Dihydroflavin-driven adenosylation of 4-coordinate Co(II) corrinoids: are cobalamin reductases enzymes or electron transfer proteins?

The identity of the source of the biological reductant needed to convert cobalamin to its biologically active form adenosylcobalamin has remained elusive. Here we show that free or protein-bound dihydroflavins can serve as the reductant of Co(2+)Cbl bound in the active site of PduO-type ATP-dependent corrinoid adenosyltransferase enzymes. Free dihydroflavins (dihydroriboflavin, FMNH(2), and FADH(2)) effectively drove the adenosylation of Co(2+)Cbl by the human and bacterial PduO-type enzymes at very low concentrations (1 microm). These data show that adenosyltransferase enzymes lower the thermodynamic barrier of the Co(2+) --> Co(+) reduction needed for the formation of the unique organometalic Co-C bond of adenosylcobalamin. Collectively, our in vivo and in vitro data suggest that cobalamin reductases identified thus far are most likely electron transfer proteins, not enzymes.