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The photochemical mechanism of a B12-dependent photoreceptor protein.

Kutta RJ, Hardman SJ, Johannissen LO, Bellina B, Messiha HL, Ortiz-Guerrero JM, Elías-Arnanz M, Padmanabhan S, Barran P, Scrutton NS, Jones AR - Nat Commun (2015)

Bottom Line: Here we describe the photochemistry in CarH that ultimately triggers tetramer dissociation; it proceeds via a cob(III)alamin intermediate, which then forms a stable adduct with the protein.This pathway is without precedent and our data suggest it is independent of the radical chemistry common to both coenzyme B12 enzymology and its known photochemistry.It provides a mechanistic foundation for the emerging field of B12 photobiology and will serve to inform the development of a new class of optogenetic tool for the control of gene expression.

View Article: PubMed Central - PubMed

Affiliation: 1] School of Chemistry, The University of Manchester, Oxford Road, Manchester M13 9PL, UK [2] Photon Science Institute, The University of Manchester, Alan Turing Building, Oxford Road, Manchester M13 9PL, UK [3] SYNBIOCHEM, Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.

ABSTRACT
The coenzyme B12-dependent photoreceptor protein, CarH, is a bacterial transcriptional regulator that controls the biosynthesis of carotenoids in response to light. On binding of coenzyme B12 the monomeric apoprotein forms tetramers in the dark, which bind operator DNA thus blocking transcription. Under illumination the CarH tetramer dissociates, weakening its affinity for DNA and allowing transcription. The mechanism by which this occurs is unknown. Here we describe the photochemistry in CarH that ultimately triggers tetramer dissociation; it proceeds via a cob(III)alamin intermediate, which then forms a stable adduct with the protein. This pathway is without precedent and our data suggest it is independent of the radical chemistry common to both coenzyme B12 enzymology and its known photochemistry. It provides a mechanistic foundation for the emerging field of B12 photobiology and will serve to inform the development of a new class of optogenetic tool for the control of gene expression.

No MeSH data available.


Related in: MedlinePlus

Proposed photochemical mechanism of CarH.The coloured boxes and letters correspond to the colour-coded ground state (GS), intermediates (A–D) and product (or ‘light state', LS) in Fig. 5. Intermediates E* and E in Fig. 5 have not been shown here because they represent the adduct between protein and the cobalamin before complete dissociation of the tetramer and therefore resemble LS. AdoCbl is represented with a simplified corrin ring for clarity, with the Co coordinated by a lower axial H177 from the protein. The involvement of residues H132 and E129 are those favoured by our molecular dynamic simulations based on our homology model of the CarH structure (Fig. 2). After photoexcitation (hν) of the GS, each step proceeds with a rate constant, ki, where i: CT, charge transfer; HomoC, homolytic cleavage; HeteroC, heterolytic cleavage; R, rearrangement; AF, adduct formation; Diss, tetramer dissociation. See main text for discussion of the mechanism.
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f6: Proposed photochemical mechanism of CarH.The coloured boxes and letters correspond to the colour-coded ground state (GS), intermediates (A–D) and product (or ‘light state', LS) in Fig. 5. Intermediates E* and E in Fig. 5 have not been shown here because they represent the adduct between protein and the cobalamin before complete dissociation of the tetramer and therefore resemble LS. AdoCbl is represented with a simplified corrin ring for clarity, with the Co coordinated by a lower axial H177 from the protein. The involvement of residues H132 and E129 are those favoured by our molecular dynamic simulations based on our homology model of the CarH structure (Fig. 2). After photoexcitation (hν) of the GS, each step proceeds with a rate constant, ki, where i: CT, charge transfer; HomoC, homolytic cleavage; HeteroC, heterolytic cleavage; R, rearrangement; AF, adduct formation; Diss, tetramer dissociation. See main text for discussion of the mechanism.

Mentions: The model in Fig. 5a was applied to the DADS resulting from the global analysis over fs–s. From this modelling we generated a SAS for each intermediate observed (Fig. 3b–e) from which we propose the photochemical mechanism in Fig. 6. Photoexcitation of CarH-GS gives an initial excited state A, the signal from which decays with the rate k1. From our proposed model, A either relaxes to the ground state with rate αk1=kD=0.471 × 1012 s−1 or decays into one of two intermediates B or C with rates βk1=kHomoC=0.012 × 1012 s−1 and γk1=kCT=0.044 × 1012 s−1, respectively (Fig. 5a). α, β and γ are branching ratios, where α+β+γ=1. The green SAS of intermediate B in Fig. 5b is distinctive of the Co−C bond homolysis product, the cob(II)alamin radical (Fig. 6, green box)18. This is a non-productive channel, with a recombination rate k2=kRPR=1.26 × 109 s−1, after which all of the radical pairs recombine to the ground state. The brown SAS of C in Fig. 5b closely resembles the estimated spectrum of the MLCT state reported previously2123, where the Co(III) effectively receives dative coordination from an anionic ligand (Fig. 6, brown box). This intermediate has a lifetime of ≫3 ns and leads to a productive channel ending in the photoproduct. The branching ratios in our model are α=0.894, β=0.022 and γ=0.084. They suggest that, following a single-fs laser pulse, the majority of excited states decay to the ground state, with only 8.4% leading to the photoproduct and tetramer dissociation. A relatively low yield of productive channel would be consistent with the need for carotenoid biosynthesis only under high light intensity conditions, when the bacteria that express CarH are likely to experience significant photo-oxidative stress511.


