Hydrogen abstraction from thiols by adenosyl radicals: chemical precedent for thiyl radical formation, the first catalytic step in ribonucleoside triphosphate reductase from Lactobacillus leichmannii

Abstract

Aqueous solutions of adenosylcobalamin (AdoCbl) were thermolyzed with excess β-mercaptoethanol under anaerobic conditions. The product studies reveal that ∼90% Co–C bond homolysis occurs, to yield Co(II)cobalamin, 5′-deoxyadenosine, and the disulfide product from the combination of two HOCH 2CH 2S radicals, 2,2′-dithiodiethanol; there is also ∼10% Co–C bond heterolysis, yielding Co(III)cobalamin, adenine, and 2,3-dihydroxy-4-pentenal. The kinetic studies show there is a first-order dependence on AdoCbl and zero-order dependence on thiol under the higher [RSH] experimental conditions used, consistent with the rate-determining step at high [RSH] being the generation of Ado . The kinetic results require that, in enzyme-free AdoCbl solution, adenosyl radical (Ado ) is formed as a discrete intermediate which then abstracts H from the added thiol. The activation parameters for Co–C bond homolysis in the presence of thiol trap are the same within experimental error as the activation parameters for Co–C bond homolysis without trap, Δ H ‡ obs=29(2) kcal mol −1 and Δ S ‡ obs=−1(5) e.u. The results, in comparison to the rate of Co–C bond homolysis in ribonucleoside triphosphate reductase (RTPR), reveal that RTPR accelerates Co–C bond cleavage in AdoCbl by ∼10 10±1. The recent literature evidence bearing on the exact mechanism of RTPR enzymic cleavage of the Co–C bond of AdoCbl is briefly discussed, notably the fact that this mechanism is presently controversial, but does involve at least coupled (and possibly concerted) Co–C cleavage, –S–H cleavage, and C–H (Ado–H) formation steps.