Williamson G, Edmondson D E
Biochemistry. 1985 Dec 17;24(26):7790-7. doi: 10.1021/bi00347a043.
The pKa values for the various ionic forms of 8 alpha-N-imidazolylriboflavin were determined in its oxidized and hydroquinone forms and estimated for its semiquinone form. The pH dependence of the absorption and fluorescence spectral properties and potentiometric titration data show the pKa values for the oxidized form to be 6.02 +/- 0.03 for the 8 alpha-imidazole nitrogen and 9.67 +/- 0.05 for the N(3) position of the flavin ring. The pH dependence of the oxidation-reduction potential was determined by spectrocoulometric titrations, and the data points were compared with computer-simulated plots. Two pKa values for the hydroquinone form of the flavin were determined and assigned. The pKa for the imidazole ring is found to be 6.9 +/- 0.1 and for the N(1) position of the flavin hydroquinone is found to be 5.5 +/- 0.1. Analysis of the pH dependence of the one-electron couples E2 (flavoquinone/flavin semiquinone, Flox/Fl.) and E1 (flavin semiquinone/flavin hydroquinone, Fl./Flred) resulted in an estimated pKa of 6.5 for the 8 alpha-imidazole ring in the flavin semiquinone form. These data show the possible involvement of the ionization of the 8 alpha-imidazole substituent in the redox chemistry of flavoenzymes containing either an 8 alpha-N1- or an 8 alpha-N3-histidyl-linked covalent flavin coenzyme. Future work on oxidation-reduction potentials of this class of enzymes must take into consideration the influence of the 8 alpha-histidyl substituent.
测定了8α-N-咪唑基核黄素各种离子形式在氧化态和对苯二酚形式下的pKa值，并估算了其半醌形式的pKa值。吸收和荧光光谱特性以及电位滴定数据的pH依赖性表明，氧化态的pKa值在8α-咪唑氮处为6.02±0.03，在黄素环的N(3)位置为9.67±0.05。通过光谱库仑滴定法测定了氧化还原电位的pH依赖性，并将数据点与计算机模拟曲线进行了比较。确定并指定了黄素对苯二酚形式的两个pKa值。发现咪唑环的pKa为6.9±0.1，黄素对苯二酚的N(1)位置的pKa为5.5±0.1。对单电子偶合E2（黄素醌/黄素半醌，Flox/Fl.）和E1（黄素半醌/黄素对苯二酚，Fl./Flred）的pH依赖性分析得出，黄素半醌形式中8α-咪唑环的估计pKa为6.5。这些数据表明，8α-咪唑取代基的电离可能参与了含有8α-N1-或8α-N3-组氨酸连接的共价黄素辅酶的黄素酶的氧化还原化学过程。未来关于这类酶氧化还原电位的研究必须考虑8α-组氨酸取代基的影响。
