Cobalamin (Cbl) is a large heterocyclic molecule in which a central cobalt ion is equatorially coordinated by four donor N atoms from a corrin ring and axially coordinated at its lower (α) position by an N atom from 5,6-dimethylbenzimidazole (DMB) as shown in Fig. 1. In the Co(III) state the upper (β) axial position can be occupied by a large number of different ligands and several liganded Cbl X-ray crystal structures have been reported (Hannibal et al.
; Gruber et al.
; Randaccio et al.
). Cbls cannot be synthesized by higher organisms. In dietary vitamin B12
(CNCbl) a cyanide group is the β-axial ligand, but this form is enzymatically hydrolyzed to cob(II)alamin [Cbl(II)] and then transformed into one of two biologically active forms, AdoCbl and MeCbl, in which the β axial ligand is either a 5′-deoxyadenosyl (Ado) group or a methyl (Me) group, respectively (Chu et al.
; Froese et al.
; Kim et al.
(a) Structural formula of Cbl(II). (b) Atom numbering and side-chain labeling used for description.
In mammals, several proteins are involved in the uptake, transport and storage of dietary Cbls (Banerjee et al.
). X-ray crystallographic structures of complexes of Cbl with the intrinsic factor (IF; Mathews et al.
) and trans-Cbl (TC; Wuerges et al.
) transport proteins have been reported. AdoCbl and MeCbl are the largest and most complex cofactors in biology and are required by two mammalian enzymes: MeCbl is the cofactor of methionine synthase (MES), whose mechanism involves generating alkyl cations by heterolytic cleavage of the Co—C bond, while AdoCbl is the cofactor of methylmalonyl coenzyme A mutase (MCAM), whose mechanism involves generating an Ado radical by homolytic dissociation of the Co—C bond. X-ray crystallography has shown that although DMB is coordinated to the α-axial position in the ‘base-on’ configuration in the free coenzyme and in complexes with dehydratase enzymes and the transport proteins IF and TC, it is found in the ‘base-off’ configuration in complexes with the enzymes MES and MCAM, displaced from the cobalt ion by a histidine amino-acid side chain of the protein (Banerjee et al.
Understanding how an enzyme facilitates controlled and reversible cleavage of the Co—C bond and therefore transition between Co(III) and Co(I,II) is an outstanding problem in biochemistry. Although early ideas from classical coordination chemistry suggested the importance of the Co—N bond to the DMB α-ligand in modulating cleavage, more recent X-ray crystallographic studies and electronic structure calculations of enzyme–Cbl complexes (Jensen & Ryde, 2009
) and similarities between the structures of MeCbl and its homolysis product Cbl(II) (Kräutler al.
) suggest that enzymes play a dominant role in pulling the adenosyl radical away from the corrin ring. However, the exact architecture of the corrin ring, with its seven amide side chains (three acetamides and four propionamides) projecting above and below the plane, and coordination with DMB are likely to contribute to stabilizing the low-spin Co(I), Co(II) and Co(III) states involved in catalysis and recombination (Jensen & Ryde, 2009
In addition to its role as a coenzyme, Cbl has also been implicated in the inhibition of nitric oxide-induced physiologies and pathologies (Hassanin et al.
). NO is a short-lived radical that is produced by tumor and host cells and is thought to function as an important modulator of tumor progression and angiogenesis. It has been suggested that in vivo
Cbl(II) directly scavenges NO to form NOCbl, which has renewed interest in the exact structure of Cbl(II) and its β-ligand binding. Recent X-ray crystal structures of NOCbls have featured remarkably long Co—N(DMB) α-axial bond lengths, suggesting that this bond may well be correlated with direct β-ligand scavenging (Hassanin et al.
). Cbls have also been implicated in the dechlorination by anaerobic organisms of the toxic compounds perchloroethylene and trichloroethylene (Randaccio et al.
