Proton-coupled electron transfer (PCET) is key to the activation of the blue light using flavin (BLUF) domain photoreceptors. Here, to elucidate the photocycle of the central FMN-Gln-Tyr motif in the BLUF domain of OaPAC, we eliminated the intrinsic interfering W90 in the mutant design. We integrated the stretched exponential function into the target analysis to account for the dynamic heterogeneity arising from the active-site solvation relaxation and the flexible H-bonding network as shown in the molecular dynamics simulation results, facilitating a simplified expression of the kinetics model. We find that, in both the functional wild-type (WT) and the nonfunctional Q48E and Q48A, forward PCET happens in the range of 105 ps to 344 ps, with a kinetic isotope effect (KIE) measured to be ∼1.8 to 2.4, suggesting that the nature of the forward PCET is concerted. Remarkably, only WT proceeds with an ultrafast reverse PCET process (31 ps, KIE = 4.0), characterized by an inverted kinetics of the intermediate FMNH˙. Our results reveal that the reverse PCET is driven by proton transfer via an intervening imidic Gln.

We present direct observation of ultrafast proton rocking in the central motif of a BLUF domain protein scaffold. The mutant design has taken consideration of modulating the proton‐coupled electron transfer (PCET) driving forces by replacing Tyr in the original motif with Trp, in order to remove the interference of a competing electron transfer pathway. Using femtosecond pump–probe spectroscopy and detailed kinetics analysis, we resolved an electron‐transfer‐coupled Grotthuss‐type forward and reverse proton rocking along the FMN–Gln–Trp proton relay chain. The rates of forward and reverse proton transfer are determined to be very close, namely 51 ps vs. 52 ps. The kinetic isotope effect (KIE) constants associated with the forward and reverse proton transfer are 3.9 and 5.3, respectively. The observation of ultrafast proton rocking is not only a crucial step towards revealing the nature of proton relay in the BLUF domain, but also provides a new paradigm of proton transfer in proteins for theoretical investigations.

Photoacoustic spectroscopy in a differential Helmholtz resonator has been employed with near-IR and red diode lasers for the detection of CO 2 , H 2 S and O 2 in 1 bar of air/N 2 and natural gas, in static and flow cell measurements. With the red distributed feedback (DFB) diode laser, O 2 can be detected at 764.3 nm with a noise equivalent detection limit of 0.60 mbar (600 ppmv) in 1 bar of air (35-mW laser, 1-s integration), corresponding to a normalised absorption coefficient α = 2.2 × 10 −8 cm −1 W s 1/2 . Within the tuning range of the near-IR DFB diode laser (6357–6378 cm −1 ), CO 2 and H 2 S absorption features can be accessed, with a noise equivalent detection limit of 0.160 mbar (160 ppmv) CO 2 in 1 bar N 2 (30-mW laser, 1-s integration), corresponding to a normalised absorption coefficient α = 8.3 × 10 −9 cm −1 W s 1/2 . Due to stronger absorptions, the noise equivalent detection limit of H 2 S in 1 bar N 2 is 0.022 mbar (22 ppmv) at 1-s integration time. Similar detection limits apply to trace impurities in 1 bar natural gas. Detection limits scale linearly with laser power and with the square root of integration time. At 16-s total measurement time to obtain a spectrum, a noise equivalent detection limit of 40 ppmv CO 2 is obtained after a spectral line fitting procedure, for example. Possible interferences due to weak water and methane absorptions have been discussed and shown to be either negligible or easy to correct. The setup has been used for simultaneous in situ monitoring of O 2 , CO 2 and H 2 S in the cysteine metabolism of microbes ( E . coli ), and for the analysis of CO 2 and H 2 S impurities in natural gas. Due to the inherent signal amplification and noise cancellation, photoacoustic spectroscopy in a differential Helmholtz resonator has a great potential for trace gas analysis, with possible applications including safety monitoring of toxic gases and applications in the biosciences and for natural gas analysis in petrochemistry. Graphical abstract

Proton relays through H-bond networks are essential in realizing the functionality of protein machines such as in photosynthesis and photoreceptors. It has been challenging to dissect the rates and energetics of individual proton-transfer steps during the proton relay. Here, we have designed a proton rocking blue light using a flavin (BLUF) domain with the flavin mononucleotide (FMN)–glutamic acid (E)–tryptophan (W) triad and have resolved the four individual proton-transfer steps kinetically using ultrafast spectroscopy. We have found that after the photo-induced charge separation forming FMN· –/E-COOH/WH· +, the proton first rapidly jumps from the bridging E-COOH to FMN– (τfPT2 = 3.8 ps; KIE = 1.0), followed by a second proton transfer from WH· + to E-COO– (τfPT1 = 336 ps; KIE = 2.6) which immediately rocks back to W· (τrPT1 = 85 ps; KIE = 6.7), followed by a proton return from FMNH· to E-COO– (τrPT2 = 34 ps; KIE = 3.3) with the final charge recombination between FMN· – and WH· + to close the reaction cycle. Our results revisited the Grotthuss mechanism on the ultrafast timescale using the BLUF domain as a paradigm protein.

Phototriggers are useful molecular tools to initiate reactions in enzymes by light for the purpose of photoenzymatic design and mechanistic investigations. Here, we incorporated the non-natural amino acid 5-cyanotryptophan (W5CN) in a polypeptide scaffold and resolved the photochemical reaction of the W5CN–W motif using femtosecond transient UV/Vis and mid-IR spectroscopy. We identified a marker band of ∼2037 cm−1 from the CN stretch of the electron transfer intermediate W5CN·− in the transient IR measurement and found UV/Vis spectroscopic evidence for the W·+ radical at 580 nm. Through kinetic analysis, we characterized that the charge separation between the excited W5CN and W occurs in 253 ps, with a charge-recombination lifetime of 862 ps. Our study highlights the potential use of the W5CN–W pair as an ultrafast phototrigger to initiate reactions in enzymes that are not light-sensitive, making downstream reactions accessible to femtosecond spectroscopic detection.