(Received for publication, March 29, 1995; and in revised form, June 20, 1995 )
From the
The addition of excess Tb to metal-depleted Escherichia coli alkaline phosphatase results in enhanced
luminescence from enzyme-bound terbium, which increases with sample
deoxygenation and exhibits a tryptophan-like excitation spectrum.
Following pulsed excitation at 280 nm, the time-resolved terbium
emission shows a negative prefactor associated with a submillisecond
rise time, which is independent of the concentration of dissolved
oxygen. The absence of a build-up phase and similarity in lifetime in
the decay kinetics of directly excited (488 nm) terbium allows for the
assignment of the submillisecond component in the 280 nm excited sample
to bound terbium. The results of the steady state and time-resolved
experiments suggest that the time evolution of alkaline
phosphatase-bound terbium emission is determined by energy transfer (k
360 and 120 s
) from
the triplet state of tryptophan to terbium followed by terbium decay.
This model is based on the observations that 1) the tryptophan
phosphorescence lifetime (previously assigned to Trp
)
corresponds to the longer component of the terbium emission and 2) the
long-lived emission is enhanced, as is the Trp
phosphorescence, by deoxygenation. An energy transfer mechanism
involving the Trp
triplet state is shown to be
inconsistent with a dipole-dipole process and is best understood as a
through-space electron exchange over a donor-acceptor distance of
9-10 Å.
Metalloenzyme research has been greatly aided by the isomorphic
replacement of intrinsic and spectroscopically silent metals (i.e. Ca, Mg
, and
Zn
) with optical and magnetic probes such as
Mn
, Co
, Cd
,
Eu
, and Tb
. Because of its similar
size and preference for strong oxygen donor groups as ligands, the use
of the extrinsic luminescent probe Tb
as a
replacement for Ca
is particularly well
established(1, 2, 3, 4) . For the
typical concentrations used in terbium-protein systems,
<10
M, the emission of free terbium in
solution is generally not observed following direct UV excitation with
conventional sources because of its low extinction coefficient
(
0.4 M
cm
for Tb
complexes of
diethylenetriaminopentaacetic acid(5) ), whereas, when bound to
a protein and in proximity to photoexcited aromatic residues, energy
transfer is very efficient(3) . The enhanced terbium
luminescence thus observed has been used analytically to determine
binding constants (6, 7) and, most importantly,
assuming that a Förster-type dipole-dipole energy
transfer mechanism is established, to extract intraprotein
donor-acceptor pair distances (for example see (8) and (9) ).
Although enhanced terbium luminescence has found
extensive use in metalloenzyme studies, the nature of the energy
transfer mechanism and the identification of the molecular donor states
involved is far from clear. For some terbium-substituted
metalloenzymes, energy transfer has been convincingly established to
proceed by way of a long-range nonradiative transfer from protein
aromatic singlet states(5, 10) . At shorter
donor-acceptor distances, however, a Dexter exchange mechanism is
suggested(2) . The observation of increased terbium
luminescence following deoxygenation of terbium containing samples of
synthetic peptides (4) and several calcium-binding proteins (11) provides strong evidence that protein triplet states may
be involved. Indeed, for nonbiological systems the involvement of
aromatic triplet states as donors has long been
known(12, 13, 14, 15) . A recent
detailed investigation (16) has considered the mechanisms of
dipole-dipole, electron exchange, superexchange, external heavy atom
effect, and electron transfer in the interpretation of fluorescence
quenching of one to one lanthanide complexes of an indolyl-EDTA ()derivative where the metal ion is held in a cofacial
geometry about 6 Å from the center of the indole ring. It was
found for terbium complexes that the majority of the observed energy
transfer derived from the indole triplet state and was not
dipole-dipole in nature. Of particular relevance to the work reported
here is the kinetic study of terbium luminescence complexed to
elastase(17) , where the observation of an oxygen-sensitive
growth of emission following UV excitation was interpreted as evidence
of energy transfer from a triplet state donor via electron exchange.
Whether or not these results are general or specific for the
terbium-elastase system is open to question.
In the current work we
extend the above observations implicating aromatic triplet states in
the enhanced luminescence of terbium-protein complexes by using a
protein that is well characterized with respect to tryptophan room
temperature phosphorescence (RTP). For this purpose the metalloenzyme Escherichia coli alkaline phosphatase (AP) was chosen. Of its
three tryptophan residues per subunit, Trp,
Trp
, and Trp
, only Trp
shows
RTP (18, 19) with a remarkably long (2 s) lifetime in
extensively deoxygenated samples. Given the proximity of Trp
to the AP metal-binding sites, we anticipated its phosphorescence
to be useful in elucidating the nature of energy transfer to AP-bound
terbium (TbAP). We note that a recent report (20) suggests that
only the singlet state of Trp
plays a role in enhancing
the luminescence from TbAP. In the present work we report direct
kinetic evidence for the enhanced luminescence in TbAP originating at
least in part via energy transfer from the triplet state of
Trp
. Our investigation broadens the important conclusions
of the terbium-elastase study and highlights the need for caution in
the application of the Förster equation for
distance determinations in terbium-substituted metalloenzymes.
