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 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 3d_z2-based molecular orbital.