Yalloway G N, Mayhew S G, Malthouse J P, Gallagher M E, Curley G P
Department of Biochemistry, University College Dublin, Belfield, Dublin 4, Ireland.
Biochemistry. 1999 Mar 23;38(12):3753-62. doi: 10.1021/bi982476p.
Photoreduction with a 5-deazaflavin as the catalyst was used to convert flavodoxins from Desulfovibrio vulgaris, Megasphaera elsdenii, Anabaena PCC 7119, and Azotobacter vinelandii to their hydroquinone forms. The optical spectra of the fully reduced flavodoxins were found to vary with pH in the pH range of 5.0-8.5. The changes correspond to apparent pKa values of 6.5 and 5.8 for flavodoxins from D. vulgaris and M. elsdenii, respectively, values that are similar to the apparent pKa values reported earlier from the effects of pH on the redox potential for the semiquinone-hydroquinone couples of these two proteins (7 and 5.8, respectively). The changes in the spectra resemble those occurring with the free two-electron-reduced flavin for which the pKa is 6.7, but they are red-shifted compared with those of the free flavin. The optical changes occurring with flavodoxins from D. vulgaris and A. vinelandii flavodoxins are larger than those of free reduced FMN. The absorbance of the free and bound flavin increases in the region of 370-390 nm (Delta epsilon = 1-1.8 mM-1 cm-1) with increases of pH. Qualitatively similar pH-dependent changes occur when FMN in D. vulgaris flavodoxin is replaced by iso-FMN, and in the following mutants of D. vulgaris flavodoxin in which the residues mutated are close to the isoalloxazine of the bound flavin: D95A, D95E, D95A/D127A, W60A, Y98S, W60M/Y98W, S96R, and G61A. The 13C NMR spectrum of reduced D. vulgaris [2,4a-13C2]FMN flavodoxin shows two peaks. The peak due to C(4a) is unaffected by pH, but the peak due to C(2) broadens with decreasing pH; the apparent pKa for the change is 6.2. It is concluded that a decrease in pH induces a change in the electronic structure of the reduced flavin due to a change in the ionization state of the flavin, a change in the polarization of the flavin environment, a change in the hydrogen-bonding network around the flavin, and/or possibly a change in the bend along the N(5)-N(10) axis of the flavin. A change in the ionization state of the flavin is the simplest explanation, with the site of protonation differing from that of free FMNH-. The pH effect is unlikely to result from protonation of D95 or D127, the negatively charged amino acids closest to the flavin of D. vulgaris flavodoxin, because the optical changes observed with alanine mutants at these positions are similar to those occurring with the wild-type protein.
以5 - 脱氮黄素作为催化剂进行光还原反应，用于将普通脱硫弧菌、埃氏巨球型菌、鱼腥藻PCC 7119和棕色固氮菌的黄素氧还蛋白转化为对苯二酚形式。发现完全还原的黄素氧还蛋白的光谱在pH值为5.0 - 8.5的范围内随pH变化。这些变化分别对应普通脱硫弧菌和埃氏巨球型菌黄素氧还蛋白的表观pKa值为6.5和5.8，这两个值与先前报道的pH对这两种蛋白质半醌 - 对苯二酚偶联的氧化还原电位影响的表观pKa值相似（分别为7和5.8）。光谱变化类似于游离双电子还原黄素发生的变化，其pKa为6.7，但与游离黄素相比发生了红移。普通脱硫弧菌和棕色固氮菌黄素氧还蛋白发生的光学变化比游离还原型FMN的变化大。随着pH升高，游离和结合黄素在370 - 390 nm区域的吸光度增加（Δε = 1 - 1.8 mM⁻¹ cm⁻¹）。当普通脱硫弧菌黄素氧还蛋白中的FMN被异FMN取代时，以及在普通脱硫弧菌黄素氧还蛋白的以下突变体中（其中突变的残基靠近结合黄素的异咯嗪）：D95A、D95E、D95A/D127A、W60A、Y98S、W60M/Y98W、S96R和G61A，会发生定性相似的pH依赖性变化。还原的普通脱硫弧菌[2,4a - ¹³C₂]FMN黄素氧还蛋白的¹³C NMR谱显示两个峰。由于C(4a)产生的峰不受pH影响，但由于C(2)产生的峰随着pH降低而变宽；变化的表观pKa为6.2。得出的结论是，pH降低会由于黄素电离状态的变化、黄素环境极化的变化、黄素周围氢键网络的变化和/或可能由于黄素沿N(5)-N(10)轴弯曲的变化而导致还原黄素的电子结构发生变化。黄素电离状态的变化是最简单的解释，质子化位点与游离FMNH⁻的不同。pH效应不太可能是由于普通脱硫弧菌黄素氧还蛋白中最靠近黄素的带负电荷氨基酸D95或D127的质子化引起的，因为在这些位置的丙氨酸突变体观察到的光学变化与野生型蛋白质发生的变化相似。
