Spectroscopic and computational studies of Co2+corrinoids: spectral and electronic properties of the biologically relevant base-on and base-off forms of Co2+cobalamin

Co(2+)cobalmain (Co(2+)Cbl) is implicated in the catalytic cycles of all adenosylcobalamin (AdoCbl)-dependent enzymes, as in each case catalysis is initiated through homolytic cleavage of the cofactor's Co-C bond. The rate of Co-C bond homolysis, while slow for the free cofactor, is accelerated by 12 orders of magnitude when AdoCbl is bound to the protein active site, possibly through enzyme-mediated stabilization of the post-homolysis products. As an essential step toward the elucidation of the mechanism of enzymatic Co-C bond activation, we employed electronic absorption (Abs), magnetic circular dichroism (MCD), and resonance Raman spectroscopies to characterize the electronic excited states of Co(2+)Cbl and Co(2+)cobinamide (Co(2+)Cbi(+), a cobalamin derivative that lacks the nucleotide loop and 5,6-dimethylbenzimazole (DMB) base and instead binds a water molecule in the lower axial position). Although relatively modest differences exist between the Abs spectra of these two Co(2+)corrinoid species, MCD data reveal that substitution of the lower axial ligand gives rise to dramatic changes in the low-energy region where Co(2+)-centered ligand field transitions are expected to occur. Our quantitative analysis of these spectral changes within the framework of time-dependent density functional theory (TD-DFT) calculations indicates that corrin-based pi --> pi transitions, which dominate the Co(2+)corrinoid Abs spectra, are essentially insulated from perturbations of the lower ligand environment. Contrastingly, the Co(2+)-centered ligand field transitions, which are observed here for the first time using MCD spectroscopy, are extremely sensitive to alterations in the Co(2+) ligand environment and thus may serve as excellent reporters of enzyme-induced perturbations of the Co(2+) state. The power of this combined spectroscopic/computational methodology for studying Co(2+)corrinoid/enzyme active site interactions is demonstrated by the dramatic changes in the MCD spectrum as Co(2+)Cbi(+) binds to the adenosyltransferase CobA.     

Spectroscopic and computational studies of the ATP:corrinoid adenosyltransferase (CobA) from Salmonella enterica: insights into the mechanism of adenosylcobalamin biosynthesis

CobA from Salmonella enterica is a member of an enzymatic system responsible for the de novo biosynthesis of adenosylcobalamin (AdoCbl), catalyzing the formation of the essential Co-C bond by transferring the adenosyl group from a molecule of ATP to a transient Co(1+)corrinoid species generated in the enzyme active site. A particularly fascinating aspect of this reaction is that the flavodoxin in vivo reducing agent that serves as the electron donor to CobA possesses a reduction potential that is considerably more positive than that of the Co(2+/1+) couple of the corrinoid substrate. To explore how CobA may overcome this challenge, we have employed electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance (EPR) spectroscopies to probe the interaction between Co(3+)- and Co(2+)corrinoids and the enzyme active site. Our data reveal that while Co(3+)corrinoids interact only weakly with CobA, Co(2+)corrinoids undergo partial conversion to a new paramagnetic species that can be obtained in nearly quantitative yield when CobA is preincubated with the co-substrate ATP. This "activated" species is characterized by a distinct set of ligand field transitions in the near-IR spectral region and EPR parameters that are unprecedented for Co(2+)corrinoids. Analysis of these data on the basis of qualitative spectral correlations and density functional theory computations reveals that this unique Co(2+)corrinoid species possesses an essentially square-planar Co(2+) center that lacks any significant axial bonding interactions. Possible implications of these findings for the mechanism of Co(2+) --> Co(1+) reduction employed by CobA and Co-C bond-forming enzymes in general are explored.     

Correction of discrepancies in dioxin quantification between immunoassay and gas chromatography–high-resolution mass spectrometry

Due to the toxicity of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/F), efforts are made to quantify their emission into the environment. Typically, this quantification is done using gas chromatography–high-resolution mass spectrometry (GC–HRMS). However, GC–HRMS is extremely expensive and time consuming, and GC–HRMS facilities are overly requested. In order to decrease the workload on GC–HRMS, another alternative is to use an enzyme-linked immunosorbent assay (ELISA) as a semi-quantitative screening tool. One problem of this solution is that ELISA measures the total PCDD/F content of a sample differently than GC–HRMS; a disparity exists between the two techniques. This paper introduces a congener correction factor that adjusts ELISA results for this incompatibility. The importance of the correction factor is explored by examining the congener profiles of 27 different dioxin sources. The congener profiles for many of these sources are such that large incompatibilities in predicted PCDD/F content would likely exist between uncorrected ELISA and GC–HRMS. The effect that the correction factor has on the correlation between ELISA and GC–HRMS for samples from a test site with dioxin-contaminated soils was also examined. The congener profile at this site was such that the inconsistencies between uncorrected ELISA and GC–HRMS results were relatively small. However, application of the congener correction factor still improved the correlation between ELISA and GC–HRMS by 11% when using sample-specific correction factors and by 5% when using an average site-wide correction factor. The findings of this paper suggest that application of the correction factor is necessary to remove incompatibilities between ELISA and GC–HRMS—particularly when the congener profile at a site would lead to incompatibilities that are large.     

Denominators in cluster algebras of affine type

The Fomin–Zelevinsky Laurent phenomenon states that every cluster variable in a cluster algebra can be expressed as a Laurent polynomial in the variables lying in an arbitrary initial cluster. We give representation-theoretic formulas for the denominators of cluster variables in cluster algebras of affine type. The formulas are in terms of the dimensions of spaces of homomorphisms in the corresponding cluster category, and hold for any choice of initial cluster.     

Tame concealed algebras and cluster quivers of minimal infinite type

The well-known list of Happel-Vossieck of tame concealed algebras in terms of quivers with relations, and the list of A. Seven of minimal infinite cluster quivers are compared. There is a 1-1 correspondence between the items in these lists, and we explain how an item in one list naturally corresponds to an item in the other list. A central tool for understanding this correspondence is the theory of cluster-tilted algebras.     

Coloured quiver mutation for higher cluster categories

We define mutation on coloured quivers associated to tilting objects in higher cluster categories. We show that this operation is compatible with the mutation operation on the tilting objects. This gives a combinatorial approach to tilting in higher cluster categories and especially an algorithm to determine the Gabriel quivers of tilting objects in such categories.