AP was purchased from Sigma (Type III-S) as a suspension in
2.5 M ammonium sulfate and exchanged into 10 mM Tris,
pH 8.0, the buffer in which all spectroscopic measurements were
performed. Protein concentrations were determined
spectrophotometrically with a Shimadzu UV-260 or Cary 2400 UV-VIS based
on A = 0.72 (21) and a molecular weight of 94,058(22) .
Metal-depleted AP (apoAP) was prepared using the method of Bosron et al.(23) using the chelating agent
8-hydroxyquinoline-5-sulfonic acid. Atomic absorption analysis
(Perkin-Elmer 7000) of apoAP found less than 0.01 g atom of zinc and
0.01-0.02 g atom of magnesium/mol of protein. ApoAP activity (24) was found to be less than 1% of native AP (holoAP),
consistent with complete metal removal. Terbium-substituted AP was made
by slowly adding, with stirring, an appropriate amount of a
concentrated stock solution of TbCl
(Aldrich). Prior to
spectroscopic measurements in the absence of oxygen, the capped sample
cuvette was allowed to attain equilibrium with a continuous stream of
purified argon(25) . The oxygen content of an air saturated
solution is taken to be 260 µM(26) .
Steady
state emission spectra were recorded using a SPEX Fluorolog II
spectrofluorimeter. Time-resolved phosphorescence measurements and
decay analysis were made using instrumentation and methods described
previously(27) . Spectra were not corrected for photomultiplier
wavelength response. The UV excitation source used for the
time-resolved experiments was the frequency-doubled output of a
neodymium ion (Nd) doped yttrium aluminum garnet
(Y
Al
O
) laser pumped dye laser.
Data were collected using photon counting techniques in which the count
rate was kept well below the instrumental bandwidth to prevent counting
errors. For experiments where direct excitation of terbium was
required, an argon ion laser operating at 488 nm was used. An optical
chopper with a small aperture running at 48 Hz was used to produce a
short pulse (60-80 µs) from this continuous wave source.
Decay kinetics of enhanced terbium luminescence were analyzed using
a simple model that assumes irreversible energy transfer from the
excited triplet state of TrpT
(Trp)
to terbium-bound AP:
where k and k
are rates for the excited state
deactivation of the donor T
(Trp)
and
acceptor Tb
, respectively, in the absence of energy
transfer, k
. The rate equations for the
de-excitation of [T
(Trp)
]
and [Tb
] are:
where the concentration C is a collection of time-independent terms,
D and A
can be
respectively identified with the initial Trp
triplet
state population, [T
(Trp)
]
(following intersystem crossing from S
(Trp)), and the initial excited terbium
population, [Tb
]
, which arises either
through direct excitation at 280 nm or via energy transfer from one or
more fast decaying protein excited state(s). Note that for A
= 0 the prefactors associated with decay
rates k
and
have equal and opposite signs, with the larger decay rate
always having the negative amplitude. In practice, must be
modified (as discussed below) to account for heterogeneity of the
system.
Figure 1:
Total luminescence spectra of 4
µM apoAP + 80 µM Tb in
10 mM Tris, pH 8.0, observed at 1.8 nm resolution using 280 nm
excitation with a 3.6 nm band pass. Filled circles (
)
refer to an air-equilibrated sample; open circles (
)
refer to a deoxygenated sample. The spectrum of the air-saturated and
deoxygenated samples have been normalized with respect to the
integrated fluorescence from 300 to 450 nm. Also shown under identical
conditions is the total luminescence spectra of deoxygenated holoAP
(
). The emission of free Tb
under the same
experimental conditions is indistinguishable from baseline at these
instrumental settings.
Deoxygenation of the TbAP sample leads to a 4-5-fold enhancement of terbium emission (Fig. 1). Previously it was suggested (4) that increases in the emission of terbium complexed to model calcium-binding peptides following deoxygenation were caused by sample precipitation (i.e. an artifact of the deoxygenation procedure). We note that the enhanced terbium emission observed here following deoxygenation cannot be ascribed to protein aggregation. This is clearly demonstrated by the similar magnitude of elastic scattering seen at 560 nm (second order of the 280 nm excitation) under the two conditions (Fig. 1).
Also shown in Fig. 1is the total luminescence spectrum of
holoAP under deoxygenated conditions. The phosphorescence (T
S
) of
Trp
, the only AP residue that shows
RTP(18, 19) , is clearly shown to arise around 415 nm.
