· Web view-carA gene cluster, which leads to carotenoid biosynthesis. In 2002 this effect of light was reported to require B 12, with the addition of CNCbl, AdoCbl or MeCbl all

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a. School of Chemistry, Photon Science Institute and Manchester Institute of Biotechnology, The University of Manchester, Manchester, UK. E-mail: [email protected]


Photochemical and Photobiological Sciences

PERSPECTIVE

Received 00th January 20xx,Accepted 00th January 20xx

DOI: 10.1039/x0xx00000x

www.rsc.org/

The photochemistry and photobiology of vitamin B12 Alex R. Jones

Biologically active derivatives of vitamin B12 are organomettalic cobalt-corrinoid complexes crucial for the healthy function of humans, animals and microbes. Their role as cofactors to numerous, thermally-driven metabolic enzymes is well described, and varies depending on the nature of the upper axial ligand. This ligand also significantly influences the photophysics and photochemistry of B12. In this Perspective I will discuss the various aspects of B12 photochemistry, from its dynamic spin chemistry to the considerable potential it has for biotechnology applications. Although for many years this photochemistry was thought to have no biological function, in recent years it has become apparent that B12

photochemistry at very least has a role in light-dependent bacterial transcriptional regulation. I will provide an overview of what has been reported about B12 photobiology to date, with particular emphasis on the mechanism of the transcriptional repressor, CarH, the subject of my Young Investigator Award Plenary Lecture at the European Society for Photobiology Congress 2015 in Aveiro, Portugal.

IntroductionAlthough the photosensitivity of vitamin B12 and its derivatives was first observed in the 1950s,1-4 it has taken over 60 years for the nature and potential extent of its role in photobiology to begin to surface.5-13 Initially the photochemistry of B12 was regarded as an irritation in a biomedical context. What we call vitamin B12 is strictly cyanocobalamin (CNCbl, Figure 1), which is administered to address B12 deficiency in the treatment of patients with haematological diseases such as pernicious anaemia or those suffering from certain types of neuropsychiatric disorder.14 Exposure of medicinal samples of CNCbl to light during storage was found to result in their contamination with the aerobic photoproduct, hydroxocobalamin (OHCbl, Figure 1).2

B12 is a member of the uroporphinoid family of cofactors, which comprise metal centres equatorially ligated by a highly conjugated tetrapyrrole macrocycle.15 Whether their natural function is light-driven (e.g., chlorophyll) or thermally-driven (e.g., haem) this structural template means that each is highly coloured. In the case of B12, a cobalt ion is coordinated by the nitrogens of a highly conjugated corrin ring, which is contracted compared to porphyrin and thus not aromatic (Figure 1). A 5,6-dimethylbenzimidazole substituent of the

corrin forms the lower axial ligand of most biologically active forms of B12, whereas the upper axial ligand is variable. Despite the fact that Dorothy Hodgkin’s famous crystal structure had an upper axial CN (i.e., CNCbl),16 this form is a stable but inactive derivative, which must be converted in vivo Figure 1. Chemical structure of select B12 derivatives with variable upper axial ligands (R). 5’-Deoxyadensoylcobalamin and

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ARTICLE Journal Name

methylcobalamin are biologically active, cyanocobalamin is vitamin B12

and hydroxocobalamin is the main aerobic photoproduct.

to either 5’-deoxyadenosylcobalamin (coenzyme B12, AdoCbl) or methylcobalamin (MeCbl).3, 4, 17-21 B12 is only fully biosynthesised by micro-organisms, where they regulate their

own production and transport by binding directly to riboswitches.22-24

As we shall see, the photophysics and photochemistry of B12 are reasonably well understood; but for many years the only known biological function of B12 was as cofactor to thermally-driven enzymes. The best described of these are

Figure 2a. Scheme depicting the general mechanism of MeCbl-dependent methyltransfereases. After Co−C bond heterolysis, a methyl cation is effectively transferred to the substrate (Sub1), with the catalytic cycle completed after a second methyl transfer to cob(I)alamin from Sub 2. b. Substrate binding to AdoCbl-dependent mutases and eliminases triggers Co−C bond homolysis, giving a cob(II)alamin / 5’-deoxyadenosyl radical pair. The alkyl radical then abstracts a hydrogen from the substrate, resulting in a radical rearrangement. c. Cob(II)alamin / alkyl (R) radical pairs are also generated after photoexcitation (h) of alkylcobalamins such as MeCbl and AdoCbl.

enzymes crucial to human, animal and microbial metabolism.25-

27 MeCbl-dependent methyltransferases catalyse the transfer of methyl groups; for example, in the conversion of homocysteine to methionine.28 Here heterolytical cleavage of the Co−C bond (Figure 2a) effectively transfers a methyl cation to the substrate via an SN2 mechanism. MeCbl is then regenerated by a subsequent methyl cation transfer to the highly nucleophilic cob(I)alamin by a second substrate (e.g., in homocysteine metabolism, N5-methyltetrahydrofolate).

By contrast, most AdoCbl-dependent enzymes act as a latent source of radicals to numerous mutase29 and eliminase30

enzymes. These enzymes have a common activation mechanism, whereby, upon substrate binding, the Co−C bond to the upper axial ligand breaks homolytically, producing a cob(II)alamin / 5’-deoxyadenosyl radical pair (Figure 2b). The 5’-deoxyadenosyl radical then abstracts a hydrogen from the substrate, resulting in a radical rearrangement. One of the major questions, not only in B12 enzymology, but enzyme catalysis in general, is how AdoCbl-dependent enzymes achieve the ~ 1012 enhancements in the rate of thermal Co−C bond homolysis.31-34 Less is known about the mechanism of B12-dependent reductive dehalogenases, which are involved in bacterial organohalide respiration.35 Recently published crystal structures suggest the formation of a halide-cobalamin complex, and the authors speculate on possible mechanisms involving either heterolytic or homolytic cleavage, this time of the Co−halide bond.36, 37

Co−C bond homolysis is also known to follow photoexcitation of alkylcobalamins (Figure 2c)38 such as MeCbl and AdoCbl. Although for many years this photochemistry was not thought to have any biological function, it has proven useful in elucidating details of thermally-driven biochemical mechanism.e.g.,39 In this Perspective, I will review what we know about the photophysics and photochemistry of various B12

derivatives and how this response to light is beginning to be exploited as a tool in biology. My intention is not to provide a comprehensive historical overview; I will instead focus on more recent mechanistic studies that have predominantly used transient absorption and other time-resolved spectroscopic methods. I will then go on to discuss in detail the recent, exciting discovery and developing mechanistic picture of how nature uses light and B12 to effect useful change.

