Analysis of the Cob(II)alamin-5'-deoxy-3',4'-anhydroadenosyl radical triplet spin system in the active site of diol dehydrase.

A common characteristic of AdoCbl1-dependent enzymes is the generation and utilization of enzyme-bound organic radicals (1–3). The Co–C5′ bond of AdoCbl is relatively weak having a bond dissociation energy of ~ 30 kcal mol−1 (4). A prevalent mechanism for AdoCbl-dependent enzymes begins with the homolytic cleavage of the Co–C5′ bond to give cob(II)alamin and the 5′-deoxyadenosyl radical (1). The 5′-deoxyadenosyl radical being an unstabilized, primary alkyl radical is expected to have a short lifetime, low concentration, and a high reactivity towards H atom abstraction. Thus far, these properties of the 5′-deoxyadenosyl radical have precluded direct observation of the radical by EPR spectroscopy. Cleavage of the Co–C5′ bond is kinetically coupled to H atom abstraction from a substrate molecule in the reactions of methylmalonyl-CoA mutase (5), glutamate mutase (6), ethanolamine ammonia-lyase (7), and diol dehydrase (8). These kinetic experiments have highlighted the transient nature of the 5′-deoxyadenosyl radical species. In the case of the AdoCbl-dependent Class II ribonucleoside triphosphate reductase the 5′-deoxyadenosyl radical abstracts an H atom from the thiol of Cys408 of the protein (9). An allosteric effector-dependent epimerization of chiral [5′-2H1]AdoCbl by the C408A and C408S mutant forms of the enzyme requires the transient existence of the 5′-deoxyadenosyl radical (10). DDH (diol dehydrase, EC:4.2.1.28) uses 2- and 3-carbon vicinal diols as substrates and catalyzes their transformation into aldehydes (8, 11). The minimal mechanism for DDH with 1,2-propanediol as the substrate is depicted in Scheme 1. Reversible cleavage of the Co–C5′ bond of AdoCbl generates the 5′-deoxyadenosyl radical a, which abstracts a hydrogen atom from C1 of 1,2-propanediol to produce 5′-deoxyadenosine b and the substrate radical c. The substrate radical undergoes isomerization to the product-related radical d by an unknown mechanism. Hypothetical mechanisms for the radical rearrangement include the formation of a radical cation (12) or a radical anion intermediate (13). Concerted mechanisms having transition state stabilization by partial proton transfer or deprotonation of the spectator −OH group have also been discussed (14, 15). Crystal structures of DDH reveal that the vicinyl −OH groups of 1,2-propanediol are coordinated to K+, and K+ may be involved in catalysis (16, 17). Magnetic interactions between active site radicals and the nuclear spins of 39K and 203,205Tl have, however, escaped detection by EPR and ENDOR (18). Moreover, site directed mutagenesis has revealed essential roles of Glu-α170 and Asp-α335 (19) in putative H-bonding contacts with the vicinal −OH groups of the substrate. Hydrogen transfer from 5′-deoxyadenosine b to the radical d regenerates the 5′-deoxyadenosyl radical, which recombines with cob(II)alamin. The enzyme catalyzes the dehydration of the gem-diol to complete the reaction (20). Reactions of the DDH-AdoCbl complex with substrates or inhibitors elicit EPR signals corresponding to organic radicals spin coupled to the low spin Co2+ of cob(II)alamin (18, 21–26). Scheme 1 AnAdoCbl, shown in Scheme 2, is an analog of AdoCbl. AnAdoCbl has a Co–C5′ bond dissociation energy which is ~ 6 kcal mol−1 weaker than the corresponding bond in AdoCbl (27). The weakened Co–C5′ bond in anAdoCbl is attributed to allylic stabilization in the anhydroadenosyl radical relative to the 5′-deoxyadenosyl radical. Structures of the two radicals are compared in Scheme 3. AnAdoCbl is a functional coenzyme for DDH, displaying a turnover rate 0.02% of that with AdoCbl as coenzyme (28). Binding of anAdoCbl to DDH induces Co–C5′ bond cleavage. The Co–C5′ bond cleavage is observed spectrophotometrically as the formation of cob(II)alamin either in the presence or absence of the substrate (28). However, the enzyme undergoes slow inactivation as observed by the formation of cob(III)alamin, irrespective of the presence of the substrate. Inactivation is a consequence of electron transfer from cob(II)alamin to the anhydroadenosyl radical and subsequent quenching of the allylic anion by a solvent derived proton (28). Scheme 2 Scheme 3 Cleavage of the Co–C5′ bond of anAdoCbl generates the anhydroadenosyl radical and cob(II)alamin in the active site of DDH. The present paper presents EPR evidence for the presence of strongly-coupled spin-triplets involving the anhydroadenosyl radical and the low spin Co2+ of cob(II)alamin in complexes of DDH with the cofactor analog in the presence and absence of substrate. Analysis of the spin-triplet EPR spectra by simulation and interpretation of the ZFS in terms of molecular geometries is presented.