Elementary Reactions in the Functional Triads of the Blue-Light Photoreceptor BLUF Domain.

The blue light using the flavin (BLUF) domain is one of the smallest photoreceptors in nature, which consists of a unique bidirectional electron-coupled proton relay process in its photoactivation reaction cycle. This perspective summarizes our recent efforts in dissecting the photocycle into three elementary processes, including proton-coupled electron transfer (PCET), proton rocking, and proton relay. Using ultrafast spectroscopy, we have determined the temporal sequence, rates, kinetic isotope effects (KIEs), and concertedness of these elementary steps. Our findings provide important implications for illuminating the photoactivation mechanism of the BLUF domain and suggest an engineering platform to characterize intricate reactions involving proton motions that are ubiquitous in nonphotosensitive protein machines.

MiR-2113 overexpression attenuates sepsis-induced acute pulmonary dysfunction, inflammation and fibrosis by inhibition of HMGB1.

Purpose: Sepsis-induced acute lung injury is related to high mortality. MiR-2113 possesses important functions in human diseases. This research aimed to clarify the role and mechanism of miR-2113 in sepsis-induced acute lung injury. Methods: The expression of miR-2113 in lipopolysaccharide (LPS)-induced MLE-12 cells, serum of sepsis patients, and cecal ligation and puncture mouse models was examined using quantitative real-time PCR. The functions of miR-2113 in LPS-treated MLE-12 cells were estimated by Cell Counting Kit-8 assay, flow cytometry, enzyme-linked immunosorbent assay, Western blot, and immunofluorescence. The influences of miR-2113 in cecal ligation and puncture-induced acute lung injury in mice were assessed by hematoxylin-eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay, acute pulmonary dysfunction analysis, lactate dehydrogenase levels and total protein concentrations in bronchoalveolar lavage fluid, and Masson staining. Also, the mechanism of miR-2113 was examined using a dual-luciferase reporter assay. Results: MiR-2113 expression was decreased in LPS-induced MLE-12 cells, serum of sepsis patients, and cecal ligation and puncture mouse models. miR-2113 overexpression restored LPS-reduced MLE-12 cell proliferation, but alleviated LPS-induced apoptosis and markers of inflammation and fibrosis in MLE-12 cells. Moreover, we found that miR-2113 mimic reduced LPS-induced MLE-12 cell injury by negatively regulating high-mobility group box 1. In vivo data further confirmed that miR-2113 overexpression alleviated acute pulmonary dysfunction, inflammation and fibrosis in cecal ligation and puncture-induced sepsis mice. Conclusion: MiR-2113 relieved sepsis-induced acute pulmonary dysfunction, inflammation and fibrosis through decreasing high-mobility group box 1.

Single-Electron Oxidation-Initiated Enantioselective Hydrosulfonylation of Olefins Enabled by Photoenzymatic Catalysis.

Controlling the enantioselectivity of hydrogen atom transfer (HAT) reactions has been a long-standing synthetic challenge. While recent advances on photoenzymatic catalysis have demonstrated the great potential of non-natural photoenzymes, all of the transformations are initiated by single-electron reduction of the substrate, with only one notable exception. Herein, we report an oxidation-initiated photoenzymatic enantioselective hydrosulfonylation of olefins using a novel mutant of gluconobacter ene-reductase (GluER-W100F-W342F). Compared to known photoenzymatic systems, our approach does not rely on the formation of an electron donor-acceptor complex between the substrates and enzyme cofactor and simplifies the reaction system by obviating the addition of a cofactor regeneration mixture. More importantly, the GluER variant exhibits high reactivity and enantioselectivity and a broad substrate scope. Mechanistic studies support the proposed oxidation-initiated mechanism and reveal that a tyrosine-mediated HAT process is involved.

Origin of the multi-phasic quenching dynamics in the BLUF domains across the species.

