A sophisticated intracellular trafficking pathway in humans is used to tailor vitamin B12 into its active cofactor forms, and to deliver it to two known B12-dependent enzymes. Herein, we report an unexpected strategy for cellular retention of B12, an essential and reactive cofactor. If methylmalonyl-CoA mutase is unavailable to accept the coenzyme B12 product of adenosyltransferase, the latter catalyzes homolytic scission of the cobalt−carbon bond in an unconventional reversal of the nucleophilic displacement reaction that was used to make it. The resulting homolysis product binds more tightly to adenosyltransferase than does coenzyme B12, facilitating cofactor retention. We have trapped, and characterized spectroscopically, an intermediate in which the cobalt−carbon bond is weakened prior to being broken. The physiological relevance of this sacrificial catalytic activity for cofactor retention is supported by the significantly lower coenzyme B12 concentration in patients with dysfunctional methylmalonyl-CoA mutase but normal adenosyltransferase activity.

This contribution describes the trapping of the hydroperoxyl radical at a pH of 4 during turnover of wild-type oxalate decarboxylase and its T165V mutant using the spin trap BMPO. Radicals were detected and identified by a combination of EPR and mass spectrometry. Superoxide, or its conjugate acid, the hydroperoxyl radical, is expected as an intermediate in the decarboxylation and oxidation reactions of the oxalate monoanion both of which are promoted by oxalate decarboxylase. Another intermediate, the carbon dioxide radical anion was also observed. The quantitative yields of superoxide trapping is similar in the wild type and the mutant while it is significantly different for the trapping of the carbon dioxide radical anion. This suggests that the two radicals are released from different sites of the protein.

The CblC and CblD chaperones are involved in early steps in the cobalamin trafficking pathway. Cobalamin derivatives entering the cytoplasm are converted by CblC to a common cob­(II)­alamin intermediate via glutathione-dependent alkyltransferase or reductive elimination activities. Cob­(II)­alamin is subsequently converted to one of two biologically active alkylcobalamins by downstream chaperones. The function of CblD has been elusive although it is known to form a complex with CblC under certain conditions. Here, we report that CblD provides a sulfur ligand to cob­(II)­alamin bound to CblC, forming an interprotein coordination complex that rapidly oxidizes to thiolato-cob­(III)­alamin. Cysteine scanning mutagenesis and EPR spectroscopy identified Cys-261 on CblD as the sulfur donor. The unusual interprotein Co–S bond was characterized by X-ray absorption spectroscopy and visualized in the crystal structure of the human CblD thiolato-cob­(III)­alamin complex. Our study provides insights into how cobalamin coordination chemistry could be utilized for cofactor translocation in the trafficking pathway.

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The electrophoretic mobility of low molecular mass oligoglycines is examined in this study using a "coarse-grained" bead modeling methodology [Pei, H., Allison, S. A., J. Chromatogr. A 2009, 1216, 1908-1916]. The advantage of focusing on these peptides is that their charge state is well known [Plasson, R., Cottet, H., Anal. Chem. 2006, 78, 5394-5402] and extensive electrophoretic mobility data are also available in different buffers [Survay, M. A., Goodall, D. M., Wren, S. A. C., Rowe, R. C., J. Chromatogr. A 1996, 741, 99-113] and over a broad range of temperatures [Plasson, R., Cottet, H., Anal Chem. 2005, 77, 6047-6054]. Except for assumptions about peptide secondary structure, the B model has no adjustable parameters. It is concluded that the oligoglycines adopt a random configuration at high temperature (50 degrees C and higher), but more compact conformations at lower temperature. It is proposed that triglycine through pentaglycine adopt compact cyclic structures at low temperature (up to about 25 degrees C) in aqueous solution. At 25 degrees C, buffer interactions are also examined and may or may not influence peptide conformation depending on the buffer species. In a borate buffer at high pH, the mobility data are consistent with complex formation between the oligoglycine and borate anion.