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Time-resolved fluorescence analysis of the mobile flavin cofactor in p-hydroxybenzoate hydroxylase

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Abstract

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.

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Abbreviations

GR:

glutathione reductase

MEM:

maximum entropy method

PHBH:

p-hydroxybenzoate hydroxylase

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Authors and Affiliations

  1. Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA, Wageningen, The Netherlands

    Petra A W Van Den Berg, Koert Grever, Willem J H Van Berkel & Antonie J W G Visser

  2. Microspectroscopy Centre, PO Box 8128, 6700 ET, Wageningen, The Netherlands

    Petra A W Van Den Berg, Arie Van Hoek & Antonie J W G Visser

  3. Department of Structural Biology, Faculty of Earth and Life Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands

    Antonie J W G Visser

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  1. Petra A W Van Den Berg
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  2. Koert Grever
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  3. Arie Van Hoek
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  4. Willem J H Van Berkel
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  5. Antonie J W G Visser
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Correspondence to Antonie J W G Visser.

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Van Den Berg, P.A.W., Grever, K., Van Hoek, A. et al. Time-resolved fluorescence analysis of the mobile flavin cofactor in p-hydroxybenzoate hydroxylase. J Chem Sci 119, 123–133 (2007). https://doi.org/10.1007/s12039-007-0019-3

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