The requirement of reductant for in vitro biosynthesis of the iron-molybdenum cofactor of nitrogenase.

A source of reductant is routinely added to the in vitro iron-molybdenum cofactor (FeMo-co) synthesis assay, although a requirement for reductant has not been established. This report demonstrates that the addition of reductant to the in vitro FeMo-co synthesis system is not required when Azotobacter vinelandii cell-free extract is prepared in buffer that lacks added reductant. The addition of reductant is required, however, if the A. vinelandii cell-free extract is chemically oxidized prior to addition to the assay. These results might suggest that extracts of A. vinelandii contain a physiological source of reductant that functions in the in vitro synthesis of FeMo-co. It is possible that the proteins required for FeMo-co biosynthesis (e.g. NIFNE and dinitrogenase reductase) are at the appropriate redox state to function in the in vitro reaction in the extract that is free of added reductant but not in the chemically oxidized extract. It is also possible that dinitrogenase reductase and/or NIFNE (both Fe-S proteins required for FeMo-co synthesis) might catalyze the reductant-dependent reaction for FeMo-co synthesis. Dithionite, Ti(III) citrate, and NADH are able to serve as the source of reductant for in vitro FeMo-co biosynthesis.