Alex R. Jones

Updated structure of Drosophila cryptochrome

Arising from B. D. Zoltowski et al. 480, 396–399 (2011)10.1038/nature10618Recently, we determined the X-ray crystal structure of full-length cryptochrome from Drosophila. Here we report an improved model of the Drosophila cryptochrome (dCRY) structure that corrects errors in the original coordinates (Protein Data Bank (PDB) accession 3TVS). Further refinement of the structure, with automated rebuilding algorithms in Phenix followed by manual building, indicated that a model of dCRY could be produced with excellent refinement statistics without taking into account the non-merohedral twinning originally reported (Table 1).

Cryptochrome-dependent magnetic field effect on seizure response in Drosophila larvae

The mechanisms that facilitate animal magnetoreception have both fascinated and confounded scientists for decades, and its precise biophysical origin remains unclear. Among the proposed primary magnetic sensors is the flavoprotein, cryptochrome, which is thought to provide geomagnetic information via a quantum effect in a light-initiated radical pair reaction. Despite recent advances in the radical pair model of magnetoreception from theoretical, molecular and animal behaviour studies, very little is known of a possible signal transduction mechanism. We report a substantial effect of magnetic field exposure on seizure response in Drosophila larvae. The effect is dependent on cryptochrome, the presence and wavelength of light and is blocked by prior ingestion of typical antiepileptic drugs. These data are consistent with a magnetically-sensitive, photochemical radical pair reaction in cryptochrome that alters levels of neuronal excitation, and represent a vital step forward in our understanding of the signal transduction mechanism involved in animal magnetoreception.

Continuous wave photolysis magnetic field effect investigations with free and protein-bound alkylcobalamins.

The activation of the Co-C bond in adenosylcobalamin-dependent enzymes generates a singlet-born Co(II)-adenosyl radical pair. Two of the salient questions regarding this process are: (1) What is the origin of the considerable homolysis rate enhancement achieved by this class of enzyme? (2) Are the reaction dynamics of the resultant radical pair sensitive to the application of external magnetic fields? Here, we present continuous wave photolysis magnetic field effect (MFE) data that reveal the ethanolamine ammonia lyase (EAL) active site to be an ideal microreactor in which to observe enhanced magnetic field sensitivity in the adenosylcobalamin radical pair. The observed field dependence is in excellent agreement with that calculated from published hyperfine couplings for the constituent radicals, and the magnitude of the MFE (<18%) is almost identical to that observed in a solvent containing 67% glycerol. Similar augmentation is not observed, however, in the equivalent experiments with EAL-bound methylcobalamin, where all field sensitivity observed in the free cofactor is washed out completely. Parallels are drawn between the latter case and the loss of field sensitivity in the EAL holoenzyme upon substrate binding (Jones et al. J. Am. Chem. Soc. 2007, 129, 15718-15727). Both are attributed to the rapid removal of the alkyl radical immediately after homolysis, such that there is inadequate radical pair recombination for the observation of field effects. Taken together, these results support the notion that rapid radical quenching, through the coupling of homolysis and hydrogen abstraction steps, and subsequent radical pair stabilization make a contribution to the observed rate acceleration of Co-C bond homolysis in adenosylcobalamin-dependent enzymes.

The photochemical mechanism of a B12-dependent photoreceptor protein.

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.