B12 photochemistryThe structural and functional diversity of vitamin B12

derivatives began to emerge in the 1950s and 1960s,3, 4, 16-21 and initial descriptions of their photosensitivity followed closely behind.1-4, 38, 40-46 This early work identified a range of photoproducts (both cobalamin and alkyl) along with their quantum yields, which were gradually refined as experimental methods improved. It was the use of transient absorption techniques, however, which began to reveal the true extent of the photophysical and photochemical dynamics of B12. Although this began in the late 1970s,47, 48 the application of femtosecond pump-probe spectroscopy to this system since around the turn of the century, principally by Roseanne Sension and co-workers at the University of Michigan, Ann Arbor, has yielded the greatest detail.49-62 This work has provided clues in the electronic structure of AdoCbl and MeCbl to why each adopts a distinct mode of Co−C bond cleavage in their respective roles as enzyme cofactors (Figure 2a&b).

The photochemistry of coenzyme B12

The photochemistry of AdoCbl (coenzyme B12) generates, transiently, the same cob(II)alamin / 5’-deoxyadenosyl radical pair that is produced upon substrate binding to AdoCbl-dependent enzymes.38 As such, photolysis of AdoCbl has

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Journal Name ARTICLE

proven a useful tool with which to probe the influence of the cofactor environment on radical pair generation and their subsequent reaction dynamics. Under anaerobic conditions, there is now broad consensus that the quantum yield of solvent-separated radical pairs following photolysis of free AdoCbl is in the order of 0.20-0.24.50, 63, 64 What this figure

obscures, however, is that in aqueous solution the Co−C bond breaks with almost unity quantum yield within about 100 ps,50 which appears to be largely independent of the excitation wavelength.51 The quantum yield of solvent-separated radical pairs is then determined by a competition between

Figure 3. The photolysis of AdoCbl. The black scheme represents a proposed mechanism for the photophysical and photochemical dynamics on the ultrafast timescale (less than about 10 ns). There is a competition between recombination ( kR) of the cob(II)alamin / 5’-deoxyadenosyl radical pair and cage escape (kE) to form solvent-separated radical pairs. Under aerobic conditions (red scheme) the reaction proceeds via peroxy intermediates to form OHCbl and adenosine-5’-aldehyde photoproducts. Under anaerobic conditions (blue scheme) the cob(II)alamin accumulates and the predominant alky photoproduct is 5’-deoxy-5’,8-cycloadenosine. Refer to main text for a full discussion of each scheme.

geminate recombination and escape from the solvent cage.51, 52

A proposed scheme of the transient intermediates that have been fully resolved by UV-visible transient absorption for the photolysis of AdoCbl in water is in Figure 3 (black scheme). The initial AdoCbl excited state decays within tens of picoseconds to an intermediate where the bond to the lower axial base is weakened or broken. This intermediate then dissociates into the close radical pair. In aqueous solution, 75-80 % of these close pairs recombine at kR = 1.43 0.02 ns–1, with the remaining 20-25 % escaping the solvent cage at kE = 0.57 0.06 ns–1.50, 52 Although kR appears to be largely independent of solvent viscosity, kE in ethylene glycol is substantially reduced to 0.11 0.03 ns–1. This solvent cage effect therefore favours close pair recombination and thus reduces the quantum yield of solvent-separated radical pairs to around 0.08.52

Coordination of the lower axial base also has a profound effect on the photolysis quantum yields.60, 63, 64 The yield of close radical pairs drops from near unity to 0.12 after photolysis at low pH, where the lower axial nitrogen is protonated and the cobalt ion is instead coordinated by a water molecule. The absence of the lower axial ligand alters the electronic structure and opens up a channel to fast non-radiative decay, which proves strong competition to bond cleavage.60 The kinetics of geminate recombination are also significantly impacted in base-off AdoCbl. Although there is no evidence of recombination on the sub-10 ns timescale, the quantum yield of solvent separated radical pairs appears to drop to 0.045,63, 64 suggesting significant recombination does occur, albeit on a slower timescale. It has been speculated that

this might be owing to triplet-born radical pairs following photolysis of base-off AdoCbl – as opposed to singlet-born for base-on – the spin state of which would have to evolve before recombination to the singlet ground state is possible.60

A similar radical pair cage effect to that observed in ethylene glycol is evident after photolysis of AdoCbl bound to its dependent enzymes.54, 56, 65 In glutamate mutase the quantum yield of solvent-separated radicals drops to 0.05, with kR = 1.0 0.02 ns–1 and kE = 0.05 0.06 ns–1.56 Similar UV-visible transient absorption experiments revealed that kE

following AdoCbl photolysis in ethanolamine ammonia lyase (EAL) is dependent on the viscosity of the solvent surrounding the protein.65 Time resolved IR detection suggested that this was most likely owing to vibrational coupling between the AdoCbl and the local protein environment.66, 67 Equivalent vibrational signals from EAL residues were shown by stopped-flow FTIR to evolve with kinetics that match those of Co−C bond homolysis following substrate-binding to EAL,67 which indicates that such vibrational coupling is relevant to catalysis. A highly-conserved glutamate residue that binds the substrate in EAL (E287), and which is essential for catalytic efficiency,68, 69 has been proposed as the origin of this vibrational coupling. It forms the basis of a proposed ‘substrate trigger’ mechanism for Co−C bond activation in AdoCbl-dependent enzymes.65

