Direct Kinetic Evidence for Triplet State Energy Transfer from Escherichia coli Alkaline Phosphatase Tryptophan 109 to Bound Terbium

Abstract

The addition of excess Tb 3؉ 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 ET ϳ360 and 120 s ؊1) 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 109 ) corresponds to the longer component of the terbium emission and 2) the long-lived emission is enhanced, as is the Trp 109 phosphorescence, by deoxygenation. An energy transfer mechanism involving the Trp 109 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 . Because of its similar size and preference for strong oxygen donor groups as ligands, the use of the extrinsic luminescent probe Tb 3ϩ as a replacement for Ca 2ϩ is particularly well established (1-4). For the typical concentrations used in terbium-protein systems, Ͻ10 Ϫ3 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 (⑀ 285 Ϸ0.4 M Ϫ1 cm Ϫ1 for Tb 3ϩ 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 dipoledipole energy transfer mechanism is established, to extract intraprotein donor-acceptor pair distances (for example see Refs. 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 terbiumsubstituted 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 inc...

Photoinduced energy transfer from the triplet state of naphthalene moiety in the Tb3+/Eu3+ complexes of a specially designed naphthalene cryptand

“…Steady state and time-resolved studies in Terbium substituted E. coli alkaline phosphatase confirmed the involvement of T 1 state of the Tryptophan residue at 109 positions in ET by exchange mechanism [11,47]. Also the Dexter exchange mechanism using the T 1 state of the Tryptophan in ET to Tb 3+ has been suggested for the Terbium-elastase system [34].…”

“…Thus the ET process in the present case occurs most likely by through space exchange mechanism. Earlier studies of Schlyer et al [9] on Tb-alkaline phosphatase system, the emphasis is given on through space ET. In our earlier studies with Tb-alkaline phosphatase system [11] and Tb 3+ complex of a tris methoxy coumarin derivative of a cryptand [48], we also emphasized that through space ET mechanism is operative.…”

“…[1][2][3][4][5][6][7][8][9][10]. Studies on ET at room temperature and at low temperature in Eu 3+ and/or Tb 3+ ion complexes of different naphtho-crown and aza-crown ethers and cryptands and some proteins have been carried out earlier in our laboratory [11][12][13].…”

Engineering a terbium-binding site into an integral membrane protein for luminescence energy transfer

“…The nature of this energy transfer obeys the Dexter mechanism of electron exchange, and the triplet excited state (phosphorescence) of the antenna is involved (39). Therefore, a Trp residue is introduced at position 7 of the EF-hand motif to serve this purpose (24,40 luminescence to 50% at millimolar concentrations, thereby illustrating a lower affinity for divalent cations (data not shown). Thus, both the affinity and specificity of the EF-hand motif engineered into LacY are appropriate for LRET studies.…”

Luminescence Dynamics of Terbium Monodipicolinate in Ethyl Ammonium Nitrate

“…Long risetimes are sometimes found in terbium complexes where a protein is the ligand. 15 A long risetime has also been found in terbium complexes of naphthalene and 1-Me-naphthalene. 16 The effect of backtransfer has been studied in Tb 3þ -doped glasses.…”