Swenson R P, Krey G D
Department of Biochemistry, Ohio State University, Columbus 43210-1292.
Biochemistry. 1994 Jul 19;33(28):8505-14. doi: 10.1021/bi00194a015.
The contributions made by tyrosine-98 in establishing the redox properties of the flavodoxin from Desulfovibrio vulgaris were investigated by substituting a number of amino acids at this position using site-directed mutagenesis. Tyr98, which makes extensive van der Waals contacts with the isoalloxazine ring of the flavin mononucleotide cofactor, is often found in the cofactor binding site of flavodoxins and related flavoproteins. Solution studies suggest that tyrosine may assist in the stabilization of the neutral flavin semiquinone through preferential complex formation relative to the other oxidation states. In this study, the midpoint potentials of the oxidized/semiquinone couple of the Y98W and Y98F mutants were found to be very similar to the wild-type flavodoxin. However, significantly more negative midpoint potentials (by 25-60 mV) were observed in the Y98A, Y98H, and Y98R mutants. These results imply that it is the general apolar environment provided by the aromatic amino acids rather than preferential affinities suggested by solution studies that is at least partially responsible for the thermodynamic stabilization of the neutral flavin semiquinone in this flavodoxin. The midpoint potential of the semiquinone/hydroquinone couple is profoundly dependent on the properties of the amino acid at this position. Compared to phenylalanine, the more electron-rich aromatic side chains of tryptophan and tyrosine decrease the midpoint potential of this couple by 30-40 mV. Greater solvent exposure of the isoalloxazine ring in the Y98A mutant increases the midpoint potential by 140 mV relative to wild type. The positively charged amino acids increase the midpoint potential of this couple by > 180 mV, most probably through favorable electrostatic interactions with the flavin hydroquinone anion. These observations strongly support the proposition that the functional role of the electron-rich, apolar aromatic amino acid residues adjacent to the flavin isoalloxazine ring is to substantially destabilize the flavin hydroquinone anion, resulting in the very low oxidation-reduction potentials for the semiquinone/hydroquinone couple that typify the flavodoxin family.
通过定点诱变在该位置替换多个氨基酸，研究了酪氨酸98对普通脱硫弧菌黄素氧还蛋白氧化还原特性的贡献。Tyr98与黄素单核苷酸辅因子的异咯嗪环有广泛的范德华接触，常见于黄素氧还蛋白和相关黄素蛋白的辅因子结合位点。溶液研究表明，相对于其他氧化态，酪氨酸可能通过优先形成复合物来协助稳定中性黄素半醌。在本研究中，发现Y98W和Y98F突变体的氧化/半醌对的中点电位与野生型黄素氧还蛋白非常相似。然而，在Y98A、Y98H和Y98R突变体中观察到明显更负的中点电位（降低25 - 60 mV）。这些结果表明，至少部分负责该黄素氧还蛋白中中性黄素半醌热力学稳定的是芳香族氨基酸提供的一般非极性环境，而非溶液研究表明的优先亲和力。半醌/氢醌对的中点电位极大地依赖于该位置氨基酸的性质。与苯丙氨酸相比，色氨酸和酪氨酸富含电子的芳香侧链使该对的中点电位降低30 - 40 mV。Y98A突变体中异咯嗪环更大程度地暴露于溶剂中，相对于野生型使中点电位增加140 mV。带正电荷的氨基酸使该对的中点电位增加>180 mV，很可能是通过与黄素氢醌阴离子的有利静电相互作用。这些观察结果有力地支持了这样的观点，即与黄素异咯嗪环相邻的富含电子的非极性芳香族氨基酸残基的功能作用是显著使黄素氢醌阴离子不稳定，导致典型的黄素氧还蛋白家族半醌/氢醌对的极低氧化还原电位。