The cofactor environment also alters the nature of the intermediates that precede bond cleavage.58 Photoexcitation of cob(III)alamins are thought to predominantly correspond to initial ππ* transitions involving the corrin ring.70, 71 The S1 state of AdoCbl is similar to that of nonalkylcobalamins (see below), and is best described as a corrin π Co 3d ligand-to-metal

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charge transfer (LMCT; π3dz2) state. Whereas prior to

homolysis in water this state first decays into an intermediate with a weakened or dissociated lower axial ligand (Figure 3),50 in ethylene glycol the S1 state proceeds directly to radicals.52 When bound to glutamate mutase (which AdoCbl does via a lower axial histidine residue) on the other hand, the S 1 state of AdoCbl more closely resembles a longer-lived corrin π Co 3d adenosyl metal-to-ligand charge transfer (MLCT) state similar to that observed during MeCbl photolysis (see below).49,

53 Here, the electrostatic environment of the protein is thought to stabilise a shift of electron density onto the upper axial adenosyl through mixing of the Co 3d orbitals with the 2p and 2s of the adenosyl C.58 Time-dependent density functional theory (TD-DFT) calculations suggest two minima on the S 1

surface of AdoCbl that might go some way to explain the experimentally observed effects of environment on its nature. One corresponds to elongated bonds to the axial ligands, which are more consistent with the pre-homolysis intermediates observed in water, whereas the other is a MLCT minimum, which could be stabilised by a protein environment.72

When focusing on the reaction dynamics of the geminate radical pair following photolysis of AdoCbl, most of the mechanistic work described above was conducted under anaerobic conditions. After radical pair separation, the main photoproducts of anaerobic photolysis are the cob(II)alamin radical and 5’-deoxy-5’,8-cycloadenosine (Figure 3, blue scheme).38, 41, 46 By contrast, aerobic photolysis produces mostly aquocobalamin (OH2Cbl) or OHCbl (depending on pH) and adenosine-5’-aldehyde (Figure 3).40, 41, 45 Reaction of the transient cob(II)alamin / 5’-deoxyadenosyl radical pair with molecular oxygen generates 5’-peroxyadenosylcobalamin, which first hydrolyses to 5’-peroxyadenosine (and OHCbl) and then the peroxyalkyl irreversibly decomposes to adenosine-5’-aldehyde.73

The photochemistry of methylcobalamin and other alkylcobalamins

MeCbl-dependent enzymes do not proceed via radical intermediates; the Co−C bond breaks heterolytically resulting in an oxidation state change from Co(III) to Co(I) (Figure 2b).28,

74 Despite this, photolysis of MeCbl generates a cob(II)alamin / methyl radical pair analogous to that encountered after AdoCbl photolysis and during catalysis in AdoCbl-enzymes (Figure 2a). There are fundamental differences, however, in the photophysics that precede Co−C bond cleavage in MeCbl and the radical pair reaction dynamics that follow it.

Unlike the photolysis of free AdoCbl, the mechanism of MeCbl photolysis is dependent on the excitation wavelength.45,

51 The quantum yield for solvent-separated radicals after excitation with green light is roughly half (~0.15)45, 51 of that after excitation with blue light (~0.3).45, 51, 63 Much like when AdoCbl is bound to its dependent enzymes,54, 56 transient absorption data are consistent with the S1 state of MeCbl being a MLCT state with electron density on the upper axial methyl ligand.49, 51, 58 This assignment has since been supported by TD-

DFT calculations.75 If this state is populated more directly using excitation wavelengths in the green (~ 520 nm), the majority decay back to the ground state via internal conversion with only ~ 15 % producing radical pairs at a rate of 1.10 0.15 ns–

1 (Figure 4, green scheme).51 On the other hand, if MeCbl is excited to a higher state than S1 using UV or blue light (~ 400 nm) then there is a branching mechanism before Co−C homolysis (Figure 4, blue scheme). Around 75 % populate the MLCT S1 state in under a picosecond, which then decay into radicals with the same rate and yield as more direct population of this state. The remaining 25 % undergo what has been termed ‘prompt homolysis’, also on the sub-picosecond timescale.

Figure 4. The photophysics of methylcobalalmin is wavelength-dependent. 100 % of molecules excited with green light (green scheme) populate the S1, MLCT, state (with electron density on the methyl) before decaying back to the ground state or dissociating into radicals. Photoexcitation in the UV or blue results in a branching mechanism (see text for full description). IC = internal conversion; E = cage escape; R = geminate recombination.

This branching mechanism explains the increased quantum yield of radicals after excitation with UV/blue light when compared to green. It should be noted that, whereas the quantum yield of solvent separated radicals following the photolysis of base-on AdoCbl is determined by competition between geminate recombination and cage escape, evidence from transient absorption data suggest geminate recombination following MeCbl photolysis in water is limited.49,





51, 53, 55, 60 Instead, much like for base-off AdoCbl the quantum yield of solvent-separated radicals following MeCbl photolysis is predominantly influenced by competition between internal conversion – from a higher lying state to S1 and S1 to the ground state – and bond homolysis. The rate constant for internal conversion from the S1 state is further increased by ~ 10-fold in base-off MeCbl.60

The generation of radical pairs makes the photochemistry of AdoCbl and MeCbl ostensibly equivalent. As we have seen, however, on closer inspection both the photophysics and radical pair reaction dynamics reveal substantial variations. It appears, therefore, that the upper axial position of the cobalamins has quite a significant impact on both electronic structure and reactivity. The nature of the upper ligand has been probed further by investigation of the photolysis of a range of other alkylcoballamins.53, 55, 58, 59 Methyl-, ethyl- and n-propylcobalamin all reveal comparable branching mechanisms after excitation at shorter wavelengths and all form similar MLCT S1 states.53, 58 The stability of the partial alkyl anion in this state decreases with increasing ligand size. This drop in stability reduces the barrier to, and hence increases the rate of, Co−C bond homolysis.53 In fact, like AdoCbl, internal conversion from S1 is only significant for base-off ethyl- and n-propylcobalamin.60 Unsurprisingly, the rate of cage escape correlates with the size of the alkyl radical (methyl > ethyl > n-propyl > n-hexylnitrile).53 Radical diffusion is dependent on viscosity,55 and other properties of the solvent such as mass and volume.59

