The PduO-type ATP:corrinoid adenosyltransferase from Lactobacillus reuteri (LrPduO) catalyzes the formation of the essential Co-C bond of adenosylcobalamin (coenzyme B12) by transferring the adenosyl group from cosubstrate ATP to a transient Co1+corrinoid species generated in the enzyme active site. While PduO-type enzymes have previously been believed to be capable of adenosylating only Co1+cobalamin (Co1+Cbl -), our kinetic data obtained in this study provide in vitro evidence that LrPduO can in fact also utilize the incomplete corrinoid Co 1+cobinamide (Co1+Cbi) as an alternative substrate. To explore the mechanism by which LrPduO overcomes the thermodynamically challenging reduction of its Co2+corrinoid substrates, we have examined how the enzyme active site alters the geometric and electronic properties of Co2+Cbl and Co2+Cbi+ by using electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopic techniques. Our data reveal that upon binding to LrPduO that was preincubated with ATP, both Co2+corrinoids undergo a partial (∼40-50%) conversion to distinct paramagnetic Co2+ species. The spectroscopic signatures of these species are consistent with essentially four-coordinate, square-planar Co2+ complexes, based on a comparison with the results obtained in our previous studies of related enzymes. Consequently, it appears that the general strategy employed by adenosyltransferases for effecting Co2+ → Co1+ reduction involves the formation of an "activated" Co 2+corrinoid intermediate that lacks any significant axial bonding interactions, to stabilize the redox-active, Co 3dz2-based molecular orbital.
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