基黄素氧还蛋白结合，伴随着与FMN结合时观察到的类似吸光度和荧光变化。1,5 - 二氢 - 8 - MeSO2FMN黄素氧还蛋白呈现出与相应游离黄素类似的光谱性质（λmax = 323、382 nm），只是由于黄素环境极性变化导致红移。从峰分辨率和强度判断，1,5 - 二氢 - FMN黄素氧还蛋白（λmax = 311、362 nm）的光谱性质似乎介于游离的1,5 - 二氢形式的FMN与8 - MeSO2FMN所观察到的光谱性质之间。这表明与游离黄素相比，蛋白质环境可能有利于增强π电子密度进入结合的1,5 - 二氢 - FMN苯环的共振离域。这个假设与先前的NMR研究以及关于电子从还原型黄素氧还蛋白转移到其他氧化还原蛋白通过环的这个区域发生的提议一致。与黄素氧还蛋白结合的8 - MeSO2FMN很容易与外源硫醇反应，但不与亚硫酸盐反应。（摘要截短至400字）