MeCbl appears to be a special case, however, which cannot be adequately explained using hydrodynamic analyses of diffusion, no matter how sophisticated they may be. As I have already discussed, when excited by 400 nm a population of MeCbl access a directly dissociative state instead of converting to S1.51 Here, excess energy from the exciting photon imparts additional translational energy to the newly formed methyl radical,55 adding to its rate of cage escape and contributing to the lack of geminate recombination. It has been proposed the geometry of the alky radical might also play a role.76 In the case of the 5’-deoxyadenosyl radical, the unpaired spin density is predominantly located on the pyramidal 5’-carbon, which ‘points’ the unpaired electron towards the cob(II)alalmin, thus favouring recombination. The methyl radical, on the other hand, is planar. The unpaired spin therefore has access to the face of the radical that points away from the cob(II)alamin, which, in combination with rapid diffusion owing to the small size of the methyl radical and its excess energy, is proposed to make significant recombination highly unlikely.76 That said, careful analysis of the kinetics of geminate recombination and cage escape following the photolysis of MeCbl via the S1 state suggests that the quantum yield of solvent-separated radicals (at 9 ns) need not account for alkyl radical geometry.53

Spin chemistry and magnetic field effects

Another feature that influences the extent of geminate recombination following the photolysis of cobalamins is the dynamic spin chemistry of the radical pair.77 Physical laws regarding the conservation of spin dictate that the initial spin

state of any radical pair formed from non-radical precursors is 100 % polarised according to the multiplicity of those precursors. In the context of a photochemical, homolytic bond cleavage, therefore, a singlet (S) excited state precursor will produce a S-born radical pair and a triplet (T) excited state a T-born pair. Because the cob(II)alamin and alkyl radicals have different hyperfine couplings, if the radicals separate sufficiently to overcome the exchange interaction between the unpaired spins, the spin state will evolve with time. The effect is that the S and T radical pair spin-states coherently interconvert (Figure 5a) before they incoherently relax to an equilibrium state. Owing to Pauli exclusion, recombination of a cob(II)alamin / alkyl radical pair will only happen from the S spin-state. The reactivity of radical pairs is therefore inherently spin-selective. Because it is magnetic in origin, the extent of coherent spin-state interconversion between the S and T radical pairs, and thus the probability of recombination, can be perturbed by the application of external magnetic fields.77-79

Figure 5a. Spin-state interconversion of a S-born cob(II)alamin / alkyl radical pair. Only the S spin states can recombine, and therefore the reactivity is spin-selective. h indicates absorption of a photon. The excited state dynamics have been ignored and diffusion has been assumed to produce solvent-separated radical pairs. b. The Zeeman effect on radical pair spin-states in an increasing magnetic field removes the T±1 spin-states in energy such that their involvement in interconversion becomes increasingly inefficient.

In 1993, magnetic field effects (MFEs) were reported on the photolysis of AdoCbl.80 In viscous solution, the observed rate of radical pair recombination increased when exposed to magnetic fields on the order of tens to hundreds of mT,





consistent with a S-born radical pair. At these field magnitudes, the T±1 spin-states – which each have bulk magnetisation along the applied field direction – are separated in energy by the Zeeman interaction such that their involvement in interconversion becomes increasingly inefficient (Figure 5b). Thus, for a S-born radical pair, the relative S population increases in an applied field and so does the probability of recombination. For a T-born pair, the T±1 spin-states are trapped in the presence of a field – separated in energy and therefore unable to covert to the S spin-state – and thus the recombination probability decreases.

The following year, the same group reported analogous MFEs on the steady-state turnover parameters Vmax / Km for the AdoCbl-dependent enzyme, ethanolamine ammonia lyase (EAL).81 This represented the first observed MFE on a wildtype protein system with its natural substrate, and they apparently identified the origin of the MFE, using stopped-flow, as geminate recombination of a S-born radical pair following activation of AdoCbl on substrate binding (Figure 2b).82 Although we are able to reproduce the MFEs on AdoCbl photolysis,83 we were unable to do so for the EAL-catalysed reaction.34 We discovered that the stopped-flow method used in [80] was insensitive to the pre-steady-state kinetics of geminate recombination, and that the difference in magnetic-sensitivity can be explained by limited geminate recombination in the enzyme-catalysed reaction. Despite this negative result, MFEs have proven a useful tool is elucidating B12-dependent enzyme mechanism, providing evidence of how both radical quenching34, 83, 84 and protein dynamics39, 65 contribute to catalytic power. I have reviewed this in detail elsewhere.39, 79, 84

The MFEs on AdoCbl are consistent with S radical pair precursors,80, 83 which is further supported by the polarisation pattern observed in time-resolved EPR data.85 Is this also true for the photolysis of MeCbl and other alkylcobalamins, and is the excited state multiplicity influenced by the different photophysics observed at different excitation wavelengths?51, 53 Relative to AdoCbl, the diminutive upper axial ligand of MeCbl means its low-lying excited states are more amenable to investigation by quantum chemical calculations like TD-DFT.72 Irrespective of the size of the upper axial ligand, however, theoretical investigation of cobalamins is computationally very expensive, and has therefore invariably been carried out on structural models where the corrin side chains are replaced by H-atoms and the lower axial base by imidazole. Initial TD-DFT calculations seemed to suggest that bond photolysis in MeCbl75

and ethylcoabalamin86 is mediated by a repulsive T state. Conservation of spin means that such a state would lead to T-born radical pairs, which is apparently inconsistent with MFEs on MeCbl photolysis that suggest, like AdoCbl, the magnetically-sensitive radical pairs are S-born.80, 83 Further TD-DFT studies appeared to reconcile with these experimental observations.87 A potential energy surface was constructed for the S1 state of base-on MeCbl that provides viable photolytic pathways, with intersystem crossing to the T excited state said to have a low probability.

