Electron Magnetic Resonance (EMR, aka, ESR, EPR) measurements were important from the very first studies of the active B12 enzyme/coenzyme system in that they demonstrated the involvement the of free radicals. Almost a blasphemy in biochemistry at the time! Both an organic radical and a Co(II) center were identified, supporting the mechanism in which the Co-C bond to the adenosyl group underwent homolytic fission.
The reaction whereby a hydrogen and a group on an adjacent carbon atom exchange places is considered to take place by way of a radical mechanism. The evidence for this comes principally from observations of EMR spectra for several of the active enzyme systems. Thus if the dioldehydrase (holoenzyme) is treated with the substrate analogue 1,2-propanediol, a radical signal believed to be due to the 5'-deoxyadenosyl radical, together with a low spin Co(II) signal is formed within milliseconds, and persists whilst there is substrate present . Similarly for glycerol-dehydrase, ethanol-ammonia lyase and the ribonucleotide reductase system.
Thus EMR spectra can be observed either from reduction of the Co(II) coenzyme or from measurements of the active enzyme+coenzyme+substrate system.
The Latest EMR Measurements
There are currently four very interesting papers from Kurt Warncke's group at the Department of Physics, Emory University, Atlanta, Georgia
Interaction of the Substrate Radical and the 5'-Deoxyadenosine-5'-Methyl Group in Vitamin B12 Coenzyme-Dependent Ethanolamine Deaminase
Kurt Warncke and Andrew S. Utada, J.A.C.S., 2001, 123(35), 8564.
"The distance and relative orientation of the C5' methyl group of 5'-deoxyadenosine and the substrate radical in vitamin B12 coenzyme-dependent ethanolamine deaminase from Salmonella typhimurium have been characterized by using X-band two-pulse electron spin-echo envelope modulation (ESEEM) spectroscopy in the disordered solid state. The (S)-2-aminopropanol-generated substrate radical catalytic intermediate was prepared by cryotrapping steady-state mixtures of enzyme in which catalytically exchangeable hydrogen sites in the active site had been labeled by previous turnover on 2H4-ethanolamine. Simulation of the time- and frequency-domain ESEEM requires two types of coupled 2H. The strongly coupled 2H has an effective dipole distance (reff) of 2.2 Å, and isotropic coupling constant (Aiso) of -0.35 MHz. The weakly coupled 2H has reff = 3.8 Å and Aiso = 0 MHz. The best 2H ESEEM time- and frequency-domain simulations are achieved with a model in which the hyperfine couplings arise from one strongly coupled hydrogen site and two equivalent weakly coupled hydrogen sites located on the C5' methyl group of 5'-deoxyadenosine. This model indicates that the unpaired electron on C1 of the substrate radical and C5' are separated by 3.2 Å and are thus at closest contact. The close proximity of C1 and C5' indicates that C5' of the 5'-deoxyadenosyl moiety directly mediates radical migration between cobalt in cobalamin and the substrate/product site over a distance of 5-7 Å in the active site of ethanolamine deaminase."
Identification of a Rearranged-Substrate, Product Radical Intermediate and the Contribution of a Product Radical Trap in Vitamin B12 Coenzyme-Dependent Ethanolamine Deaminase Catalysis
by Kurt Warncke, Jennifer C. Schmidt, and Shyue-Chu Ke, J.A.C.S.,1999 121, 10522.
"The radical intermediate present during steady-state turnover of substrate aminoethanol by ethanolamine deaminase from Salmonella typhimurium has been characterized by using X-band electron paramagnetic resonance (EPR) spectroscopy. The radical intermediate was prepared by cryotrapping enzyme, aminoethanol substrate, and vitamin B12 coenzyme (adenosylcob(III)alamin) immediately following mixing. Natural abundance, 1,1,2,2-2H4-, 2-13C-, and 1,2-13C2-aminoethanol were used as substrates. The EPR spectrum obtained for natural abundance aminoethanol shows a broad feature at approximately g = 2.3 that arises from CoII in cob(II)alamin, and a feature from an organic radical that has an absorption maximum at g = 2.02 and a line width of 10.8 mT. The EPR line shape is characteristic of a relatively weakly electron spin-coupled CoII-organic radical system. The EPR line shapes for the 2H- and 13C-labeled substrates were narrowed and broadened by 0.7 and 2.4 mT, respectively, demonstrating that the radical is substrate-based. The comparable line widths of the 2-13C- and 1,2-13C-labeled radicals show that the unpaired spin density is localized primarily at the C2 carbon atom. This identifies the radical intermediate as a rearranged substrate radical, or product radical. The results are consistent with either the 1-aminoethanol-2-yl radical or the ethanal-2-yl radical, which have been proposed as intermediates in, respectively, the amine migration and amine elimination mechanisms of rearrangement. A qualitative reaction free energy profile for the CoII-radical pair intermediate states on the enzyme is constructed, based on the EPR results and previous isotope exchange and kinetic isotope effect studies. The results and analysis reveal that a product radical trap strategy contributes to the stabilization of the radical pair state, which enhances catalytic performance of ethanolamine deaminase."
