Proton Transfer and Hydrogen-Bond Interactions Determine the Fluorescence Quantum Yield of Bacteriophytochrome, a Novel Deep-Tissue Fluorescent Probe

Abstract

Phytochromes are red-light photoreceptors that regulate a variety of responses and cellular processes. The phytochrome light activation mechanism involves isomerization around the C15=C16 double bond of an open-chain tetrapyrrole chromophore, resulting in a flip of its D-ring. In an important new development, bacteriophytochrome (Bph) has been engineered for use as a fluorescent marker in mammalian tissues (Shu et al. Science 2009). Bph fluoresces at ∼720 nm, a wavelength less prone to scattering that can penetrate more deeply into tissue than light emitted by GFP-derived fluorescent proteins. The Bph chromophore biliverdin is a naturally occurring cofactor in mammalian tissue that covalently binds to a conserved cysteine in Bph, and hence BPhs can readily be genetically encoded. BPh photochemistry is thus of considerable significance for biomedical technology. Here we report that an unusual Bph, P3 from Rps. palustris, is highly fluorescent. We identify the factors that determine the fluorescence and isomerization quantum yields of P3 through the application of ultrafast spectroscopy to wild-type and mutants of P3 and a classical Bph, P2. The excited-state lifetime of biliverdin in P3 was significantly longer at 330 - 500 ps than in P2, and accompanied by a significantly reduced isomerization quantum yield. H/D exchange reduces the rate of decay from the biliverdin excite state by a factor of 1.4 and increases the isomerization quantum yield. Comparison of the properties of the P2 and P3 variants in relation to X-ray structures shows that the quantum yields of fluorescence and isomerization are determined by excited-state deprotonation of biliverdin at the pyrrole rings, in competition with hydrogen-bond rupture between the biliverdin D-ring and the apoprotein. This work provides a basis for structure-based conversion of BPh into an efficient near-IR fluorescent marker.