Maeda-Yorita K, Massey V
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.
J Biol Chem. 1993 Feb 25;268(6):4134-44.
Steps in the hydroxylation pathway of the flavoprotein phenol hydroxylase with resorcinol as substrate have been studied by a combination of fluorescence and absorbance stopped flow techniques. In the presence of azide, a series of highly fluorescent oxygenated flavin intermediates has been observed, corresponding to those previously detected by absorbance measurements (Detmer, K., and Massey, V. (1985) J. Biol. Chem. 260, 5998-6005). In addition, yet another intermediate has been found as the immediate product of the reaction of the reduced enzyme with O2. This new species is non-fluorescent in the presence of azide, but fluorescent in the absence of monovalent anions and had escaped detection in previous absorbance studies because of the similarity in its rates of formation and conversion to the next intermediate and similarity in their spectra. These two early intermediates are tentatively identified as the anionic and protonated species of the flavin C4a-hydroperoxide or, alternatively, as two conformationally different forms of the enzyme hydroperoxide. The next intermediate, previously referred to as intermediate II, is also highly fluorescent and so is considered unlikely to be due to a complex of a flavin alkoxyl radical and a substituted cyclohexadienyl radical, as proposed by Anderson et al. (Anderson, R. F., Patel, K. B., and Stratford, M. R. L. (1990) J. Biol. Chem. 265, 1952-1957). The conversion of intermediate II to the next intermediate, intermediate III (the C4a-hydroxyflavin), is characterized by a large substrate deuterium isotope effect in the 320-390 nm range, but not by fluorescence or by absorbance at wavelengths > 400 nm. This is ascribed to dissociation from the enzyme of a cyclohexadienone product, leaving the enzyme in its C4a-hydroxyflavin form. The latter eliminates H2O to re-form oxidized flavin, but in a competing reaction, in the presence of excess substrate, forms a very stable complex, which decays orders of magnitude more slowly than the uncomplexed enzyme.
以间苯二酚为底物，采用荧光和吸光度停流技术相结合的方法，研究了黄素蛋白苯酚羟化酶羟基化途径中的各个步骤。在叠氮化物存在的情况下，观察到一系列高荧光的氧化黄素中间体，与先前通过吸光度测量检测到的中间体相对应（Detmer, K., and Massey, V. (1985) J. Biol. Chem. 260, 5998 - 6005）。此外，还发现了另一种中间体，它是还原态酶与O₂反应的直接产物。这种新物质在叠氮化物存在时无荧光，但在一价阴离子不存在时具有荧光，并且由于其形成和转化为下一个中间体的速率相似以及光谱相似，在先前的吸光度研究中未被检测到。这两种早期中间体初步被鉴定为黄素C4a - 氢过氧化物的阴离子和质子化形式，或者是酶氢过氧化物的两种构象不同的形式。下一个中间体，先前称为中间体II，也具有高荧光，因此不太可能如Anderson等人所提出的那样（Anderson, R. F., Patel, K. B., and Stratford, M. R. L. (1990) J. Biol. Chem. 265, 1952 - 1957），是由黄素烷氧基自由基和取代环己二烯基自由基形成的复合物。中间体II向下一个中间体中间体III（C4a - 羟基黄素）的转化在320 - 390 nm范围内具有较大的底物氘同位素效应，但在荧光或波长> 400 nm的吸光度方面没有表现。这归因于环己二烯酮产物从酶上解离，使酶处于其C4a - 羟基黄素形式。后者消除H₂O以重新形成氧化黄素，但在竞争反应中，在过量底物存在下，会形成一种非常稳定的复合物，其衰变速度比未复合的酶慢几个数量级。