The photochemical mechanism of a B12-dependent photoreceptor protein.

Kutta RJ, Hardman SJ, Johannissen LO, Bellina B, Messiha HL, Ortiz-Guerrero JM, Elías-Arnanz M, Padmanabhan S, Barran P, Scrutton NS, Jones AR - Nat Commun (2015)

Proposed photochemical mechanism of CarH.The coloured boxes and letters correspond to the colour-coded ground state (GS), intermediates (A–D) and product (or ‘light state', LS) in Fig. 5. Intermediates E* and E in Fig. 5 have not been shown here because they represent the adduct between protein and the cobalamin before complete dissociation of the tetramer and therefore resemble LS. AdoCbl is represented with a simplified corrin ring for clarity, with the Co coordinated by a lower axial H177 from the protein. The involvement of residues H132 and E129 are those favoured by our molecular dynamic simulations based on our homology model of the CarH structure (Fig. 2). After photoexcitation (hν) of the GS, each step proceeds with a rate constant, ki, where i: CT, charge transfer; HomoC, homolytic cleavage; HeteroC, heterolytic cleavage; R, rearrangement; AF, adduct formation; Diss, tetramer dissociation. See main text for discussion of the mechanism.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4557120&req=5

f6: Proposed photochemical mechanism of CarH.The coloured boxes and letters correspond to the colour-coded ground state (GS), intermediates (A–D) and product (or ‘light state', LS) in Fig. 5. Intermediates E* and E in Fig. 5 have not been shown here because they represent the adduct between protein and the cobalamin before complete dissociation of the tetramer and therefore resemble LS. AdoCbl is represented with a simplified corrin ring for clarity, with the Co coordinated by a lower axial H177 from the protein. The involvement of residues H132 and E129 are those favoured by our molecular dynamic simulations based on our homology model of the CarH structure (Fig. 2). After photoexcitation (hν) of the GS, each step proceeds with a rate constant, ki, where i: CT, charge transfer; HomoC, homolytic cleavage; HeteroC, heterolytic cleavage; R, rearrangement; AF, adduct formation; Diss, tetramer dissociation. See main text for discussion of the mechanism.
Mentions: The model in Fig. 5a was applied to the DADS resulting from the global analysis over fs–s. From this modelling we generated a SAS for each intermediate observed (Fig. 3b–e) from which we propose the photochemical mechanism in Fig. 6. Photoexcitation of CarH-GS gives an initial excited state A, the signal from which decays with the rate k1. From our proposed model, A either relaxes to the ground state with rate αk1=kD=0.471 × 1012 s−1 or decays into one of two intermediates B or C with rates βk1=kHomoC=0.012 × 1012 s−1 and γk1=kCT=0.044 × 1012 s−1, respectively (Fig. 5a). α, β and γ are branching ratios, where α+β+γ=1. The green SAS of intermediate B in Fig. 5b is distinctive of the Co−C bond homolysis product, the cob(II)alamin radical (Fig. 6, green box)18. This is a non-productive channel, with a recombination rate k2=kRPR=1.26 × 109 s−1, after which all of the radical pairs recombine to the ground state. The brown SAS of C in Fig. 5b closely resembles the estimated spectrum of the MLCT state reported previously2123, where the Co(III) effectively receives dative coordination from an anionic ligand (Fig. 6, brown box). This intermediate has a lifetime of ≫3 ns and leads to a productive channel ending in the photoproduct. The branching ratios in our model are α=0.894, β=0.022 and γ=0.084. They suggest that, following a single-fs laser pulse, the majority of excited states decay to the ground state, with only 8.4% leading to the photoproduct and tetramer dissociation. A relatively low yield of productive channel would be consistent with the need for carotenoid biosynthesis only under high light intensity conditions, when the bacteria that express CarH are likely to experience significant photo-oxidative stress511.

Bottom Line: Here we describe the photochemistry in CarH that ultimately triggers tetramer dissociation; it proceeds via a cob(III)alamin intermediate, which then forms a stable adduct with the protein.This pathway is without precedent and our data suggest it is independent of the radical chemistry common to both coenzyme B12 enzymology and its known photochemistry.It provides a mechanistic foundation for the emerging field of B12 photobiology and will serve to inform the development of a new class of optogenetic tool for the control of gene expression.

View Article: PubMed Central - PubMed

Affiliation: 1] School of Chemistry, The University of Manchester, Oxford Road, Manchester M13 9PL, UK [2] Photon Science Institute, The University of Manchester, Alan Turing Building, Oxford Road, Manchester M13 9PL, UK [3] SYNBIOCHEM, Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.

ABSTRACT
The coenzyme B12-dependent photoreceptor protein, CarH, is a bacterial transcriptional regulator that controls the biosynthesis of carotenoids in response to light. On binding of coenzyme B12 the monomeric apoprotein forms tetramers in the dark, which bind operator DNA thus blocking transcription. Under illumination the CarH tetramer dissociates, weakening its affinity for DNA and allowing transcription. The mechanism by which this occurs is unknown. Here we describe the photochemistry in CarH that ultimately triggers tetramer dissociation; it proceeds via a cob(III)alamin intermediate, which then forms a stable adduct with the protein. This pathway is without precedent and our data suggest it is independent of the radical chemistry common to both coenzyme B12 enzymology and its known photochemistry. It provides a mechanistic foundation for the emerging field of B12 photobiology and will serve to inform the development of a new class of optogenetic tool for the control of gene expression.

No MeSH data available.


Related in: MedlinePlus