Fluorescent labeling of biomacromolecules enjoys increasing popularity for structural, mechanistic, and microscopic investigations. Its success hinges on the ability of the dye to alternate between bright and dark states. Förster resonance energy transfer (FRET) is an important source of fluorescence modulation. Photo-induced electron transfer (PET) may occur as well, but is often considered only when donor and acceptor are in van der Waals contact. In this study, PET is shown between a label and redox centers in oxidoreductases, which may occur over large distances. In the small blue copper protein azurin, labeled with ATTO655, PET is observed when the label is at 18.5Å, but not when it is at 29.1Å from the Cu. For CuII, PET from label to Cu occurs at a rate of (4.8±0.3)×104s-1 and back at (0.7±0.1)×103s-1. With CuI the numbers are (3.3±0.7)×106s-1 and (1.0±0.1)×104s-1. Reorganization energies and electronic coupling elements are in the range of 0.8-1.2eV and 0.02-0.5cm-1, respectively. These data are compatible with electron transfer (ET) along a through-bond pathway although transient complex formation followed by ET cannot be ruled out. The outcome of this study is a useful guideline for experimental designs in which oxidoreductases are labelled with fluorescent dyes, with particular attention to single molecule investigations. The labelling position for FRET can be optimized to avoid reactions like PET by evaluating the structure and thermodynamics of protein and label.
- Copper protein
- Electron transfer
- Fluorescence correlation spectroscopy
- Redox enzymes