When terbium is substituted for the native AP metals the
phosphorescence is quenched.
Figure 2: Terbium emission transient observed at 544 nm following pulsed excitation at 280 nm. A, air-equilibrated solution. B, deoxygenated sample.
Figure 3:
Phosphorescence decay kinetics of
Trp in TbAP and AP as well as of terbium. A, B, and C show the phosphorescence of Trp
observed at 440 nm following 280 nm excitation of TbAP in
deoxygenated (A) and air-equilibrated samples (C) and
of native AP in an air saturated solution (B). Shown in D and E are terbium transients observed at 544 nm following
pulsed excitation at 488 nm with TbAP (D) or a
TbCl
-containing solution (E). For D and E the decay rates are not affected by dissolved
oxygen.
A further check of our assignment
of the 280 nm excitation-induced transient is afforded by observing the
terbium decay at 544 nm following direct excitation of this cation into
the weakly allowed transition at 488 nm. The results of this experiment (Fig. 3D) can be fit to 2 lifetimes, 430 (minor
component) and 930 µs (Table 1). The 430-µs lifetime is
assigned to free Tb(aq) because it is in
good agreement with the 420 µs found for a TbCl
solution (Fig. 3E) and with literature
values(30) . Because the lifetime of terbium is linearly
related to the number of hydroxyl oscillators in its ligand
field(30, 31) , the longer lifetime of terbium in TbAP
relative to terbium free in solution is evidence for complexation. The
notable absence of a decay component associated with a negative
prefactor for directly excited terbium in TbAP is further proof of the
participation of a protein aromatic residue in the excitation process
serving as energy donor.
Inspection of according to the
above decay component assignment shows that the decay associated with
the negative prefactor arises from terbium (k in ), implying that C in our model is positive and
therefore greater than A
. This is consistent with
its increased contribution to the decay following deoxygenation (Fig. 2) because under these conditions k
,
and hence
, decreases.
The functional homodimer of AP contains two Zn and one Mg
in each of two active
sites(23) . Catalytic activity in the native enzyme requires
Zn
at site M1 (32) . Although the role of the
second Zn
(site M2) and Mg
(site
M3) were once believed to be largely structural in nature(32) ,
their close proximity to one another lends itself to the idea of a
cocatalytic site(33) .
The experiments presented here do not
allow us to determine which metal-binding site contains the terbium(s)
involved in triplet state energy transfer from Trp.
Experiments where only one equivalent of terbium was added to apoAP
(data not shown) still show the distinctive early rise of terbium
luminescence upon pulsed excitation indicative of a triplet state
donor. The analysis of the decay data again requires three lifetime
components, one associated with the build-up and attributed to terbium
decay and two with the decay of the enhanced emission fed by the donor
tryptophan. We attribute the multiplicity of donor lifetimes in these
experiments to a difference in terbium-Trp
distances
resulting in different energy transfer rates. This may occur when
multiple terbium ions occupy distinct metal-binding sites or
alternately when a single terbium residing at a single site adopts
multiple distances with respect to Trp
due to different
protein conformations. The distances (obtained from the atomic
coordinates deposited in the Brookhaven Protein Data Bank(34) )
from the indole ring center (taken to be the midpoint of the
C
-C
bond in the following discussion)
to sites M1, M2, and M3 are 13.6, 9.7, and 9.4 Å. Below we show
how the subtle differences in distance between Trp
and
terbium in M2 and M3 are consistent, when interpreted in terms of an
exchange mechanism, with the observed multiple donor decay kinetics.
It should be noted that the 280 nm induced decays shown in Fig. 2clearly have nonzero values at t = 0. Thus the contribution from either directly excited terbium or from energy transfer to terbium from fast decaying protein excited states, most likely singlet states, is significant.
Because the rate of energy transfer
according to a dipole-dipole (Förster) mechanism is
proportional to the overlap of the normalized donor emission and
unnormalized acceptor absorbance (J), we anticipate that its
contribution to energy transfer in TbAP will be small given the
negligible absorbance of terbium. To verify this we have estimated the
energy transfer rate using the Förster
equation(36) , k =
8.785
10
k
R
J s
, where
is the refractive index of the
medium (taken as 1.36), k
is the radiative decay
rate of the unquenched tryptophan triplet state (0.087 s
(37) ), and the donor-acceptor distance (R) is
taken to be 9.55 Å, an average of the two nearest metal-binding
sites, M2 and M3. The orientation factor,
, is assumed
to be 2/3. The overlap integral (J) was determined from the
phosphorescence spectrum of AP and the absorbance spectrum of a
standard TbCl
solution. With J = 1.84
10
M
cm
, we determine k
= 3.6
10
s
,
which is more than six orders of magnitude less than the experimental
result. As anticipated, the triplet state is not significantly quenched
via a dipole-dipole process, and therefore an exchange mechanism is
indicated.