Although one might expect data from magnetic resonance studies to provide clarity, those conducted to date serve only

to muddy the waters. Chemically induced dynamic electron polarisation (CIDEP) signals from EPR experiments are consistent with the photolysis of both the MeCbl (at 77 K) 88

and its analogue, methylaquocobaloxime (at room temperature),89 proceeding via a excited T state. Chemically induced dynamic nuclear polarisation (CIDNP) signals from 1H NMR spectra, on the other hand, indicate that photolysis of B 12

model compounds proceed via a S state.90 The authors of [89] speculate that the CIDEP signals might be dominated by triplet mechanism polarisation, whereas the CIDNP and MFE data by a reaction channel that proceeds predominantly via S-born radical pairs. Such branching would be consistent with the wavelength-dependence now known to occur from transient absorption (Figure 4).51 More recent, room temperature TR-EPR data from the photolysis of MeCbl also show a slight wavelength-dependence on the polarisation pattern.85

Different mechanisms are discussed that could feasibly explain the data: i) T radical pair precursors with CIDEP features from ST0 mixing; ii) S radical pair precursors with CIDEP features from ST–1

mixing. Because of closer agreement with the CIDNP and MFE data, the latter (i.e., S-born pairs) was favoured for both wavelengths. The slight variation in polarisation was instead attributed to differences in hyperfine field resulting from each branch in Figure 4 (blue scheme) proceeding via a different coordination structure at the point of homolysis. They speculate that the precursor to ‘prompt’ homolysis is base-on MeCbl, whereas the S1, MLCT state is base-off. Interestingly, TD-DFT calculations of the low-lying excited states of base-off MeCbl again allude to the possibility of intersystem crossing91

facilitated by spin-orbit coupling92 to give T-born radical pairs.From these data, I tentatively draw two conclusions that

borrow heavily from the analyses and discussions in the papers summarised above. First, I think it is clear that the axial ligation profoundly affects the electronic structure of cobalamins, and one should therefore be cautious when drawing parallels between results from cobalamins and their analogues. This is obviously an important conclusion, however, in the context of why AdoCbl and MeCbl enzymes employ such different mechanisms (Figures 2a and b). Second, it seems increasingly likely that MeCbl, and perhaps other alkylcobalamins, produce both S- and T-born radical pairs. A definitive effort is now needed to finalise the underlying mechanism.

The photophysics and photochemistry of non-alkylcobalamins

Decreasing either the size of the upper axial ligand or the solvent pH (to dissociate the lower axial base) reduces the photolysis yield by increasing the relative rate of internal conversion to the ground state. This effect is enhanced still further in non-alkylcobalamins, the photoresponse of which are revealed by transient absorption data to be relatively simple.57, 58, 61, 62, 66 In most cases, photoexcitation causes a ππ* transition, which relaxes on the sub-ps timescale to a LMCT S1

state similar to that observed for AdoCbl in water.57

Assignment of this π3dz2 state is supported by TD-DFT

calculations for CNCbl93, 94 and is consistent with an elongation of the axial bonds.57, 58 Its lifetime is typically < 10 ps, which decreases with the extent of -donation from the upper axial





ligand (CNCbl > N3Cbl > H2OCbl) and increasing solvent polarity.57 The S1 lifetime of H2OCbl (~ 3 ps)57 is therefore roughly half that of OHCbl (~ 5.5 ps), the pKa of which is about 8.62 Consequently, where the photoresponse has been investigated around neutral pH,66 it is likely that the kinetics reflect a mixture of those from H2OCbl and OHCbl. At most excitation wavelengths the S1 state of typical non-alkylcobalamins decays to the ground state via internal conversion without significant cleavage of the upper axial bond. The rationale from TD-DFT is that elongation of the CoCN bond in the S1 state doesn’t result in it breaking, but in level crossing with the S0 state leading to internal conversion.94

Most non-alkylcobalamins investigated are therefore highly photostable. Although to some degree the same is true of OHCbl, for excitation wavelengths of less than 350 nm there is evidence for a modest yield of radicals.62, 95 After photoexcitation at 269 nm, transient absorption data indicate a long-lived spectral component consistent with cob(II)alamin and a photolysis yield of ~1.5 %. TD-DFT calculations suggest more complex excited state dynamics for OHCbl62 than for CNCbl93, 94 that would enable bond dissociation following photoexcitation at shorter wavelengths. The potential energy surface for the S1 state of OHCbl has three minima, each with varying extents of pOH/d π* and pOH/d * character. The two with a significant proportion of pOH/d π* tend to decay to the ground state via internal conversion mechanisms similar to those proposed for CNCbl and MeCbl, respectively. The state where pOH/d * dominates, however, is only accessible from higher excited states and has longer CoOH bond lengths, some of which can result in bond cleavage.

Example applications of B12 photochemistry

The fact that the photolysis of various cobalamins releases the upper axial ligand has led to an increasing interest in using B 12

photochemistry as a biological tool. As we have already encountered, the cob(II)alalmin radical will readily react with molecular oxygen to ultimately form OHCbl (Figure 3).73 The aerobic photolysis of OHCbl using short wavelengths described above has therefore been exploited as a photocatalytic method for the in situ generation of hydroxyl radicals (Figure 6a).95 This method can be applied to the study of DNA structure and binding and offers greater spatial and temporal control than the commonly used chemical reduction of H2O2

(e.g., by the Fenton reaction).It is the various alkylcobalamins, however, that have the

greatest potential for the in situ release of molecular cargo. They are significantly more amenable to photolytic bond cleavage – with a substantially higher quantum yield of radicals than OHCbl, at a broader range of excitation wavelengths – and there is more scope for chemical modification.96 Although cobalamins can be directly excited to trigger photolysis using wavelengths shorter than ~ 600 nm, there are considerable benefits of using longer wavelengths in terms of penetration into biological samples and the mitigation of collateral photodamage. Cobalamins have therefore been functionalised with commercial fluorophores either at the upper axial position or the 5’-OH group of the lower axial 5,6-

dimethylbenzimidazole.97 These fluorophores act as antennae for red and near IR light, and have emission spectra that overlap with the absorption spectra of cob(III)alalmins. Following photoexcitation, energy transfer between the fluorophore and corrin chromophore can lead to CoC cleavage, with the potential for the targeted release of drugs or an alternative to optogenetic control over cellular behaviour.