Cbls possess a number of functional groups found in biomacromolecules and can be crystallized in forms that contain several ordered solvent molecules and which diffract X-rays to high resolution. They have therefore been suggested as particularly useful models for studying the interaction of water with proteins and nucleic acids (Bouquiere et al.
; Randaccio et al.
; Savage et al.
; Savage, 1986
). Neutrons offer important advantages for determining solvent structures hydrating biomacromolecules because the relatively strong scattering power of hydrogen (H) and deuterium (D) can allow the location of all atoms in a water molecule even at medium resolution (>1.5 Å; Blakeley et al.
); D atoms (neutron scattering length 6.67 × 10−15
m) appear as strong peaks in neutron scattering density maps, thereby revealing the location of isotopically exchanged H atoms, while H atoms (−3.74 × 10−15
m) appear as negative troughs.
The water structure in crystals of vitamin B12
has been studied in detail previously using high-resolution monochromatic neutron data collected at 279 K to a resolution of 0.95 Å on the former instrument D8 with a point detector at the high-flux reactor neutron source run by the Institute Laue–Langevin (ILL; Savage et al.
). This study revealed intricate networks of partially disordered solvent running throughout the crystal (Savage, 1986
). A subsequent high-resolution study on the original D19 at 15 K extended the resolution to 0.90 Å (Bouquiere et al.
). We have previously studied the water structure in Cbl(II) at room temperature using quasi-Laue neutron data collected using the original LADI diffractometer also at ILL (Langan et al.
). LADI has recently been significantly upgraded (to LADI-III) in order to improve its performance (Blakeley et al.
). Limitations set by the wavelength band used on the original LADI (which had a peak at 2.8 Å and a FWHH of 19%) restricted the resolution of the data to d
> 1.42 Å. The final nominal resolution (at which the completeness fell below 70%) of the data set collected in 36 h was around 1.8 Å. Even at this medium resolution we were able to obtain coordinates for all of the atoms involved in seven ordered water and two acetone solvent molecules. From these coordinates, hydrogen-bonding parameters were determined that contributed to an improved general understanding and modeling of the hydration of biomacromolecules.
In addition to the data set collected on LADI, another more complete neutron data set was collected from Cbl(II) at room temperature on the original D19 with its small prototype 4° × 64° area detector. We were able to collect data to a higher resolution with a data-collection time of 28 d. The resolution of the data was still limited to d > 0.92 Å because of the use of a wavelength of 1.54 Å and the geometry of the original D19. Recently, a new single-crystal diffractometer called TOPAZ has been built at the Spallation Neutron Source (SNS) run by Oak Ridge National Laboratory. At the SNS neutrons are produced by bombarding a mercury target with pulses of high-energy protons. Neutrons produced by proton pulses are ‘time-stamped’ and travel as a function of their energy so that neutrons of different energies are detected at different arrival times. By recording the time-of-flight (TOF) information, the corresponding energy and wavelength of each neutron can be calculated. TOF techniques therefore allow wavelength-resolved Laue patterns to be collected using all of the available useful neutrons. In particular, the tunable wavelength range on TOPAZ in combination with an array of position-sensitive detectors will offer the possibility of collecting data to ultrahigh resolution from biological molecules with unit-cell parameters of up to about 70 Å in length.
In this study, we report the structure of Cbl(II) determined from the monochromatic neutron data set collected on the original D19 at the H11 thermal beam at the ILL to 0.92 Å resolution. We also report preliminary studies of the feasibility of extending the resolution of this structure even further by collecting TOF neutron data during a short commissioning experiment on TOPAZ. Particular attention is given to a description of the data-collection and integration process on TOPAZ since this is a new instrument and it has not been reported in full before. The results from D19 provide detailed hydrogen-bonding parameters for the hydration of different functional groups in a biological molecule at room temperature to unprecedented resolution and accuracy. The features of the Cbl(II) structure may promote the capture and scavenging of small molecules such as NO. The results from TOPAZ demonstrate that neutron data can be collected from biological molecules rapidly to high resolution but that there is also the potential to collect to ultrahigh resolution with longer data-collection times.