Ultrafast infrared spectral fingerprints of Vitamin B12 and related cobalamins

Vitamin B(12) (cyanocobalamin, CNCbl) and its derivatives are structurally complex and functionally diverse biomolecules. The excited state and radical pair reaction dynamics that follow their photoexcitation have been previously studied in detail using UV-visible techniques. Similar time-resolved infrared (TRIR) data are limited, however. Herein we present TRIR difference spectra in the 1300-1700 cm(-1) region between 2 ps and 2 ns for adenosylcobalamin (AdoCbl), methylcobalamin (MeCbl), CNCbl, and hydroxocobalamin (OHCbl). The spectral profiles of all four cobalamins are complex, with broad similarities that suggest the vibrational excited states are related, but with a number of identifiable variations. The majority of the signals from AdoCbl and MeCbl decay with kinetics similar to those reported in the literature from UV-visible studies. However, there are regions of rapid (<10 ps) vibrational relaxation (peak shifts to higher frequencies from 1551, 1442, and 1337 cm(-1)) that are more pronounced in AdoCbl than in MeCbl. The AdoCbl data also exhibit more substantial changes in the amide I region and a number of more gradual peak shifts elsewhere (e.g., from 1549 to 1563 cm(-1)), which are not apparent in the MeCbl data. We attribute these differences to interactions between the bulky adenosyl and the corrin ring after photoexcitation and during radical pair recombination, respectively. Although spectrally similar to the initial excited state, the long-lived metal-to-ligand charge transfer state of MeCbl is clearly resolved in the kinetic analysis. The excited states of CNCbl and OHCbl relax to the ground state within 40 ps with few significant peak shifts, suggesting little or no homolysis of the bond between the Co and the upper axial ligand. Difference spectra from density functional theory calculations (where spectra from simplified cobalamins with an upper axial methyl were subtracted from those without) show qualitative agreement with the experimental data. They imply the excited state intermediates in the TRIR difference spectra resemble the dissociated states vibrationally (the cobalamin with the upper axial ligand missing) relative to the ground state with a methyl in this position. They also indicate that most of the TRIR signals arise from vibrations involving some degree of motion in the corrin ring. Such coupling of motions throughout the ring makes specific peak assignments neither trivial nor always meaningful, suggesting our data should be regarded as IR spectral fingerprints.

Is there a dynamic protein contribution to the substrate trigger in coenzyme B12-dependent ethanolamine ammonia lyase?

Coenzyme B12, or 5’-deoxyadenosylcobalamin (AdoCbl), acts as cofactor to a number of enzymes from a range of organisms. 2] In all cases, the Co C bond in the cofactor undergoes homolysis upon substrate binding, generating a singlet-born, Cbl/adenosyl radical pair (RP) and thus initiating radical-mediated catalysis. When compared to thermal homolysis of the free cofactor in solution, rate increases achieved by these enzymes are in the region of 10– 10, the precise origin of which is not yet fully understood. To date, the protein contribution to this catalytic power has been discussed either in terms of ground-state destabilization and a “strain” hypothesis, or transition state stabilization by electrostatic factors. However, there may be another contribution to consider—protein dynamics. Using a unique combination of spin-chemical and photochemical techniques we present evidence for coupling between RP reaction dynamics and protein dynamics in AdoCbl-dependent ethanolamine ammonia lyase (EAL). The adenosyl radical has never been observed directly during turnover in an AdoCbl-dependent enzyme under ambient conditions. In EAL it is rapidly quenched by Habstraction from the substrate to give the more stable substrate radical. 9, 10] The Co C bond can be photolyzed, however, enabling investigation of the singlet-born geminate pair dynamics at room temperature both in the free and protein-bound cofactor. The spin-state of this RP can coherently interconvert between the singlet and the triplet sublevels (Scheme 1). If the geminate RP re-encounter, only those in the singlet state will recombine, whereas triplet pairs will separate again. The extent to which the spin-states mix can be altered by the application of external magnetic fields (MFs). For increasing MFs of moderate strength (tens to hundreds of mT) the T 1 levels are gradually removed in energy and ultimately only S and T0 interconvert. With a singlet-born RP, this process increases the relative S population and hence the probability of recombination. Such magnetic field effects (MFEs) have been observed in the rate of anaerobic, continuous wave (cw) photolysis of both free and EAL-bound AdoCbl. 16] Under continuous illumination the reactive adenosyl radicals are ultimately and irreversibly quenched to yield an accumulated Cbl signal, and the MFE manifests as a decrease in the apparent rate of this accumulation. The magnitude of the MFE was viscositydependent for unbound AdoCbl, the viscogen acting as a RP “cage”. Likewise, the protein limits RP diffusion, thus enhancing the MFE over that observed in buffered water. The magnetic sensitivity of homolysis is removed in EAL, however, when the Co C bond is broken thermally by substrate binding. The observation of a significant kinetic isotope effect in the pre-steady-state signal representing the conversion of AdoCbl to Cbl suggests kinetic coupling of homolysis to subsequent H-abstraction from the substrate. The effect of this coupling is to rapidly quench the adenosyl radical, generating the substrate radical (which accumulates during turnover), thus stabilizing against recombination of the geminate pair and removing the MFE. However, this does not preclude the possibility of MF-sensitivity in the recombination step after product release. While the chemistry that immediately follows homolysis in the EAL-catalyzed reaction appears to favor RP dissociation, what of the protein contribution? The role of protein dynamics in enzyme function 19] is commonly probed by varying solvent viscosity (see, e.g. Ref. [20,21]) and assessing the extent to which this variation at the protein surface is transmitted to the active site. We therefore investigated the influence of viscosity on the cw-photolysis rate, and its MFE, of both free and EAL-bound AdoCbl at 298 K, using a specially configured MFE stopped-flow spectrophotometer. 22] The aim was to isolate the effect of protein dynamics on the geminate RP. Typical traces acquired at 525 nm are shown in the Supporting Information (Figure S1a and b). Scheme 1. The reaction and spin dynamics of the separated Cbl/ adenosyl radical pair following anaerobic photolysis of AdoCbl. During cw-photolysis, the adenosyl radicals are irreversibly quenched yielding an accumulated Cbl signal.

