Electron transfer reaction in a single protein molecule observed by total internal reflection fluorescence microscopy

To observe an electron transfer (ET) process in a single protein molecule, we constructed a model system, Alexa-HCytb₅, in which cytochrome b₅ (Cytb₅) is modified with a fluorescent probe, Alexa Fluor 647 dye. In this model system, intramolecular transfer of an electron from the Alexa dye to heme in Cytb₅ is supposed to oxidize the probe and quench its fluorescence, and the ET reaction at the single-molecule level can be monitored as the intermittent change in the fluorescence intensity. Alexa-HCytb₅ was fixed on the glass surface, and illumination of laser light by the total internal reflection resulted in blinking of the fluorescence from the single Alexa-HCytb₅ molecule in the time scale of several hundred milliseconds. Each Alexa-HCytb₅ molecule is characterized by its own rate constant of the blinking, corresponding to the ET rate constant at the single-molecule level, and its variation ranges between 1 and 10 s^-1. The current system thus enables us to visualize the ET reaction in the single protein molecule, and the protein ET reaction was found to be explained by the distribution of the rate constants. On the basis of the Marcus theory, we suggest that the origin of this rate distribution is the distance change associated with the structural fluctuation in the protein molecule.