In recent years, B12-derivatives have been synthesised that act as so-called ‘anti-vitamins’.98-100 Unlike CNCbl (used in vitamin supplements to treat B12-deficiency), derivatives such as Co-4-ethylphenylcob(III)alamin (EtPhCbl) are resistant to metabolic conversion to the biological active cofactors, AdoCbl and MeCbl. The administration of B12 anti-vitamins can therefore serve as a non-surgical means by which to induce B12-deficiency in laboratory animals for the study how such a deficiency causes related pathologies.98, 99 The fact that EtPhCbl is light-sensitive introduces the idea of a ‘conditional anti-vitamin B12’, which can be selectively converted to an active form of B12 by the photochemical generation of cob(II)alamin (Figure 6b), the in vivo precursor to AdoCbl and MeCbl.100 Figure 6. Applications of cob(III)alamins photolysis. a. The photocatalytic generation of hydroxyl radicals by the aerobic photolysis

of OHCbl. The hydroxyl radicals interact with, e.g., DNA for structure and binding studies. b. The in vivo photoactivation of the inactive B12

anti-vitamin, EtPhCbl, via cob(II)alamin to the active MeCbl, which can then bind to a dependent enzyme.

B12 photobiologyThe potential for biotechnology applications using B12

photochemistry is significant. There is an increasing list of possible chemical modifications to B12,96, 101 and thus broad scope for functionalisation, caging, and other attachment, e.g., to surfaces or nanoparticles. With such potential versatility, it would be surprising if Nature hadn’t found a way to exploit the photochemistry of B12. This is particularly true for AdoCbl,





which can, in principle, release the bulky and reactive 5’-deoxyadenosyl radical with unity quantum yield after absorbing a photon.50 And indeed, since the turn of the century a photobiological role has begun to emerge for B12, in the photoreception of both photosynthetic and non-photosynthetic bacteria.

Bacterial carotenoid biosynthesis

Certain bacteria respond to high levels of short-wavelength ambient light by producing carotenoids,102-104 potent antioxidants that help protect against photo-oxidative stress. For example, in the non-photosynthetic, Gram-negative bacterium, Myxococcus xanthus, the light-inducible promoter, PB, drives the carB-carA gene cluster, which leads to carotenoid biosynthesis. In 2002 this effect of light was reported to require B12, with the addition of CNCbl, AdoCbl or MeCbl all sufficient for function in vivo.5 The binding of the transcriptional repressor, CarH, to the PB promoter was later found to be B12-dependent,6 and in vitro studies of CarH from Thermus thermophilus revealed that it is specifically AdoCbl that regulates its activity.7 In the dark, AdoCbl binds to CarH, inducing formation of a homotetramer, which in turn binds to the PB promoter (Figure 7a), thus blocking transcription of the genes responsible for carotenogenesis. Under light of wavelengths that are absorbed by AdoCbl (but not red light), CarH returns to its monomeric state, thereby releasing DNA and thus acting as a genetic ‘on’ switch.7

The observations in [7] gave the strongest indication yet that Nature had indeed found a way to exploit the photochemistry of AdoCbl to effect useful change. AdoCbl binds to the CarH monomer with the lower axial 5,6-dimethylbenzimidazole base dissociated and the cobalt instead coordinated by a histidine residue (in Thermus thermophilus CarH, H177) from a C-terminal Rossmann fold domain.7, 11 It has this mode of binding in common with several B12-dependent enzymes, such as MeCbl-dependent methionine synthase105 and AdoCbl-dependent methylmalonyl CoA mutase.106 The chromophore is sandwiched between this cobalamin-binding domain and a four-helix bundle, which interacts with the 5’-deoxyadnosyl. This overall sensory domain is kept in an extended configuration that allows for tetramer formation by a steric interaction between the 5’-deoxyadnosyl and a tryptophan from the helical bundle (W131). A separate, N-terminal domain is dedicated to binding of DNA. Strikingly, in the CarH tetramer from Thermus thermophilus only three out of four of these domains appear to interact with DNA.11

The photolysis of AdoCbl bound to CarH results in a B 12

photoproduct with a UV-visible spectrum typical of a cob(III)alamin, with a more structured band than AdoCbl and a prominent -band similar to that for CNCbl and OHCbl (Figure 7b). Although it was initially speculated that the photoproduct might be OHCbl or similar,7 one would not expect OHCbl to bind particularly tightly and attempts to remove the cobalamin species bound to the photoconverted CarH monomer were in vain.10 Native mass spectrometry was used to confirm the formation of a highly stable adduct

between the protein and cobalamin photoproduct.10 This was identified by X-ray crystallography as a covalent bond between the metal and a histidine residue at the upper axial position.11

The photochemical displacement of the 5’-deoxyadnosyl produces a cavity that the W131 moves into (Figure 7c), which results in a substantial change in relative orientation between the cobalamin-binding domain and the helical bundle.10, 11 This structural change undermines the interactions that maintain the CarH tetramer, which therefore dissociates, resulting in release of DNA. It also places H132 from the helical bundle directly above the cobalt, with which it forms a covalent adduct (Figure 7c).

