Ultrafast infrared spectroscopy reveals a key step for successful entry into the photocycle for photoactive yellow protein

Abstract

Photoactive proteins such as PYP (photoactive yellow protein) are generally accepted as model systems for studying protein signal state formation. PYP is a blue-light sensor from the bacterium Halorhodospira halophila. The formation of PYP's signaling state is initiated by trans-cis isomerization of the p-coumaric acid chromophore upon the absorption of light. The quantum yield of signaling state formation is Ϸ0.3. Using femtosecond visible pump͞mid-IR probe spectroscopy, we investigated the structure of the very short-lived ground state intermediate (GSI) that results from an unsuccessful attempt to enter the photocycle. This intermediate and the first stable GSI on pathway into the photocycle, I0, both have a mid-IR difference spectrum that is characteristic of a cis isomer, but only the I0 intermediate has a chromophore with a broken hydrogen bond with the backbone N atom of Cys-69. We suggest, therefore, that breaking this hydrogen bond is decisive for a successful entry into the photocycle. The chromophore also engages in a hydrogen-bonding network by means of its phenolate group with residues Tyr-42 and Glu-46. We have investigated the role of this hydrogen bond by exchanging the H bond-donating residue Glu-46 with the weaker H bond-donating glutamine (i.e., Gln-46). We have observed that this mutant exhibits virtually identical kinetics and product yields as WT PYP, even though during the I0-to-I1 transition, on the 800-ps time scale, the hydrogen bond of the chromophore with Gln-46 is broken, whereas this hydrogen bond remains intact with Glu-46.ground state intermediate ͉ hydrogen bond ͉ quantum yield ͉ picosecond ͉ vibrational P YP (photoactive yellow protein) belongs to the Xanthopsins, a family of blue-light photoreceptors that contain 4-hydroxycinnamic acid as their photoactive chromophore (see refs. 1-3 for a review). PYP is a small protein and therefore an attractive model system for exploring how a chromophore and protein interact to sense light and send a biological signal. Its photocycle has been characterized by various experimental techniques, such as fluorescence (4, 5), (time-resolved) FTIR (6-8), (timeresolved) x-ray crystallography (9-12), NMR (13), Stark spectroscopy (14), and pump(-dump)-probe spectroscopy (15, 16). X-ray diffraction on PYP crystals has demonstrated that the PYP chromophore is covalently linked (see Fig. 1) to the protein backbone by means of . It is further embedded in a hydrogen-bonding network consisting of . In the ground state, the chromophore is in a deprotonated trans form, negatively charged, and possibly stabilized by the positive Arg-52 residue (12). After photoexcitation, the chromophore forms a red-shifted intermediate, referred to as I 0 , within a few picoseconds. This intermediate has a shifted absorption maximum from 446 to 500 nm. The second intermediate, I 1 (or pR or PYP L ), absorbs maximally at 480 nm and is formed in 1-3 ns (15-20). This intermediate is followed by protonation of the chromophore and a large structural change of the protein on a mil...

Watching a signaling protein function in real time via 100-ps time-resolved Laue crystallography

“…The earliest structural event, the trans-to-cis photoisomerization of pCA, occurs in ∼1-3 ps (13)(14)(15)(16)(17) and is too fast to track with 150-ps time resolution. Nevertheless, the structural changes arising from this transition are captured in the 100-ps snapshot shown in Fig.…”

Populations and coherence in femtosecond time resolved X-ray crystallography of the photoactive yellow protein

“…The time-constant for photoisomerisation of the PYP chromophore has been reported with values ranging from ~400 fs to 1.4 ps, depending on the details of optical conditions and spectroscopic probe, and also sample conditions [19][20][21][22]. Notably, excited state decay has been shown to be multi-phasic from femtosecond fluorescence and transient absorption spectroscopy [8,20].…”

“…Second, transient visible absorption as well as transient mid-IR absorption has revealed the presence of an early intermediate which has been assigned to an electronic ground state species [19,23,30]. This ground state intermediate (GSI) is characterised by distinctive induced absorption at 490 nm, while the primary photoproduct absorbs maximally at ~510 nm [23,30].…”

“…It is emphasized that the transient concentration of the GSI is sensitive to the optical parameters [8], and it has been argued on the basis of transient absorption spectroscopy [8] done under comparable optical conditions that femtosecond and picosecond delays of the recent TR-SFX study should include only small contributions from the GSI [12]. An alternative modelling of ultrafast measurements, using infrared detection, assumed that all population resulting in ground-state re-filling passes through the GSI, which represents 72% of the total decay amplitude [19]. However based on the amplitude of the distinguishing transient absorption at 490 nm, the assumption of nearly equal branching between direct ground state recovery and passage through the GSI is more probable (figure 4c) [8,29].…”

Direct observation of ligand transfer and bond formation in cytochrome c oxidase by using mid-infrared chirped-pulse upconversion

“…IR pump-probe spectroscopy approaches have relied either on timeconsuming frequency (3,9) or delay scanning (12) or, more recently, on expensive mercury cadmium teluride (HgCdTe) detector arrays (10,11,13,14) for detection of the IR probe. Here, we measure the transmitted mid-IR pulse using the recently demonstrated technique of upconversion into the visible range by sumfrequency generation with a chirped 800-nm pulse (15).…”