Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 781
Figure 23.41. Single-molecule intensity and lifetime images for a mixture of Cy5 and JF9 on a glass surface. Reprinted with permission from [78].
Copyright © 2002, American Chemical Society.
Menten rate constants to be determined from the reactioninduced
blinking. 79
Another example of single-molecule enzyme kinetics
is shown in Figure 23.44. The enzyme dihydroorotate dehydrogenase
(DHOD) catalyses a reaction in the first step of
de-novo pyrimidine synthesis. In this reaction cycle FMN is
reduced and oxidized. Unfortunately, the flavin is quenched
by a tyrosine residue in the wild-type E. coli protein (Fig-
Figure 23.42. Blinking of FAD emission during oxidation of cholesterol
by Cox. The top panel shows the chemical reaction. Revised
from [79].
ure 23.44). To obtain a useful signal the nearby tyrosine
residue responsible for the quenching was mutated to a
leucine. 80 The enzyme molecules were immobilized in a 1%
agarose gel and localized by the flavin emission. However,
the emission quickly disappeared (Figure 23.45), and the
disappearing signal was due to dissociation of FMN from
the protein and not photobleaching. Because the rate of disappearance
was not dependent on incident power, in the
presence of substrates the FMN displayed rapid blinking in
addition to disappearance that was the result of the oxidation–reduction
cycles occurring prior to dissociation (Figure
23.45). The distribution of on and off times could be
used to determine kinetic constants for the reaction.
Figure 23.43. Single-molecule images of cholesterol oxidase as seen
from the emission of FAD. Reprinted from [79].