Figure 7a. Binding of AdoCbl (red circle) to the CarH monomer (sensory domain in green; DNA binding domain in brown) in the dark, followed by the formation of the CarH tetramer and DNA-binding. Photolysis of AdoCbl (h) causes the CarH tetramer to dissociate, releasing DNA and allowing transcription. Cobalamin (red crescent) remains bound to CarH. b. UV-visible spectra of the AdoCbl bound to the CarH tetramer in the dark (black) and the CarH monomer following photolysis (red). c. Following photoexcitation of AdoCbl, the 5’-deoxyadenolsyl is displaced, forming 4′,5′- anhydroadenosine and allowing a tryptophan (W131) to move into the cavity. This results in a structural change that leads to tetramer dissociation and an adduct between cob(III)alamin and a histidine residue (H132).

When one considers the ubiquity of radical chemistry in the bio- and photo-chemistry of AdoCbl, one would be forgiven for assuming radical pair intermediates here as well. The alkyl photoproduct following the photolysis of CarH-bound AdoCbl





has been identified as 4′,5′- anhydroadenosine.13 Two plausible mechanisms were offered: i) generation of the 5’-deoxyadenolsyl radical followed by -hydrogen





Figure 8. Proposed mechanism of AdoCbl photolysis and adduct formation in CarH. AdoCbl is bound to CarH from Thermus thermophilus via a lower axial His177. Following photoexcitation (h) there is evidence of a branching mechanism. There are two deactivation channels: i) internal conversion to the ground state; ii) a small yield of radical pairs, which all recombine. The third, productive channel proceeds via a Type II S1 state, with electron density transferred to the upper axial ligand (MLCT), which could trigger -hydride elimination to form the 4′,5′- anhydroadenosine alkyl photoproduct. Displacement results in a structural change (Figure 7c), placing His132 above the cob(III)alamin to enable adduct formation.

elimination; ii) generation of the 5’-deoxyadenosyl anion followed by -hydride elimination. Owing to both the overwhelming precedent for radical intermediates and the fact that the 5’-deoxyadenosyl radical appears to accumulate under anaerobic conditions, the authors of [13] not unreasonably favoured the former mechanism. It was argued that rapid -hydrogen elimination would protect against the potentially deleterious effects of releasing a primary carbon radical so close to DNA. Data from transient absorption investigations, however, suggest that the productive photochemical pathway in CarH avoids radical intermediates altogether.10

Time-resolved data in [10] were acquired over almost 15 orders of magnitude, and reveal a biochemical mechanism without precedent for a B12 molecule (Figure 8). Almost 90 % of photoexcited AdoCbl decay to the ground state via internal conversion within a few ps. Such a high level of deactivation could reflect that significant levels of CarH activation are only required under conditions of photooxidative stress and thus high levels of light. Signal consistent with the cob(II)alamin radical is evident, but with a very low yield (~ 2 %) and all radical pairs appear to recombine within 1 ns. This is therefore considered a non-productive channel, and is perhaps a legacy from the photochemistry of free AdoCbl. Another population of excited states goes on to produce a signal within ~ 20 ps

that closely resembles the MLCT S1 state observed previously following the photoexcitation of MeCbl49 and AdoCbl bound to glutamate mutatase.56 It has been argued that a protein environment might stabilise the transfer of electron density to the upper axial ligand through mixing of the Co 3d orbitals with the 2p and 2s of the adenosyl C.58 This mixing changes the nature of the S1 state from one with a weakened bond to the axial ligand observed for free AdoCbl (Type I) to one with a -donating alkyl anion ligand (Type II). The MLCT states in MeCbl and glutamate mutase-bound AdoCbl do go on to form radicals within hundreds of picoseconds. What is striking about CarH, is that it seems to stabilise this Type II S1 state to such an extent that its lifetime is ~ 10 ns and radicals are not subsequently produced. Indeed, the species spectra that follow are all consistent with cob(III)alamin species and not cob(II)alamin (or, indeed, cob(I)alamin). In the absence of any evidence to the contrary, it was proposed in [10] that the MLCT state resulted in CoC heterolysis. There is some contention about this assignment, however, with the stability of the 5’-deoxyadenolsyl anion that would result being questioned.107 The fact remains that there is no evidence for the cob(II)alamin radical following the MLCT state.10 It is possible that concerted -hydride elimination might generate the 4′,5′- anhydroadenosine photoproduct (Figure 8), or that the 5’-deoxyadenolsyl anion is only very short-





lived much like the 5’-deoxyadenosyl radical in AdoCbl-enzymes (which has never been directly observed during turnover).108 More investigation is necessary to resolve this point.

With the 5’-deoxyadenolsyl displaced, W131 can move into the cavity produced (Figure 7c), resulting in the structural change discussed earlier, which triggers tetramer dissociation and places H132 above the cob(III)alamin.11 These changes occur on the ms timescale, with the H132 / cobalamin adduct formed with a rate of ~ 0.1 103 s–1.10 Transient absorption spectral changes continue up to around 55 s that are consistent with the chromophore in a changing environment. Two further kinetic phases are evident, which might provide a clue about the kinetics and mechanism of tetramer dissociation. The crystal structure of CarH reveals the quaternary structure to be a dimer of head-to-tail dimers.11 The two kinetic phases observed in [10] could therefore conceivably represent the conversion of the tetramer into dimers, which then dissociate into monomers.

This unusual photochemical mechanism for AdoCbl in CarH is a good example of how proteins can fine-tune the chemistry of small molecules. But questions remain, not least about precisely how CarH stabilises the Type II, MLCT S1 state to the extent that the productive reaction channel avoids radical pair intermediates completely. Might molecular oxygen play a role? After all, illumination of CarH under anaerobic conditions appears to result in accumulation of the cob(II)alamin radical.13 Moreover, what effect does the excitation wavelength have? Although the photolysis of free AdoCbl is wavelength-independent, the photophysics of AdoCbl bound to CarH more closely resembles that of MeCbl, which is wavelength-dependent.51 Moreover, are four photons required to dissociate each CarH tetramer (i.e., one per bound AdoCbl), or will fewer suffice? Finally, why does such a stable covalent adduct form between cob(III)alamin and a upper axial histidine? One might speculate that it is a way of recycling the biosynthetically expensive cobalamin, but there is nothing yet reported to rule out a more mechanistic role, for instance, in regulating tetramer dissociation.

