Kinetic and spectroscopic studies of the ATP:: Corrinoid adenosyltransferase PduO from Lactobacillus reuteri:: Substrate specificity and insights into the mechanism of Co(II)corrinoid reduction

The PduO-type ATP:corrinoid adenosyltransferase from Lactobacillus reitteri (LrPduO) catalyzes the formation of the essential Co-C bond of adenosylcobalamin (coenzyme B-12) 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 Collcobalamin (Co(1+)Cbi), our kinetic data obtained in this study provide in vitro evidence that LrPduO can in fact also utilize the incomplete corrinoid Co1+ cobinamide (Co(1+)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 Co(2+)Cbl and Co(2+)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 (similar to 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 outprevious 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 3d(Z)(2)-based molecular orbital.