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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

Decay-associated difference spectra of the ultrafast photoresponse.Comparison of DADS that result from SVD and global analysis of ultrafast transient absorption data acquired between −1.5 ps and ∼3 ns following photoexcitation of CarH-GS (a–d) and free AdoCbl (e–h). The constant functions (a,e) describe the baseline. The remaining black lines show the dynamic components from fresh sample with lifetimes τ1–3 (b–d: 1.9 ps, 794 ps and ≫3 ns, respectively), for CarH and τ1–4 (f–h: 10 ps, 120 ps, 467 ps (dashed) and ≫3 ns, respectively) for AdoCbl. Blue lines are DADS from sample that has mostly converted to the monomeric light state (CarH-LS); they show one constant (a) and one dynamic component with lifetime τ=3.2 ps (b).
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f4: Decay-associated difference spectra of the ultrafast photoresponse.Comparison of DADS that result from SVD and global analysis of ultrafast transient absorption data acquired between −1.5 ps and ∼3 ns following photoexcitation of CarH-GS (a–d) and free AdoCbl (e–h). The constant functions (a,e) describe the baseline. The remaining black lines show the dynamic components from fresh sample with lifetimes τ1–3 (b–d: 1.9 ps, 794 ps and ≫3 ns, respectively), for CarH and τ1–4 (f–h: 10 ps, 120 ps, 467 ps (dashed) and ≫3 ns, respectively) for AdoCbl. Blue lines are DADS from sample that has mostly converted to the monomeric light state (CarH-LS); they show one constant (a) and one dynamic component with lifetime τ=3.2 ps (b).

Mentions: The effect of CarH on the ultrafast photoresponse of AdoCbl is well illustrated by comparing the decay-associated difference spectra (DADS) from the CarH data to those from an equivalent data set from free AdoCbl (black DADS in Fig. 4a–d,e–h, respectively). DADS were identified by globally fitting the raw data (for CarH, the data in Fig. 3a) to a sum of exponentials. The number of exponentials used was determined by using SVD-based rank analysis25. A good global fit to the data from CarH-GS required one constant component (Fig. 4a) and three dynamic components with lifetimes, τ1–3 (Fig. 4b–d). The resulting DADS represent the amplitudes of the exponential components and are used to generate species-associated spectra (SAS) for the various reaction intermediates using a model. The lifetime associated with each DADS is used to calculate the rate constant of each chemical conversion (for SAS and rates see the next section). The DADS with the shortest lifetime from the CarH data set (Fig. 4bτ1=1.9 ps) is quite distinct from that of free AdoCbl (Fig. 4f) and more closely resembles the transient difference spectra observed following photoexcitation of non-alkyl cobalamins such as CNCbl (ref. 16). The homolysis product, cob(II)alamin, is formed (Fig. 4c) but not via a base-off intermediate as observed for free AdoCbl (Fig. 4g). Moreover, these radicals are much shorter lived (τ2=794 ps) than those observed for free AdoCbl photolysis (Fig. 4h), with no evidence of any remaining beyond ∼1 ns. In fact, the DADS with a lifetime beyond the data acquisition window (Fig. 4d, τ3≫3 ns) closely resembles the difference spectrum that corresponds to the MLCT state known to precede homolysis during the photolysis of MeCbl (ref. 23) and AdoCbl bound to glutamate mutase21. By contrast, there is no evidence within the 3ns time window of Co−C bond homolysis following the formation of this MLCT state in AdoCbl bound to CarH.


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)

Decay-associated difference spectra of the ultrafast photoresponse.Comparison of DADS that result from SVD and global analysis of ultrafast transient absorption data acquired between −1.5 ps and ∼3 ns following photoexcitation of CarH-GS (a–d) and free AdoCbl (e–h). The constant functions (a,e) describe the baseline. The remaining black lines show the dynamic components from fresh sample with lifetimes τ1–3 (b–d: 1.9 ps, 794 ps and ≫3 ns, respectively), for CarH and τ1–4 (f–h: 10 ps, 120 ps, 467 ps (dashed) and ≫3 ns, respectively) for AdoCbl. Blue lines are DADS from sample that has mostly converted to the monomeric light state (CarH-LS); they show one constant (a) and one dynamic component with lifetime τ=3.2 ps (b).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Decay-associated difference spectra of the ultrafast photoresponse.Comparison of DADS that result from SVD and global analysis of ultrafast transient absorption data acquired between −1.5 ps and ∼3 ns following photoexcitation of CarH-GS (a–d) and free AdoCbl (e–h). The constant functions (a,e) describe the baseline. The remaining black lines show the dynamic components from fresh sample with lifetimes τ1–3 (b–d: 1.9 ps, 794 ps and ≫3 ns, respectively), for CarH and τ1–4 (f–h: 10 ps, 120 ps, 467 ps (dashed) and ≫3 ns, respectively) for AdoCbl. Blue lines are DADS from sample that has mostly converted to the monomeric light state (CarH-LS); they show one constant (a) and one dynamic component with lifetime τ=3.2 ps (b).
Mentions: The effect of CarH on the ultrafast photoresponse of AdoCbl is well illustrated by comparing the decay-associated difference spectra (DADS) from the CarH data to those from an equivalent data set from free AdoCbl (black DADS in Fig. 4a–d,e–h, respectively). DADS were identified by globally fitting the raw data (for CarH, the data in Fig. 3a) to a sum of exponentials. The number of exponentials used was determined by using SVD-based rank analysis25. A good global fit to the data from CarH-GS required one constant component (Fig. 4a) and three dynamic components with lifetimes, τ1–3 (Fig. 4b–d). The resulting DADS represent the amplitudes of the exponential components and are used to generate species-associated spectra (SAS) for the various reaction intermediates using a model. The lifetime associated with each DADS is used to calculate the rate constant of each chemical conversion (for SAS and rates see the next section). The DADS with the shortest lifetime from the CarH data set (Fig. 4bτ1=1.9 ps) is quite distinct from that of free AdoCbl (Fig. 4f) and more closely resembles the transient difference spectra observed following photoexcitation of non-alkyl cobalamins such as CNCbl (ref. 16). The homolysis product, cob(II)alamin, is formed (Fig. 4c) but not via a base-off intermediate as observed for free AdoCbl (Fig. 4g). Moreover, these radicals are much shorter lived (τ2=794 ps) than those observed for free AdoCbl photolysis (Fig. 4h), with no evidence of any remaining beyond ∼1 ns. In fact, the DADS with a lifetime beyond the data acquisition window (Fig. 4d, τ3≫3 ns) closely resembles the difference spectrum that corresponds to the MLCT state known to precede homolysis during the photolysis of MeCbl (ref. 23) and AdoCbl bound to glutamate mutase21. By contrast, there is no evidence within the 3ns time window of Co−C bond homolysis following the formation of this MLCT state in AdoCbl bound to CarH.

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