title: Evidence from electron paramagnetic resonance spectroscopy of the participation of radical intermediates in the reaction catalyzed by methylmalonyl-coenzyme A mutase.
abstract: Recombinant methylmalonyl-coenzyme A (CoA) mutase from Propionibacterium shermanii has been purified 20-fold to near homogeneity in a highly active form. Neither the apoenzyme (the form in which the enzyme is isolated) nor the holoenzyme (reconstituted with the cofactor, adenosylcobalamin) has an electron paramagnetic resonance (EPR) spectrum associated with it. However, the addition of either the substrate, methylmalonyl-CoA, or the product, succinyl-CoA, results in the appearance of a transient EPR signal. The signal has hyperfine features that indicate coupling of the unpaired electron to the cobalt nucleus. In the presence of [CD3]methylmalonyl-CoA, an EPR signal is also seen and is similar to that obtained in the presence of protiated substrate. Power saturation studies reveal the presence of two components, a slow relaxing species (with an apparent g value of 2.11) and a fast relaxing species (with an apparent g value of 2.14) that can be partially resolved at low temperature and high power. The EPR-active intermediate is observed under catalytic conditions and is approximately midway in its resonance position between a free radical and cob(II)alamin. It is postulated to represent an exchange-coupled cob(II)alamin ... free radical pair. The signal bears close resemblance to those observed with partially dehydrated polycrystalline adenosylcobalamin following laser photolysis (Ghanekar, V.D., Lin, R.J., Coffman, R.E., and Blakley, R.L. (1981) Biochem. Biophys. Res. Commun. 101, 215-221) and with the adenosylcobalamin-dependent ribonucleotide reductase under freeze-quench conditions (Orme-Johnson, W.H., Beinert, H., and Blakley, R.L. (1974) J. Biol. Chem. 249, 2338-2343). When cob(II)alamin is generated under noncatalytic conditions (i.e. in the presence of propionyl-CoA or by electrochemical reduction of enzyme-bound hydroxocob-(III)alamin), a different EPR signal is observed with g = 2.26 and g = 2.00, typical of base-on cob(II)alamin.
authors.affiliation: Biochemistry Department, University of Nebraska, Lincoln 68583-0718, USA.
authors.forename: R
authors.initials: R
authors.lastname: Padmakumar
authors.forename: R
authors.initials: R
authors.lastname: Banerjee
mesh_terms: D003038:Cobamides / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D008765:Methylmalonyl-CoA Mutase / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature
chemical_list: D003038:Cobamides; D005609:Free Radicals; D008765:Methylmalonyl-CoA Mutase; C000913:cobamamide