Human cobalophilin: The structure of bound methylcobalamin and a functional role in protecting methylcobalamin from photolysis

Abstract

The interactions of methylcobalamin with cobalophilin from human serum were analyzed using extended X-ray absorption fine structure (EXAFS) spectroscopy, photolysis of the cobalt-carbon bond of methylcobalamin, and a pKa determination of the protonation of the coordinated nitrogen of 5,6-dimethylbenzimidazole (DMB). These results are consistent with the idea that the DMB nitrogen is still coordinated when protein is bound; however, the ability of a methyl radical (generated by photolysis) to escape the geminate cage of the protein is considerably reduced. For methylcobalamin in solution, the DMB nitrogen ligand is at a distance of 2.20 +/- 0.03 A from cobalt [Sagi, I., & Chance, M. R. (1992) J. Am. Chem. Soc. 114, 8061-8066]. This distance to the lower axial ligand does not change when protein binds (2.20 +/- 0.04 A), nor do the optical spectra exhibit any base-off character. The average of the distance from cobalt to the four equatorial nitrogens of the corrin plane is also unchanged. The pKa for the conversion of the "base-on" to the "base-off" form of methylcobalamin, where the above DMB nitrogen becomes protonated and the Co-N axial bond is cleaved, does not deviate from the free cobalamin value of 2.7 when methylcobalamin is bound to cobalophilin. These results indicate that replacement of the DMB ligand with a ligand from the protein is unlikely. Although the background-subtracted EXAFS data sets for free methylcobalamin and for the protein complex are extremely similar, more accurate data with explicit higher shell analysis would be required to entirely rule out ligand replacement. The chemical and electronic nature of the ligand changes little.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequence, S–S Bridges, and Spectra of Bovine Transcobalamin Expressed in Pichia pastoris

“…The binding of Cbl to bovine TC decreased the mobility of the ␤-group in the Cbl molecule according to velocities of H 2 O 7 azide exchange. A similar effect had been observed for another protein HC-Cbl (40,41). The mechanism of protein-enforced partial immobilization of the ␤-group is not clear.…”

Thermolysis of Neopentylcobalamin Analogs Complexed to Haptocorrin: Side Chain Entropy and Activation of Organocobalamins for Carbon−Cobalt Bond Homolysis

“…(eq 1), for the case where one or both of the free radical products are rapidly trapped (as here, by O 2 ), the observed rate constant for homolysis, k obs , is given by eq 2, where F c , the fractional cage efficiency, is given by eq 3 Frisbie and Chance have reported a large decrease in the quantum yield for CH 3 Cbl photolysis upon complexation of CH 3 Cbl to human HC and attributed this effect to an increase in the rate constant, k c , for geminate recombination within the solvent cage. While such an effect is possible, it occurs to us that complexation of an RCbl to a protein the size of HC should also result in a reduction of the rate constant, k d , for diffusional separation of the caged radical pair.…”

Time-Resolved Spectroscopic Studies of B12Coenzymes: The Identification of a Metastable Cob(III)alamin Photoproduct in the Photolysis of Methylcobalamin

“…39,40 In addition, protection from photolysis has been proposed as a function of the transport protein cobalophilin. 46 Photolysis can be inhibited in the protein environment in several ways, four of which are enumerated here: (1) The cage effect of the protein will increase the recombination probability by preventing escape of the methyl radical. (2) The protein may exclude oxygen from the cobalamin binding pocket, preventing oxidation of the methyl radical, and thereby increasing the relative yield of methyl recombination.…”

“…The Photolysis of Methylcobalamin in Methionine Synthase. Photolysis of methylcobalamin has been used to probe the effects of mutations on the structure and reactivity of methionine synthase. , In addition, protection from photolysis has been proposed as a function of the transport protein cobalophilin . Photolysis can be inhibited in the protein environment in several ways, four of which are enumerated here: (1) The cage effect of the protein will increase the recombination probability by preventing escape of the methyl radical.…”