In vitro incorporation of nascent molybdenum cofactor into human sulfite oxidase

We were able to reconstitute molybdopterin (MPT)free sulfite oxidase in vit ro with the molybdenum cofactor (Moco) synthesized de novo from precursor Z and molybdate. MPT-free human sulfite oxidase apoprotein was obtained by h eterologous expression in an Escherichia coil mutant with a defect in the e arly steps of MPT biosynthesis. In vitro reconstitution of the purified apo protein was achieved using an incubation mixture containing purified precur sor Z, purified MPT synthase, and sodium molybdate. In vitro synthesized MP T generated from precursor Z by MPT synthase remains bound to the synthase. Surprisingly, MPT synthase was found capable of donating bound MPT to MPT- free sulfite oxidase. MPT was not released from MPT synthase when either b ovine serum albumin or Moco-containing sulfite oxidase was used in place of aposulfite oxidase. After the inclusion of sodium molybdate in the reconst itution mixture, active sulfite oxidase was obtained, revealing that in vit ro MPT synthase and aposulfite oxidase are sufficient for the insertion of MPT into sulfite oxidase and the conversion of MPT into Moco in the presenc e of high concentrations of molybdate. The conversion of MPT into Moco by m olybdate chelation apparently occurs concomitantly with the insertion of RI FT into sulfite oxidase.