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首页> 外文期刊>Journal of Molecular Biology >Comparisons of wild-type and mutant flavodoxins from Anacystis nidulans. Structural determinants of the redox potentials.
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Comparisons of wild-type and mutant flavodoxins from Anacystis nidulans. Structural determinants of the redox potentials.

机译：比较野生无花果黄酮和突变黄酮毒素。氧化还原电位的结构决定因素。

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摘要

The long-chain flavodoxins, with 169-176 residues, display oxidation-reduction potentials at pH 7 that vary from -50 to -260 mV for the oxidized/semiquinone (ox/sq) equilibrium and are -400 mV or lower for the semiquinone/hydroquinone (sq/hq) equilibrium. To examine the effects of protein interactions and conformation changes on FMN potentials in the long-chain flavodoxin from Anacystis nidulans (Synechococcus PCC 7942), we have determined crystal structures for the semiquinone and hydroquinone forms of the wild-type protein and for the mutant Asn58Gly, and have measured redox potentials and FMN association constants. A peptide near the flavin ring, Asn58-Val59, reorients when the FMN is reduced to the semiquinone form and adopts a conformation ("O-up") in which O 58 hydrogen bonds to the flavin N(5)H; this rearrangement is analogous to changes observed in the flavodoxins from Clostridium beijerinckii and Desulfovibrio vulgaris. On further reduction to the hydroquinone state, the Asn58-Val59 peptide in crystalline wild-type A. nidulans flavodoxin rotates away from the flavin to the "O-down" position characteristic of the oxidized structure. This reversion to the conformation found in the oxidized state is unusual and has not been observed in other flavodoxins. The Asn58Gly mutation, at the site which undergoes conformation changes when FMN is reduced, was expected to stabilize the O-up conformation found in the semiquinone oxidation state. This mutation raises the ox/sq potential by 46 mV to -175 mV and lowers the sq/hq potential by 26 mV to -468 mV. In the hydroquinone form of the Asn58Gly mutant the C-O 58 remains up and hydrogen bonded to N(5)H, as in the fully reduced flavodoxins from C. beijerinckii and D. vulgaris. The redox and structural properties of A. nidulans flavodoxin and the Asn58Gly mutant confirm the importance of interactions made by N(5) or N(5)H in determining potentials, and are consistent with earlier conclusions that conformational energies contribute to the observed potentials.The mutations Asp90Asn and Asp100Asn were designed to probe the effects of electrostatic interactions on the potentials of protein-bound flavin. Replacement of acidic by neutral residues at positions 90 and 100 does not perturb the structure, but has a substantial effect on the sq/hq equilibrium. This potential is increased by 25-41 mV, showing that electrostatic interaction between acidic residues and the flavin decreases the potential for conversion of the neutral semiquinone to the anionic hydroquinone. The potentials and the effects of mutations in A. nidulans flavodoxin are rationalized using a thermodynamic scheme developed for C. beijerinckii flavodoxin. Copyright 1999 Academic Press.
机译：长链黄酮毒素具有169-176个残基，在pH 7时表现出氧化还原电位，对于氧化/半醌（ox / sq）平衡而言，其变化范围为-50至-260 mV，对于半醌为-400 mV或更低/氢醌（sq / hq）平衡。为了检查蛋白相互作用和构象变化对长无瓣念珠菌（Synechococcus PCC 7942）长链黄酮毒素中FMN电位的影响，我们确定了野生型蛋白的半醌和对苯二酚形式以及突变型Asn58Gly的晶体结构，并测量了氧化还原电势和FMN关联常数。当FMN还原为半醌形式时，黄素环附近的肽Asn58-Val59会重新定向，并采用其中O 58氢键连接至黄素N（5）H的构象（“ O-up”）;这种重排类似于在拜氏梭状芽胞杆菌和寻常型脱硫弧菌中发现的黄素毒素的变化。在进一步还原为对苯二酚状态时，结晶野生型构巢曲霉黄酮毒素中的Asn58-Val59肽从黄素旋转到氧化结构的“ O-下”位置。这种在氧化状态下还原为构象的现象是不寻常的，在其他黄酮毒素中尚未观察到。当FMN减少时，在构象变化的位点的Asn58Gly突变有望稳定在半醌氧化态下发现的O-up构象​​。此突变将ox / sq电位提高了46 mV至-175 mV，将sq / hq电位降低了26 mV至-468 mV。在Asn58Gly突变体的对苯二酚形式中，C-O 58保持向上状态，氢键合至N（5）H，就像完全还原的拜氏梭状芽胞杆菌和寻常型D.黄酮毒素一样。构巢曲霉黄酮毒素和Asn58Gly突变体的氧化还原和结构特性证实了N（5）或N（5）H在确定电势中相互作用的重要性，并且与构象能量有助于观察到的电势的早期结论一致。设计突变Asp90Asn和Asp100Asn来探测静电相互作用对蛋白结合黄素电位的影响。在位置90和100处用中性残基取代酸性不会干扰结构，但会对sq / hq平衡产生实质性影响。该电势增加了25-41 mV，表明酸性残基与黄素之间的静电相互作用降低了中性半醌向阴离子对苯二酚转化的可能性。利用为拜氏梭菌黄酮毒素开发的热力学方案，合理地确定构巢曲霉黄酮毒素中的突变潜力和影响。版权所有1999，学术出版社。

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