Energy transfer between terbium (III) and cobalt (II) in thermolysin: a new class of metal--metal distance probes.
The visible fluorescence of terbium(III) when bound to a calcium binding site of thermolysin is greatly enhanced with an excitation maximum at 280 nm but substitution of cobalt(II) for zinc at the active site decreases the intensity by 89.5%. Treatment with N-bromosuccinimide quenches enzyme tryptophan and Tb(III) fluorescence to a similar extent and suggests the operation of tryptophan vector Tb(III) vector Co(II) energy relay system in the enzyme. Dipoledipole radiationless energy transfer between the Tb(III) donor and the Co(II) acceptor can account for this quenching. The inherent characteristics of the metal pair limits the value of the orientation factor, K2, of the Förster equation, thereby reducing uncertainties in distance measurements by energy transfer compared with other systems. A quantum yield of 0.51 yields a value of R0, the distance for 50% energy transfer, of 19.6 A, and a distance, R, between Tb(III) and Co(II) of 13.7 A, a value identical to that measured for the distance between the active site zinc atom and calcium atom number 1 by x-ray analysis in native thermolysin crystals. The limits of confidence of this measurement are discussed. Energy transfer between two different metal atom sites of a protein provides a new class of probes to measure intramolecular distances of biological macromolecules in solution.
D001665:Binding Sites; D001974:Bromosuccinimide; D003035:Cobalt; D004735:Energy Transfer; D008433:Mathematics; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013725:Terbium; D013820:Thermolysin
D013725:Terbium; D003035:Cobalt; D013820:Thermolysin; D001974:Bromosuccinimide