Enhancement of Photophysical and Photosensitizing Properties of Flavin Adenine Dinucleotide by Mutagenesis of the C-Terminal Extension of a Bacterial Flavodoxin Reductase

The role of the mobile C‐terminal extension present in Rhodobacter capsulatus ferredoxin–NADP(H) reductase (RcFPR) was evaluated using steady‐state and dynamic spectroscopies for both intrinsic Trp and FAD in a series of mutants in the absence of NADP(H). Deletion of the six C‐terminal amino acids b...

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Published in:Chemphyschem 2015-03, Vol.16 (4), p.872-883
Main Authors: Valle, Lorena, Abatedaga, Inés, Vieyra, Faustino E. Morán, Bortolotti, Ana, Cortez, Néstor, Borsarelli, Claudio D.
Format: Article
Language:English
Subjects:
Amino acids
Bacteria
Bacteriology
Ferredoxin-NADP Reductase - genetics
Ferredoxin-NADP Reductase - metabolism
flavin triplet state
Flavin-Adenine Dinucleotide - chemistry
Flavin-Adenine Dinucleotide - metabolism
flavoproteins
fluorescence
oxidoreductases
Photochemical Processes
Photosensitizing Agents - chemistry
Photosensitizing Agents - metabolism
Rhodobacter capsulatus
Rhodobacter capsulatus - enzymology
singlet oxygen
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Summary:The role of the mobile C‐terminal extension present in Rhodobacter capsulatus ferredoxin–NADP(H) reductase (RcFPR) was evaluated using steady‐state and dynamic spectroscopies for both intrinsic Trp and FAD in a series of mutants in the absence of NADP(H). Deletion of the six C‐terminal amino acids beyond Ala266 was combined with the replacement A266Y to emulate the structure of plastidic reductases. Our results show that these modifications of the wild‐type RcFPR produce subtle global conformational changes, but strongly reduce the local rigidity of the FAD‐binding pocket, exposing the isoalloxazine ring to the solvent. Thus, the ultrafast charge‐transfer quenching of 1FAD* by the conserved Tyr66 residue was absent in the mutant series, producing enhancement of the excited singlet‐ and triplet‐state properties of FAD. This work highlights the delicate balance of the specific interactions between FAD and the surrounding amino acids, and how the functionality and/or photostability of redox flavoproteins can be modified. A little more sensitivity: C‐terminal scission or site‐specific mutation of Rhodobacter capsulatus ferredoxin–NADP(H) reductase (RcFPR) prevents the charge‐transfer quenching of 1FAD* by the conserved Tyr66, producing enhancement of the excited singlet‐ and triplet‐state properties of FAD. This in turn drives the photosensitization of 1O2, challenging the photostability and hence the functionality of the redox flavoprotein.
ISSN:1439-4235
1439-7641
DOI:10.1002/cphc.201402774