Co-C bond activation in B12-dependent enzymes:: Cryogenic resonance Raman studies of methylmalonyl-coenzyme A mutase

The determination of its crystal structure, as well as extensive biochemical characterizaion, has focused attention on methylmalonyl-coenzymeA (MM-CoA) mutase as a paradigm for AdoCbl (adenosylcobalamin)-dependent enzymes. These enzymes catalyze carbon skeleton rearrangement reactions via a radical mechanism, which originates in homolysis of the Co-C bond. The determinants of this mechanism are of great chemical and biochemical interest. We report resonance Raman (RR) spectra of MM-CoA, in the presence of substrates and inhibitors, using a cryogenic technique to prevent laser-induced photolysis of the Co-C bond. RR spectroscopy provides an in-site probe of cobalamin structure. Although the spectra are dominated by the corrin ring, four RR bands arising from Go-bound adenosine can be detected via isotope editing. These are assigned to the Go-C bond stretch, the Co-C-C angle bend, a 5'-C-coupled ribose deformation, and a hindered rotation of the adenosine about the Co-C bond. The RR spectra confirm enzyme-catalyzed H exchange between substrate, but not inhibitors, and the adenosyl 5'-C atom. The RR enhancement pattern is affected in different ways by binding inhibitors or slow substrates on the one hand and product or substrate on the other. Since product dissociation is apparently rate-limiting and the rearrangement equilibrium lies toward product, the adducts with product or substrate represent the product state (P state), whereas those with inhibitors or the slow substrates represent the substrate state (S state). The RR enhancement changes (activation of delta CoCC and weakening and downshift of nu CoC) indicate Co-Ado tilting, to a small extent in the resting enzyme and to a larger extent in the S state. These changes are reversed in the P state. Thus Co-Ado tilting is identified as a contributor to activation of the Co-C bond in the S state. The steric forces that induce tilting are apparently relaxed in the P state, thus promoting the rearrangement of substrate to product. The corrin vibrational modes are responsive to the ring conformation but are mostly unaffected by substrate or inhibitor binding, indicating that Co-C activation does not involve corrin conformation changes. However, two modes, whose frequencies (423 and 437 cm(-1)) are consistent with contributions from Co-N(corrin) stretching, shift down in the P state, suggesting displacement of the Co from the corrin plane. This effect may result from lengthening of the Co-N(histidine) bond, as seen in the crystal structure.