A R T I C L E S
Shi et al.
of evacuation and equilibration over an atmosphere of purified
argon.13 Substrate solutions were made anaerobic by bubbling
with purified argon within the syringes that were to be loaded
onto the stopped-flow instrument.
To investigate whether the abrupt disappearance of fluorescence
of single DHOD molecules was the result of photobleaching,
the laser power was increased from 1 to 2.5 µW. Exponential
fits of the on-time distributions for 1 and 2.5 µW laser power
gave a similar koff, the rate constant of fluorescence loss, around
0.8 s-1 (Figure 2b), showing that photobleaching is not the origin
for the loss in fluorescence. A more plausible mechanism is
that FMN dissociates from the protein and rapidly diffuses away
from the field of observation.14 This is consistent with our
observation that the fluorescence signal would occasionally
return after the molecule being observed lost its initial fluores-
cence during the 30-s data collection time.
The rapid loss of FMN from the holoenzyme suggests that
our single-molecule samples contained a mixture of free FMN,
holoenzyme, and apoenzyme in a dynamic equilibrium. To better
quantify the equilibrium, the concentration of holoenzyme was
estimated by comparing to a fluorescent standard. At a
concentration of 1 nM, the number of fluorescent DHOD
molecules was comparable to that observed for 0.1 nM
plasminogen activator inhibitor 1 covalently labeled with
fluorescein. Therefore, the amount of apoenzyme due to FMN
dissociation is significant at nanomolar concentrations compared
to holoenzyme. By comparing the number of fluorescent DHOD
molecules to the number of fluorescent plasminogen activator
inhibitor 1 molecules, we estimate that there was ∼0.1 nM holo-
DHOD in a ∼1 nM total protein sample. This allows the
estimation of a Kd of FMN from DHOD of ∼10-8 M. By
assumption of a simple one-step dissociation of FMN from the
holoenzyme and use of the observed dissociation rate constant
of ∼0.8 s-1, an association rate constant of 8 × 107 M-1 s-1
can be estimated, which is near the diffusion limit. Since the
free FMN concentration is subnanomolar, the reassociation of
FMN is a rare event.
In the presence of DHO and DCIP, FMN fluorescence blinked
on and off as the flavin cycled between the oxidized and reduced
states during turnover. However, eventually FMN dissociated
from the holoenzyme, as indicated by the long time scale of
loss of fluorescence. A histogram of the duration of the
fluorescence signal before the final loss of FMN gave an FMN
dissociation rate constant of 0.3 s-1 (Figure 2c), which is 2.5-
fold slower than that without substrates.
Steady-State Turnover. Steady-state turnover of the
Tyr318Leu mutant was measured in assays at 22 °C, by
monitoring the reduction of DCIP. A turnover rate of 5.7 s-1
was measured with 500 µM DHO and 100 µM DCIP (both
saturating concentrations). With the addition of 0.1% reduced
Triton X-100, a turnover number of 9.3 s-1 was measured.
Stopped-Flow Studies on the Reductive Half-Reaction. The
reduction of the enzyme-bound flavin was studied by mixing
anaerobic solutions of enzyme and DHO at 22 °C in a stopped-
flow instrument and recording the loss of either the absorbance
at 450 nm (characteristic of flavin), or the absorbance at 550
nm (characteristic of a reduced flavin-orotate charge-transfer
complex), or the flavin fluorescence (excitation at 450 nm,
emission at >520 nm). The fluorescence traces were fit well to
Confocal Fluorescence Microscopy. Fluorescence time
traces of single DHOD molecules were collected by use of an
inverted confocal microscope designed and built by our group.
It was equipped with a 1.65 NA ×100 oil-immersion objective
(Optical Analysis APO 1-UB615). Light at 457 nm from an
Ar+-ion laser (Melles-Griot 532-AP) was passed through a
dichroic beam splitter and then focused on a glass slide coated
with the sample. The fluorescence emission was detected by a
single-photon-counting avalanche photodiode (Perkin-Elmer
Optoelectronics SPCM-AQ 161) after a notch-plus filter (Kaiser
38988) and a 520 nm band-pass filter (Chroma D520/60M).
The image in this geometry was built up by raster-scanning. A
count rate threshold was set in the control program for initiating
the collection of trajectories. Time traces were collected in time
steps of either 20 or 10 ms.
Preparation of Single-Molecule Samples. To physically
confine enzyme molecules in the pores of a 1% agarose gel,8
agarose was melted in 50 mM phosphate buffer, pH 7.0 (with
or without substrates) with gentle stirring and heating. DHOD
was diluted into the agarose solution to 1 nM just above the
gelling temperature (∼30 °C), and the mixture was spin-coated
onto a glass slide, forming a smooth thin layer of gel containing
the DHOD molecules. After the slide was mounted on the
microscope stage, a small volume of buffer (∼60 µL) was
applied on the sample to keep it moist, which also helped to
reduce background noise. In turnover experiments, the substrates
were premixed with the agarose solution with or without 0.1%
reduced Triton X-100. On average, at enzyme concentrations
on the order of 1 nM, no more than one molecule would reside
in the confocal field of view.
Simulation of the Stochastic Enzymatic Turnover. A
program for the stochastic simulation of enzyme turnover was
written in Mathematica. Turnover was assumed to consist of a
reductive and an oxidative half-reaction controlled by the
experimentally determined rate constants. The rate constants k
were used to compute the probability of reduction or oxidation
in unit time, R ) [1 - exp(-k∆t)], where ∆t is the time step
of simulated data collection. R was compared to a random
number p, generated by the Random function in Mathematica,
whose value ranged between 0 and 1. Reactions occurred when
p < R; if p g R, the molecule remained unreacted and another
value of p was generated. The number of iterations of random
number generation before the occurrence of a reaction, N, is a
random variable and has a geometric probability distribution
as R(1 - R)N. The on- or off-time was approximated by N∆t.
3. Results
FMN Dissociation. Figure 2a presents typical fluorescence
trajectories of single DHOD molecules, in which the fluores-
cence signal was constant over time until it dropped to
background level. The one-step fluorescence loss suggested we
were observing single molecules, not aggregates of several
molecules. The histogram of fluorescence on-times of 110
DHOD molecules fits a single-exponential decay (Figure 2b).
(14) If we assume the diffusion rate of FMN in agarose gel is similar to that in
sucrose (whose molecular weight is similar), D ) 4.59 × 10-6 cm2 s-1
.
The pore size of 1% agarose gel, x , is around 0.5 µm in diameter.
Therefore, the time t for FMN to diffuse away from our field of observation
can be derived as 0.3 ms with t ) x 2/2D, which is much faster than the
resolution in our data collection time (10 ms). So the drop of fluorescence
is very sharp.
(13) Williams, C. H., Jr.; Arscott, D. L.; Matthews, R. G.; Thorpe, C.; Wilkinson,
K. D. Methods Enzymol. 1979, 62, 185-198.
9
6916 J. AM. CHEM. SOC. VOL. 126, NO. 22, 2004