Protein Motions Are Coupled to the Reaction Chemistry in Coenzyme B12‐Dependent Ethanolamine Ammonia Lyase

The role of protein dynamics in promoting catalysis is hotly debated. Infrared data from both ultrafast flash photolysis and stopped-flow studies show that not only does there appear to be vibrational coupling between the cofactor and protein in B(12) -dependent ethanolamine ammonia lyase, but also that there are significant protein motions coupled to the reaction that follows substrate binding.

Human cryptochrome-1 confers light independent biological activity in transgenic drosophila correlated with flavin radical stability

Cryptochromes are conserved flavoprotein receptors found throughout the biological kingdom with diversified roles in plant development and entrainment of the circadian clock in animals. Light perception is proposed to occur through flavin radical formation that correlates with biological activity in vivo in both plants and Drosophila. By contrast, mammalian (Type II) cryptochromes regulate the circadian clock independently of light, raising the fundamental question of whether mammalian cryptochromes have evolved entirely distinct signaling mechanisms. Here we show by developmental and transcriptome analysis that Homo sapiens cryptochrome - 1 (HsCRY1) confers biological activity in transgenic expressing Drosophila in darkness, that can in some cases be further stimulated by light. In contrast to all other cryptochromes, purified recombinant HsCRY1 protein was stably isolated in the anionic radical flavin state, containing only a small proportion of oxidized flavin which could be reduced by illumination. We conclude that animal Type I and Type II cryptochromes may both have signaling mechanisms involving formation of a flavin radical signaling state, and that light independent activity of Type II cryptochromes is a consequence of dark accumulation of this redox form in vivo rather than of a fundamental difference in signaling mechanism.

Time resolved studies of radical pairs

The effect of magnetic fields on chemical reactions through the RP (radical pair) mechanism is well established, but there are few examples, in the literature, of biological reactions that proceed through RP intermediates and show magnetic field-sensitivity. The present and future relevance of magnetic field effects in biological reactions is discussed.

Relating localized protein motions to the reaction coordinate in coenzyme B12‐dependent enzymes

The classical picture of enzyme catalysis relies on controlling the entropic and enthalpic contributions by manipulating reaction barriers and co‐locating reactants and cofactors to facilitate the reaction chemistry. Catalysis is linked inextricably to the geometry of the enzyme–substrate complex and the chemical/physical properties of the active site, and probably to dynamical contributions that guide reactants along the desired reaction coordinate. Coenzyme B12‐dependent enzymes have remarkable catalytic power and unique properties that enable detailed analysis of the reaction chemistry and associated dynamics. Here we discuss recent developments that are beginning to provide atomistic insight into how coenzyme B12‐dependent enzymes steer reactants along the reaction coordinate. Such insight will ultimately generate ‘movies’ of the catalytic process across all relevant time scales. In the longer term, this will enable more predictive engineering of this class of enzyme to achieve new and desirable chemical outcomes.