Journal
PHYSICAL CHEMISTRY CHEMICAL PHYSICS
Volume 24, Issue 25, Pages 15397-15405Publisher
ROYAL SOC CHEMISTRY
DOI: 10.1039/d2cp00311b
Keywords
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Funding
- Heidrun Neubert and Dominik Goldbach (HZDR)
- Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) [392552271, 201269156 - SFB 1032]
- Ludwig-Maximilian University, Munich via the Center for NanoScience (CeNS)
- LMUinnovativ initiative BioImaging Network (BIN)
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In this study, the effects of pH and temperature on the distribution of different PQQ species were investigated using NMR, UV-Vis spectroscopy, and TRLFS. The binding of Eu(iii) to the active sites of MDH enzymes was monitored using two techniques, TRLFS and antenna effect.
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 (H(3)PQQ to PQQ(3-) 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.
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