Bradley L H, Swenson R P
Department of Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
Biochemistry. 2001 Jul 31;40(30):8686-95. doi: 10.1021/bi010571j.
The role of the hydrogen bonding interaction with the N(3)H of the flavin cofactor in the modulation of the redox properties of flavoproteins has not been extensively investigated. In the flavodoxin from Clostridium beijerinckii, the gamma-carboxylate group of glutamate-59 serves as a dual hydrogen bond acceptor with the N(3)H of flavin mononucleotide (FMN) cofactor and the amide hydrogen of the adjacent polypeptide backbone in all three oxidation states. This "bridging" interaction serves to anchor the FMN in the binding site, which, based on the E59Q mutant, indirectly affects the stability of the neutral flavin semiquinone by facilitating a strong and critical interaction at the FMN N(5)H [Bradley, L. H., and Swenson, R. P. (1999) Biochemistry 38, 12377-12386]. In this study, the specific role of the N(3)H interaction itself was investigated through the systematic replacement of Glu59 by aspartate, asparagine, and alanine in an effort to weaken, disrupt, and/or eliminate this interaction, respectively. Just as for the E59Q mutant, each replacement significantly weakened the binding of the cofactor, particularly for the semiquinone state, affecting the midpoint potentials of each one-electron couple in opposite directions. (1)H-(15)N HSQC nuclear magnetic resonance (NMR) spectroscopic studies revealed that not only was the N(3)H interaction weakened as anticipated, but so also was the hydrogen bonding interaction with the N(5)H. Using the temperature coefficients of the N(5)H to quantify and correct for changes in this interaction, the contribution of the N(3)H hydrogen bond to the binding of each redox state of the FMN was isolated and estimated. Based on this analysis, the N(3)H hydrogen bonding interaction appears to contribute primarily to the stability of the oxidized state (by as much as 2 kcal/mol) and to a lesser extent the reduced states. It is concluded that this interaction contributes only modestly (<45 mV) to the modulation of the midpoint potential for each redox couple in the flavodoxin. These conclusions are generally consistent with ab initio calculations and model studies on the non-protein-bound cofactor.
黄素辅因子的N(3)H的氢键相互作用在黄素蛋白氧化还原性质调节中的作用尚未得到广泛研究。在拜氏梭菌黄素氧还蛋白中，谷氨酸-59的γ-羧基在所有三种氧化态下均作为与黄素单核苷酸（FMN）辅因子的N(3)H以及相邻多肽主链酰胺氢的双氢键受体。这种“桥连”相互作用有助于将FMN锚定在结合位点，基于E59Q突变体，通过促进FMN N(5)H处的强关键相互作用间接影响中性黄素半醌的稳定性[布拉德利，L.H.，和斯文森，R.P.（1999年）《生物化学》38卷，12377 - 12386页]。在本研究中，通过分别用天冬氨酸、天冬酰胺和丙氨酸系统取代谷氨酸-59来研究N(3)H相互作用本身的具体作用，以分别削弱、破坏和/或消除这种相互作用。正如E59Q突变体一样，每次取代都显著削弱了辅因子的结合，特别是对于半醌态，以相反方向影响每个单电子偶的中点电位。(1)H-(15)N HSQC核磁共振（NMR）光谱研究表明，不仅N(3)H相互作用如预期那样被削弱，而且与N(5)H的氢键相互作用也被削弱。利用N(5)H的温度系数来量化并校正这种相互作用的变化，分离并估计了N(3)H氢键对FMN每种氧化还原态结合的贡献。基于此分析，N(3)H氢键相互作用似乎主要对氧化态的稳定性有贡献（高达2千卡/摩尔），对还原态的贡献较小。得出的结论是，这种相互作用对黄素氧还蛋白中每个氧化还原偶中点电位的调节贡献不大（<45毫伏）。这些结论与关于非蛋白结合辅因子的从头计算和模型研究总体一致。
