Direct Observation of Ultrafast Proton Rocking in the BLUF Domain

Ding, Bei; Kang, Xiu‐Wen; Chen, Zijing; Zhou, Zhongneng; Zhou, Yalin; Tang, Siwei; Zhang, Yifei; Zhang, Tianyi; Zhong, Dongping

doi:10.1002/anie.202114423

Cited by 7 publications

(46 citation statements)

References 42 publications

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“…S3 ) ( 31 ). Most interestingly, in comparison with the FMNH˙/W˙ SADS of our recently reported Y6W/W90F mutant which is a W analog of WT ( 37 ), we discover clear spectral signatures of YO˙ ( 38 , 39 ) around 410 nm to 420 nm for WT, which is also observed in the Q48A and Q48E mutants ( SI Appendix , Fig. S10 ).…”

Section: Resultssupporting

confidence: 60%

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

Zhou

Chen

Kang

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Section: Resultssupporting

confidence: 60%

“…4 A and C ) show typical characteristics of FMN* for flavoproteins ( SI Appendix , Fig. S3 ) ( 37 ). The blue SADS component has positive broad absorptions in 500 nm to 650 nm and a negative peak at 384 nm that are attributed to FMNH˙ ( SI Appendix , Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Zhou

Chen

Kang

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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“…The role of proton transfer in the function of OaPAC BLUF was examined in a recent paper by the Zhong group where the authors replaced the tyrosine by a tryptophan (Y6W). 48 Kang et al observed a sequential electron transfer process: formation of the flavin anionic radical, followed by the formation of the neutral semiquinone. The authors proposed that they observed proton rocking in which the tryptophan transiently donated a proton to the flavin to form the neutral semiquinone followed by a reverse PT; the rates of the forward and reverse PT were very close, being 51 ps and 52 ps, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…The role of proton transfer in the function of OaPAC BLUF was examined in a recent paper by the Zhong group where the authors replaced the tyrosine by a tryptophan (Y6W) . Kang et al observed a sequential electron transfer process: formation of the flavin anionic radical, followed by the formation of the neutral semiquinone.…”

Section: Discussionmentioning

confidence: 99%

Unraveling the Photoactivation Mechanism of a Light-Activated Adenylyl Cyclase Using Ultrafast Spectroscopy Coupled with Unnatural Amino Acid Mutagenesis

et al. 2022

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The hydrogen bonding network that surrounds the flavin in blue light using flavin adenine dinucleotide (BLUF) photoreceptors plays a crucial role in sensing and communicating the changes in the electronic structure of the flavin to the protein matrix upon light absorption. Using time-resolved infrared spectroscopy (TRIR) and unnatural amino acid incorporation, we investigated the photoactivation mechanism and the role of the conserved tyrosine (Y6) in the forward reaction of the photoactivated adenylyl cyclase from Oscillatoria acuminata (OaPAC). Our work elucidates the direct connection between BLUF photoactivation and the structural and functional implications on the partner protein for the first time. The TRIR results demonstrate the formation of the neutral flavin radical as an intermediate species on the photoactivation pathway which decays to form the signaling state. Using fluorotyrosine analogues to modulate the physical properties of Y6, the TRIR data reveal that a change in the p K a and/or reduction potential of Y6 has a profound effect on the forward reaction, consistent with a mechanism involving proton transfer or proton-coupled electron transfer from Y6 to the electronically excited FAD. Decreasing the p K a from 9.9 to <7.2 and/or increasing the reduction potential by 200 mV of Y6 prevents proton transfer to the flavin and halts the photocycle at FAD •- . The lack of protonation of the anionic flavin radical can be directly linked to photoactivation of the adenylyl cyclase (AC) domain. While the 3F-Y6 and 2,3-F 2 Y6 variants undergo the complete photocycle and catalyze the conversion of ATP into cAMP, enzyme activity is abolished in the 3,5-F 2 Y6 and 2,3,5-F 3 Y6 variants where the photocycle is halted at FAD •- . Our results thus show that proton transfer plays an essential role in initiating the structural reorganization of the AC domain that results in AC activity.

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“…The mechanisms of further signal transduction to acceptor proteins are not completely clear. The reverse process of electron transfer to tyrosine (PCET) leads to the restoration of the original form of the photoreceptor and the closure of the photocycle [ 227 , 228 , 229 , 230 , 231 ].…”

Section: Biochemical and Physiological Application Of Pterin Photoche...mentioning

confidence: 99%

Insights into Molecular Structure of Pterins Suitable for Biomedical Applications

Buglak

Kapitonova

Vechtomova

et al. 2022

IJMS

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Pterins are an inseparable part of living organisms. Pterins participate in metabolic reactions mostly as tetrahydropterins. Dihydropterins are usually intermediates of these reactions, whereas oxidized pterins can be biomarkers of diseases. In this review, we analyze the available data on the quantum chemistry of unconjugated pterins as well as their photonics. This gives a comprehensive overview about the electronic structure of pterins and offers some benefits for biomedicine applications: (1) one can affect the enzymatic reactions of aromatic amino acid hydroxylases, NO synthases, and alkylglycerol monooxygenase through UV irradiation of H4pterins since UV provokes electron donor reactions of H4pterins; (2) the emission properties of H2pterins and oxidized pterins can be used in fluorescence diagnostics; (3) two-photon absorption (TPA) should be used in such pterin-related infrared therapy because single-photon absorption in the UV range is inefficient and scatters in vivo; (4) one can affect pathogen organisms through TPA excitation of H4pterin cofactors, such as the molybdenum cofactor, leading to its detachment from proteins and subsequent oxidation; (5) metal nanostructures can be used for the UV-vis, fluorescence, and Raman spectroscopy detection of pterin biomarkers. Therefore, we investigated both the biochemistry and physical chemistry of pterins and suggested some potential prospects for pterin-related biomedicine.

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Direct Observation of Ultrafast Proton Rocking in the BLUF Domain

Cited by 7 publications

References 42 publications

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

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

Unraveling the Photoactivation Mechanism of a Light-Activated Adenylyl Cyclase Using Ultrafast Spectroscopy Coupled with Unnatural Amino Acid Mutagenesis

Insights into Molecular Structure of Pterins Suitable for Biomedical Applications

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