Photosystem gene expression

There is evidence to suggest that B12 photochemistry is also involved in the regulation of carotenoid biosynthesis for their perhaps more familiar role as chromophore in photosynthetic light-harvesting complexes.9 In the mid-nineteen nineties, exogenous addition of B12 was observed to rescue the synthesis of wildtype levels of photosynthetic complexes in mutant strains of the photosynthetic bacterium Rhodobacter capsulatus.109, 110 Around the same time an aerobic transcriptional repressor, CtrJ, was identified that regulates the synthesis of carotenoids, bacteriochlorophyll and photosystem proteins.111 Immediately upstream of the genes that encode CrtJ are those that encode AerR,112 an aerobic activator113 homologous to PpaA from Rhodobacter sphaeroides

previously identified as having a cobalamin-binding domain.114 AerR with B12 bound inhibits binding of CrtJ to DNA, thus acting as an antirepressor for the genes that control photosystem formation.9 The idea that B12 might control the biosynthesis of bacteriochlorophyll presents an interesting evolutionary twist, when one considers that these two tetrapyrroles share common early intermediates.115

In contrast to CarH, AerR appears to have little or no affinity for AdoCbl.9 It does show tight binding for OHCbl, however, also via a lower axial histidine (H145). This is true whether OHCbl is added directly or generated by the aerobic photolysis of AdoCbl. This latter observation led the authors of [9] to suggest a surprising mechanism, whereby B12 photochemistry occurs before binding to AerR. They propose that the aerobic photolysis of either AdoCbl or MeCbl generates OHCbl, which subsequently binds to AerR, with the upper axial OH displaced thermally by H10 to produce a bis-His adduct of similar stability to that found in photo-converted CarH.10 The AeR/cobalamin complex then binds to CrtJ with an affinity of ~ 3 µM, which was shown to inhibit the binding of CrtJ to DNA in vitro. Moreover, addition of 10 µM B12 under photosynthetic conditions rescues the expression levels of the genes responsible for bacteriochlorophyll biosynthesis in vivo when compared to B12 limiting conditions.9

Summary, conclusions and future prospectsMany of the investigations into B12 photochemistry to date have been motivated by what they might tell us about B12

enzyme mechanism. These studies have provided invaluable information about the electronic structure of various cobalamins and how that structure is influenced by the nature of the axial ligands and their environment. We have seen that a combination of the large upper axial 5’-deoxyadenosyl and coordination of a lower axial base (be that from the 5,6-dimethylbenzimidazole or a histidine residue) seems to favour high yields of radical pairs. These radical pairs are preceded by a Type I S1 state, defined by weakened bonds to the axial ligands. The fact that AdoCbl appears to be optimised towards high radical yields is consistent with the widespread radical mechanism of AdoCbl-dependent enzymes (Figure 2b), and suggests that similar cobalamin derivatives have strong potential for the targeted release of molecular cargo in vivo using light. We have also seen that smaller upper axial alkyl and nonalkyl ligands, or conditions that result in dissociation of the lower axial base, open up rapid internal conversion channels that to varying extents compete with homolysis. Moreover, as the size of the upper axial alkyl ligand gets smaller an alternative MLCT (Type II) S1 state becomes increasingly stable. Interestingly, similar transfer of electron density on to the 5’-deoxyadenosyl is also stabilised to some extent by binding AdoCbl to its dependent enzymes. It is perhaps unsurprising, therefore, that the combination of an upper axial methyl and a tailored protein environment can serve





to alter the electronic structure of cobalamin sufficiently to avoid radical chemistry altogether (Figure 2a).

The relatively detailed picture we now have of B12

photochemistry provides an excellent foundation for the burgeoning field of B12 photobiology. Based on current reports, it appears that the influence of B12 photochemistry can be both direct and indirect. In the transcriptional repressor, CarH, AdoCbl serves as a light-triggered structural switch within the protein. Here, the CarH manages to avoid radical chemistry and again appears to do so through stabilisation of the Type II S1 state. The anti-repressor, AerR, appears to require OHCbl for activity, which could well be formed by the aerobic photolysis of either AdoCbl or MeCbl before binding. The fact that the systems described to date are involved in transcriptional regulation immediately brings to mind possible optogenetic applications. Either CarH or AerR could feasibly act as an in vivo switch for target genes, and the light-triggered domain dissociation of CarH could be used to control other biomolecular interactions. An added benefit of the B12 in this context is that it absorbs green light, which is unusual for a biological chromophore. B12-based tools, optogenetic or otherwise, therefore have great potential to provide bio-orthogonal control. Interest in B12 photobiology is therefore growing,107, 116-119 and numerous CarH and AerR homologues have been found in various bacteria,7 some of which are beginning to be characterised (e.g., LitR from Bacillus megaterium).12 It will be interesting to see how extensive and versatile B12 photobiology proves to be.

AcknowledgementsI thank the European Society for Photobiology for honouring me with the 2015 Young Investigator Award, and inviting me to contribute this Perspective. Particular thanks go to Montserrat Elías-Arnanz and S. Padmanabhan for introducing me to CarH, and for their continued collaboration, and to Roger Kutta and Linus Johannissen for their excellent work towards describing the transient CarH mechanism. Finally, I must acknowledge Roseanne Sension and co-workers at the University of Michigan, Ann Arbor, who have done more than anyone else to elucidate the photoresponse of this fascinating family of molecules.

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· Web view-carA gene cluster, which leads to carotenoid biosynthesis. In 2002 this effect of light was reported to require B 12, with the addition of CNCbl, AdoCbl or MeCbl all