Pyrroloquinoline quinone (PQQ) is a redox cofactor in calcium- and lanthanide-dependent alcohol dehydrogenases that has been known and studied for over 40 years. Despite its long history, many questions regarding its fluorescence properties, speciation in solution and in the active site of alcohol dehydrogenase remain open. Here we investigate the effects of pH and temperature on the distribution of different PQQ species (H₃PQQ to PQQ³⁻ in addition to water adducts and in complex with lanthanides) with NMR and UV-Vis spectroscopy as well as time-resolved laser-induced fluorescence spectroscopy (TRLFS). Using a europium derivative from a new, recently-discovered class of lanthanide-dependent methanol dehydrogenase (MDH) enzymes, we utilized two techniques to monitor Ln binding to the active sites of these enzymes. Employing TRLFS, we were able to follow Eu(III) binding directly to the active site of MDH using its luminescence and could quantify three Eu(III) states: Eu(III) in the active site of MDH, but also in solution as PQQ-bound Eu(III) and in the aquo-ion form. Additionally, we used the antenna effect to study PQQ and simultaneously Eu(III) in the active site.

中文翻译：

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溶液和镧系元素依赖性甲醇脱氢酶中吡咯并喹啉醌物质的研究

吡咯并喹啉醌 (PQQ) 是钙和镧系元素依赖性醇脱氢酶中的一种氧化还原辅助因子，其已知和研究已有 40 多年。尽管其历史悠久，但关于其荧光特性、溶液中的物种形成和乙醇脱氢酶活性位点的许多问题仍然悬而未决。在这里，我们研究了 pH 和温度对不同 PQQ 物种分布的影响（H ₃ PQQ 到 PQQ ³⁻除了水加合物和与镧系元素的络合物）与核磁共振和紫外可见光谱以及时间分辨激光诱导荧光光谱（TRLFS）。使用来自最近发现的一类新的镧系元素依赖性甲醇脱氢酶 (MDH) 的铕衍生物，我们利用两种技术来监测 Ln 与这些酶的活性位点的结合。使用 TRLFS，我们能够使用其发光跟踪 Eu( III ) 直接与 MDH 的活性位点结合，并且可以量化三种 Eu( III ) 状态：在 MDH 的活性位点中的 Eu( III )，也可以在溶液中作为 PQQ结合的Eu( III ) 和水离子形式。此外，我们利用天线效应研究了 PQQ，同时研究了 Eu( III) 在活动站点中。