Time-resolved fluorescence analysis of the mobile flavin cofactor in p-hydroxybenzoate hydroxylase
| Autor: | Willem J. H. van Berkel, Petra A. W. van den Berg, Koert Grever, Antonie J. W. G. Visser, Arie van Hoek |
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| Přispěvatelé: | Structural Biology |
| Rok vydání: | 2007 |
| Předmět: |
Chemistry(all)
Stereochemistry Biophysics maximum-entropy method Biochemie Flavin group Photochemistry Biochemistry glutathione-reductase Cofactor nadph binding pseudomonas-fluorescens VLAG wild-type adenine-dinucleotide Quenching (fluorescence) biology 4-hydroxybenzoate hydroxylase Chemistry Tryptophan crystal-structure Active site Substrate (chemistry) General Chemistry Fluorescence Biofysica effector specificity biology.protein NADPH binding SDG 6 - Clean Water and Sanitation protein nanospace |
| Zdroj: | Journal of Chemical Sciences, 119(2), 123-133 van den Berg, P A W, Grever, K, van Hoek, A, van Berkel, W & Visser, A J W G 2007, ' Time-resolved fluorescence analysis of the mobile flavin cofactor in p-hydroxybenzoate hydroxylase. ', Proceedings of the Indian Academy of Sciences. Chemical Sciences, vol. 119, pp. 123-133 . https://doi.org/10.1007/s12039-007-0019-3 Journal of Chemical Sciences 119 (2007) 2 Proceedings of the Indian Academy of Sciences. Chemical Sciences, 119, 123-133. Indian Academy of Sciences |
| ISSN: | 0973-7103 0253-4134 0974-3626 |
| DOI: | 10.1007/s12039-007-0019-3 |
| Popis: | Conformational heterogeneity of the FAD cofactor in p-hydroxybenzoate hydroxylase (PHBH) was investigated with time-resolved polarized flavin fluorescence. For binary enzyme/substrate (analogue) complexes of wild-type PHBH and Tyr222 mutants, crystallographic studies have revealed two distinct flavin conformations; the 'in' conformation with the isoalloxazine ring located in the active site, and the 'out' conformation with the isoalloxazine ring disposed towards the protein surface. Fluorescence-lifetime analysis of these complexes revealed similar lifetime distributions for the 'in' and 'out' conformations. The reason for this is twofold. First, the active site of PHBH contains various potential fluorescence-quenching sites close to the flavin. Fluorescence analysis of uncomplexed PHBH Y222V and Y222A showed that Tyr222 is responsible for picosecond fluorescence quenching free enzyme. In addition, other potential quenching sites, including a tryptophan and two tyrosines involved in substrate binding, are located nearby. Since the shortest distance between these quenching sites and the isoalloxazine ring differs only little on average, these aromatic residues are likely to contribute to fluorescence quenching. Second, the effect of flavin conformation on the fluorescence lifetime distribution is blurred by binding of the aromatic substrates: saturation with aromatic substrates induces highly efficient fluorescence quenching. The flavin conformation is therefore only reflected in the small relative contributions of the longer lifetimes. © Indian Academy of Sciences. |
| Databáze: | OpenAIRE |
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