In electron exchange the rate of energy transfer is given
by k =
(2
/h)Z
J`, where J` is the
spectral overlap integral normalized with respect to both donor
emission and acceptor extinction and Z is the exchange
integral(38) . Assuming hydrogenic wave functions, Dexter
arrived at the approximation k
= KJ`exp(-2R/L), where K is a constant not related to experimental parameters and L is the average orbital radius involved in the initial and
final states for the donor-acceptor pair separated by distance R.
As suggested above the exponential distance dependence
of exchange is consistent with the observation of two donor lifetimes
in TbAP if terbium ions in sites M2 and M3 act as independent
quenchers. For example, assuming that K, J`, and L are of similar magnitude for both sites and taking L as one (39) , the difference in distance between the two
sites to Trp (0.3 Å) translates into an expected
ratio in rate of energy transfer of 2. This is in reasonable agreement
with the experimental ratios of k
360 and 120
s
(ratio = 3) and k
and
470 and 130 s
(ratio = 4) for deoxygenated
and oxygenated samples, respectively. We note that such an analysis is
inconsistent if the most distant site M1 and either M2 (ratio =
2400) or M3 (ratio = 4400) are considered. Thus if distance is
the main determinant of k
, the contribution of
terbium in M1 would not be observed in the present experiment, being
instead overwhelmed by terbium in site(s) M2 and/or M3. Furthermore the
above interpretation suggests that terbium is partitioned between sites
such that the likely occupancy is TbAP (M1, M2) and TbAP (M1, M3), the
binding of metal in M1 being a prerequisite for
phosphorescence(35) .
An alternative explanation for the
multiexponential donor decay based on the presence of a single terbium
acceptor is that small conformational heterogeneity exists in the TbAP
sample. Given the distance sensitivity of the exchange mechanism, the
postulated conformers would therefore show differences in
Trp-terbium distance of 0.3-0.4 Å. It clearly
will be necessary to conduct a more detailed study to determine the
exact terbium binding-site distribution in this system.
It is
interesting to compare our results with those obtained for the
terbium-elastase system (17) in which energy transfer was
assigned to an exchange process with a rate constant of 8300
s, 17-70 times larger than that found for
TbAP. From the atomic coordinates for porcine pancreatic
elastase(40) , we find a distance of 8.4 Å from the
single metal binding site to the center of the putative energy donor
Trp
. Assuming, as above, that K and J` are of similar magnitude and L = 1 Å for
both systems and using average donor-acceptor distances of 8.4 and 9.65
Å for terbium-elastase and TbAP, respectively, we calculate the
rate of energy transfer in the terbium-elastase sample should be
12-fold larger than that in the TbAP sample. All things considered this
is in good agreement with the experimental results. An additional
consideration that may modulate the rate of electron exchange is the
geometric orientation of the donor
(
)
wave functions, orthogonal to and electron-deficient in the indole
plane. In AP both metal-binding sites M2 and M3 are elevated 7 and 10
°, respectively, with reference to the center of the indole plane,
whereas in elastase this angle to the calcium site is 35 °. Thus a
contributing factor to the different energy transfer rates observed may
be an increase in favorable donor-acceptor orbital interaction in
terbium-elastase relative to TbAP. Previous studies of exchange
interactions in bichromophoric molecules (41) have demonstrated
the importance of orientation of the interacting orbitals on k
.
Finally we note that Kirk and co-workers (16) have suggested that through-bond energy transfer with an exponential dependence on the number of interconnecting bonds is important in terbium-indolyl-EDTA complexes. Because the through-bond pathway between donor and acceptor is smaller in TbAP than in terbium-elastase, the above mechanism is not applicable, rather the energy transfer is best understood in terms of a through-space exchange mechanism.
We anticipate that the contribution of protein triplet
states to enhanced terbium luminescence is a general phenomenon. Many
proteins show RTP from buried tryptophan residues with lifetimes
greater than 0.5 ms(42) , whereas those in which
phosphorescence is not easily observed probably have triplet state
lifetimes on the order of 20 µs, the value observed for free
tryptophan in deoxygenated solution(43) . Although tyrosine RTP
has yet to be observed, the increased terbium emission in model
tyrosine-containing peptides upon sample deoxygenation (4) suggests that its triplet state may also contribute to the
enhanced luminescence process. Given the common occurrence of protein
triplet states following photoexcitation, we reiterate the previously
stated caution (2, 17) to investigators employing the
Förster equation for the determination of
intraprotein distances in terbium-substituted systems. An experiment
comparing the enhanced emission of deoxygenated and air-equilibrated
samples would in principle allow the researcher to gauge the
contribution of protein triplet states to the enhanced luminescence.