Jenna S. Trommel, Kurt Warncke, and Luigi G. Marzilli, J.A.C.S., 2001, 123, 3358.
Protein control of cobalt-axial nitrogen ligand bond length has been proposed to modulate the reactivity of vitamin B12 coenzyme during the catalytic cycle of B12-dependent enzymes. In particular, hyper-long Co-N bonds may favor homolytic cleavage of the trans-cobalt-carbon bond in the coenzyme. X-ray crystallographic studies point to hyper-long bonds in two B12 holoenzymes; however, mixed redox and ligand states in the crystals thwart clear conclusions. Since EPR theory predicts an increase in Co(II) hyperfine splitting as donation from the axial N-donor ligand decreases, EPR spectroscopy could clarify the X-ray results. However, the theory is apparently undermined by the similar splitting reported for the 2-picoline (2-pic) and pyridine (py) adducts of Co(II) cobinamide (Co(II)Cbi+), adducts thought to have long and normal Co-N axial bond lengths, respectively. Cobinamides, with the B12 5,6-dimethylbenzimidazole loop removed, are excellent B12 models. We studied Co(II)Cbi+ adducts of unhindered 4-substituted pyridines (4-X-py's) in ethylene glycol to separate orbital size effects from Co-N axial distance effects on these splittings. The linear increase in splitting with the decrease in 4-X-py basicity found is consistent with the theoretically predicted increase in unpaired electron spin density as axial N lone pair donation to Co(II) decreases. No adduct (and hence no hyper-long Co(II)-N axial bond) was formed even by 8 M 2-pic, if the 2-pic was purified by a novel Co(III)-affinity distillation procedure designed to remove trace nitrogenous ligand impurities present in 2-pic distilled in the regular manner. Adducts formed by impurities in 2-pic and other hindered pyridines misled previous investigators into attributing results to adducts with long Co-N bonds. We find that many 2-substituted py's known to form adducts with simple synthetic Co models do not bind Co(II)Cbi+. Thus, the equatorial corrin ring sterically impedes binding, making Co(II)Cbi+ a highly selective binding agent for unhindered sp2 N-donor ligands. Our results resolve the apparent conflict between EPR experiment and theory. The reported Co(II) hyperfine splitting of the enzyme-bound cofactor in five B12 enzymes is similar to that of the relevant free cofactor. The most reasonable interpretation of this similarity is that the Co-N axial bond of the bound cofactor is not hyper-long in any of the five cases.
Interactions of Substrate and Product Radicals with Co(II) in Cobalamin and with the Active Site in Ethanolamine Deaminase, Characterized by ESE-EPR and 14N ESEEM Spectroscopies
by Shyue-Chu Ke and Kurt Warncke, J.A.C.S., 1999, 121, 9922.
Interactions of the cryotrapped 2-aminopropanol-1-yl substrate radical and aminoethanol-derived product radical catalytic intermediates with the active site of vitamin B12 coenzyme-dependent ethanolamine deaminase from Salmonella typhimurium in disordered frozen aqeuous solution have been characterized by using X-band electron spin-echo electron paramagnetic resonance (ESE-EPR) and electron spin-echo envelope modulation (ESEEM) spectroscopies performed at 6 K. ESE-EPR spectra show that the doublet splitting of the radical line shape by electron-electron exchange and dipolar interactions with CoII in cob(II)alamin is stronger for the substrate radical (11.1 mT) than for the product radical (7.1 mT). The aminoethanol-derived product radical unpaired spin density at C2 is therefore positioned closer to CoII than the 2-aminopropanol-1-yl substrate radical unpaired spin density at C1. Multifrequency three-pulse ESEEM spectra obtained at g = 2.00 for each radical display the same - and magnetic field-dependent five-line pattern of narrow features. This indicates that the substrate and product radicals are comparably coupled to the same 14N nucleus. ESEEM simulations give the following best-fit principal hyperfine tensor and nuclear quadrupole coupling parameters: A = [-1.1, -0.8, -0.8] MHz; e2qQ/h = 3.05 MHz; = 0.51. The quadrupole parameters are consistent with a secondary amide nitrogen in a polypeptide bond or with the N10 amino nitrogen in the adenine ring of 5'-deoxyadenosine. The equivalent 14N coupling for each radical indicates that either the nitrogen nucleus lies along the C1-C2 bond bisector or the interaction responsible for the coupling moves to remain in register with the center of unpaired spin density as it shifts 1.5 Å from C1 to C2 in the substrate-to-product radical transformation. The longer separation distance from CoII of the product versus substrate radical indicates that the principle of radical pair stabilization by separation operates continuously through the rearrangement reaction.