Nogués Isabel, Campos Luis Alberto, Sancho Javier, Gómez-Moreno Carlos, Mayhew Stephen G, Medina Milagros
Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias and Institute of Biocomputation and Physics of Complex Systems (BIFI), Universidad de Zaragoza, 50009-Zaragoza, Spain.
Biochemistry. 2004 Dec 7;43(48):15111-21. doi: 10.1021/bi0483256.
Flavodoxins (Flds) are electron transfer proteins that carry a noncovalently bound flavin mononucleotide molecule (FMN) as a redox active center. A distinguishing feature of these flavoproteins is the dramatic change in the E(sq/rd) reduction potential of the FMN upon binding to the apoprotein (at pH 8.0, from -269 mV when free in solution to -438 mV in Anabaena Fld). In this study, the contribution of three neighboring FMN residues, Thr56, Asn58, and Asn97, and of three negatively charged surface residues, Glu20, Asp65, and Asp96, to modulate the redox properties of FMN upon its binding to the apoprotein has been investigated. Additionally, the role of these residues in the apoflavodoxin:FMN interaction has been analyzed. Concerning the redox potentials, the most noticeable result was obtained for the Thr56Gly mutant. In this Fld variant, the increased accessibility of FMN leads to an increase of +63 mV in the E(sq/rd) value. On the other hand, a correlation between the electrostatic environment of FMN and the E(sq/rd) has been observed. The more positive residues or the less negative residues present in the surroundings of the FMN N(1) atom, then the less negative the value for E(sq/rd). With regard to FMN binding to apoflavodoxin, breaking of hydrophobic interactions between FMN and residues 56, 58, and 97 seems to increase the K(d) values, especially in the Thr56Gly Fld. Such results suggest that the H-bond network in the FMN environment influences the FMN affinity.
黄素氧还蛋白（Flds）是一类电子传递蛋白，其携带一个非共价结合的黄素单核苷酸分子（FMN）作为氧化还原活性中心。这些黄素蛋白的一个显著特征是，FMN与脱辅基蛋白结合后，其E(sq/rd)还原电位会发生显著变化（在pH 8.0时，从溶液中游离时的-269 mV变为鱼腥藻黄素氧还蛋白中的-438 mV）。在本研究中，研究了三个相邻的FMN残基（苏氨酸56、天冬酰胺58和天冬酰胺97）以及三个带负电荷的表面残基（谷氨酸20、天冬氨酸65和天冬氨酸96）在FMN与脱辅基蛋白结合时对调节FMN氧化还原特性的贡献。此外，还分析了这些残基在脱辅基黄素氧还蛋白：FMN相互作用中的作用。关于氧化还原电位，苏氨酸56甘氨酸突变体得到了最显著的结果。在这种黄素氧还蛋白变体中，FMN可及性的增加导致E(sq/rd)值增加了63 mV。另一方面，观察到FMN的静电环境与E(sq/rd)之间存在相关性。FMN N(1)原子周围存在的正电荷残基越多或负电荷残基越少，E(sq/rd)的值就越不呈负性。关于FMN与脱辅基黄素氧还蛋白的结合，FMN与残基56、58和97之间疏水相互作用的破坏似乎会增加K(d)值，尤其是在苏氨酸56甘氨酸黄素氧还蛋白中。这些结果表明，FMN环境中的氢键网络会影响FMN的亲和力。
