Flavoprotein Photochemistry: Fundamental Processes and Photocatalytic Perspectives

Zhuang, B. A.; Liebl, Ursula; Vos, Marten H.

doi:10.1021/acs.jpcb.2c00969

Cited by 22 publications

(19 citation statements)

References 73 publications

(155 reference statements)

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“…The fast ET leads to rapid quenching of FMN*, causing the steady-state fluorescence to be too weak to detect. Tyrosine and tryptophan residues are well-known electron donors that quench FMN*. − There are two tyrosine residues near FMN: Y44 and Y152. We reasoned that mutation of these two tyrosine residues could shut down ET pathways and significantly increase the lifetime of FMN*.…”

Section: Resultsmentioning

confidence: 99%

Mechanism and Dynamics of Photodecarboxylation Catalyzed by Lactate Monooxygenase

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Zanetti-Polzi

et al. 2023

J. Am. Chem. Soc.

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Photoenzymes are a rare class of biocatalysts that use light to facilitate chemical reactions. Many of these catalysts utilize a flavin cofactor to absorb light, suggesting that other flavoproteins might have latent photochemical functions. Lactate monooxygenase is a flavin-dependent oxidoreductase previously reported to mediate the photodecarboxylation of carboxylates to afford alkylated flavin adducts. While this reaction holds a potential synthetic value, the mechanism and synthetic utility of this process are unknown. Here, we combine femtosecond spectroscopy, sitedirected mutagenesis, and a hybrid quantum-classical computational approach to reveal the active site photochemistry and the role the active site amino acid residues play in facilitating this decarboxylation. Light-induced electron transfer from histidine to flavin was revealed, which has not been reported in other proteins. These mechanistic insights enable the development of catalytic oxidative photodecarboxylation of mandelic acid to produce benzaldehyde, a previously unknown reaction for photoenzymes. Our findings suggest that a much wider range of enzymes have the potential for photoenzymatic catalysis than has been realized to date.

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Section: Resultsmentioning

confidence: 99%

Mechanism and Dynamics of Photodecarboxylation Catalyzed by Lactate Monooxygenase

Page

Zanetti-Polzi

et al. 2023

J. Am. Chem. Soc.

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“…They are often employed as cofactors by so-called flavoproteins, whose genes constitute a notable fraction of all genes found in the genomes of variable organisms (3,4). On their own, flavins display complex photochemistry and photophysics, and have been the focus of numerous studies (5)(6)(7)(8). However, while the spectra of other biological chromophores, such as retinal (9)(10)(11) or tetrapyrroles (12)(13)(14), are strongly influenced by their protein environment, the positions of absorption and fluorescence maxima of RF, FMN and FAD are vastly similar between different protein families (5,6).…”

Section: Introductionmentioning

confidence: 99%

Fine spectral tuning of a flavin-binding fluorescent protein for multicolor imaging

Николаев

Yudenko

Smolentseva

et al. 2022

Preprint

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Flavin-binding fluorescent proteins (FbFPs) are promising genetically encoded tags for microscopy. However, spectral properties of their chromophores (riboflavin, flavin mononucleotide and flavin adenine dinucleotide) are notoriously similar even between different protein families, which limits applications of flavoproteins in multicolor imaging. Here, we present a palette of twenty-two finely tuned fluorescent tags based on the thermostable LOV domain from Chloroflexus aggregans (CagFbFP). We performed site saturation mutagenesis of three amino acid positions in the flavin-binding pocket, including the photoactive cysteine, to obtain variants with fluorescence emission maxima uniformly covering the wavelength range from 486 to 512 nm. We demonstrate three-color imaging based on spectral separation and two-color fluorescence lifetime imaging using the proteins from the palette. These results highlight the possibility of fine spectral tuning of flavoproteins and pave the way for further applications of FbFPs in fluorescence microscopy.

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“…However, time-resolved spectroscopic studies of fully reduced flavins in proteins 8−14 are scarce compared to those in the oxidized state. 15 Fully reduced flavins naturally occur either in the anionic (FADH − or FMNH − ) or protonated, neutral (FADH 2 or FMNH 2 ) form. 1 It is commonly assumed that fully reduced (both neutral and anionic) flavins are virtually nonfluorescent in aqueous solution, but they can have long-lived, and therefore highly fluorescent, excited states in a protein environment or at low temperatures.…”

mentioning

confidence: 99%

“…For enzymes participating in two-electron redox processes, the (two-electron) fully reduced hydroquinone form can also be viewed as a quasi-stable active state that reacts with various substrates (usually external electron acceptors) to complete the catalytic cycle. − In recent years, inspired by the catalytic cycle of the photoenzyme DNA photolyase, employing fully reduced flavins as the functional state has become a promising strategy in the development of bioengineered flavin-dependent photocatalysts. − Therefore, fundamental knowledge of the properties of protein-bound fully reduced flavins is required both for understanding the catalytic mechanism of many natural flavoenzymes, and for enabling the rational design of flavoprotein variants as photocatalysts. However, time-resolved spectroscopic studies of fully reduced flavins in proteins − are scarce compared to those in the oxidized state …”

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confidence: 99%

Excited-State Properties of Fully Reduced Flavins in Ferredoxin–NADP⁺ Oxidoreductase

Zhuang

Aleksandrov

Seo

et al. 2023

J. Phys. Chem. Lett.

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The fully reduced flavin cofactor (FAD red ) in ferredoxin−NADP + oxidoreductase (FNR) is a functional intermediate that displays different catalytic and steady-state spectral properties for enzymes from Bacillus subtilis (BsFNR), Chlorobaculum tepidum (CtFNR), and Rhodopseudomonas palustris (RpFNR). Using ultrafast spectroscopy, we reveal that at physiological pH, photoexcited FAD red in BsFNR and RpFNR exhibits unprecedentedly fast decays (dominantly in 6 and 8 ps, respectively), whereas in CtFNR the decay is much slower (∼400 ps), as in other flavoproteins. Correlating these observations with the protonation states of FAD red and the dynamic properties of the protein environment, we conclude that the excited state of neutral FAD red can be intrinsically short-lived even in proteins, contrasting with the well-documented behavior of the anionic form that systematically displays markedly increased excited-state lifetime upon binding to proteins. This work provides new insight into the photochemistry of fully reduced flavins, which are emerging as functional initial states in bioengineered photocatalysts.

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Flavoprotein Photochemistry: Fundamental Processes and Photocatalytic Perspectives

Cited by 22 publications

References 73 publications

Mechanism and Dynamics of Photodecarboxylation Catalyzed by Lactate Monooxygenase

Mechanism and Dynamics of Photodecarboxylation Catalyzed by Lactate Monooxygenase

Fine spectral tuning of a flavin-binding fluorescent protein for multicolor imaging

Excited-State Properties of Fully Reduced Flavins in Ferredoxin–NADP⁺ Oxidoreductase

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Flavoprotein Photochemistry: Fundamental Processes and Photocatalytic Perspectives

Cited by 22 publications

References 73 publications

Mechanism and Dynamics of Photodecarboxylation Catalyzed by Lactate Monooxygenase

Mechanism and Dynamics of Photodecarboxylation Catalyzed by Lactate Monooxygenase

Fine spectral tuning of a flavin-binding fluorescent protein for multicolor imaging

Excited-State Properties of Fully Reduced Flavins in Ferredoxin–NADP+ Oxidoreductase

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Excited-State Properties of Fully Reduced Flavins in Ferredoxin–NADP⁺ Oxidoreductase