Blue light using flavin (BLUF) photoreceptors respond to light via one of nature's smallest photo-switching domains. Upon photo-activation, the flavin cofactor in the BLUF domain exhibits multi-phasic dynamics, quenched by a proton-coupled electron transfer reaction involving the conserved Tyr and Gln. The dynamic behavior varies drastically across different species, the origin of which remains controversial. Here, we incorporate site-specific fluorinated Trp into three BLUF proteins, i.e., AppA, OaPAC and SyPixD, and characterize the percentages for the Wout, WinNHin and WinNHout conformations using 19F nuclear magnetic resonance spectroscopy. Using femtosecond spectroscopy, we identify that one key WinNHin conformation can introduce a branching one-step proton transfer in AppA and a two-step proton transfer in OaPAC and SyPixD. Correlating the flavin quenching dynamics with the active-site structural heterogeneity, we conclude that the quenching rate is determined by the percentage of WinNHin, which encodes a Tyr-Gln configuration that is not conducive to proton transfer.

An ultrafast phototrigger of the Trp5CN-Trp motif in a ß-hairpin peptide.

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.

Dissecting the Ultrafast Stepwise Bidirectional Proton Relay in a Blue-Light Photoreceptor.

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.

The nature of proton-coupled electron transfer in a blue light using flavin domain.

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.

Direct Observation of Ultrafast Proton Rocking in the BLUF Domain.

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.

Effects of extracellular vesicles from mesenchymal stem cells on oxygen-glucose deprivation/reperfusion-induced neuronal injury.

BACKGROUND: Small extracellular vesicles (sEVs) from bone marrow mesenchymal stem cells (BMSCs) have shown therapeutic potential for cerebral ischemic diseases. However, the mechanisms by which BMSC-derived sEVs (BMSC-sEVs) protect neurons against cerebral ischemia/reperfusion (I/R) injury remain unclear. In this study, we explored the neuroprotective effects of BMSC-sEVs in the primary culture of rat cortical neurons exposed to oxygen-glucose deprivation and reperfusion (OGD/R) injury. METHODS: The primary cortical neuron OGD/R model was established to simulate the process of cerebral I/R in vitro. Based on this model, we examined whether the mechanism through which BMSC-sEVs could rescue OGD/R-induced neuronal injury. RESULTS: BMSC-sEVs (20 µg/mL, 40 µg/mL) significantly decreased the reactive oxygen species (ROS) productions, and increased the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx). Additionally, BMSC-sEVs prevented OGD/R-induced neuronal apoptosis in vivo, as indicated by increased cell viability, reduced lactate dehydrogenase (LDH) leakage, decreased terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining-positive cells, down-regulated cleaved caspase-3, and up-regulated Bcl-2/Bax ratio. Furthermore, Western blot and flow cytometry analysis indicated that BMSC-sEV treatment decreased the expression of phosphorylated calcium/calmodulin-dependent kinase II (p-CaMK II)/CaMK II, suppressed the increase of intracellular calcium concentration ([Ca2+]i) caused by OGD/R in neurons. CONCLUSIONS: These results demonstrate that BMSC-sEVs have significant neuroprotective effects against OGD/R-induced cell injury by suppressing oxidative stress and apoptosis, and Ca2+/CaMK II signaling pathways may be involved in this process.

Diode laser photoacoustic spectroscopy of CO2, H2S and O2 in a differential Helmholtz resonator for trace gas analysis in the biosciences and petrochemistry.

Photoacoustic spectroscopy in a differential Helmholtz resonator has been employed with near-IR and red diode lasers for the detection of CO2, H2S and O2 in 1 bar of air/N2 and natural gas, in static and flow cell measurements. With the red distributed feedback (DFB) diode laser, O2 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 s1/2. Within the tuning range of the near-IR DFB diode laser (6357-6378 cm-1), CO2 and H2S absorption features can be accessed, with a noise equivalent detection limit of 0.160 mbar (160 ppmv) CO2 in 1 bar N2 (30-mW laser, 1-s integration), corresponding to a normalised absorption coefficient α = 8.3 × 10-9 cm-1 W s1/2. Due to stronger absorptions, the noise equivalent detection limit of H2S in 1 bar N2 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 CO2 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 O2, CO2 and H2S in the cysteine metabolism of microbes (E. coli), and for the analysis of CO2 and H2S 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.