THE PROCESSING OF MOLYBDENUM DURING THE SYNTHESIS OF THE MOLYBDENUM-IRON PROTEIN OF NITROGENASE

The accumulation of (\u2799)Mo (from (\u2799)MoO(,4)(\u27=)) into molybdenum-containing species in Clostridium pasteurianum was investigated to determine the molybdoprotein(s) involved in Mo metabolism. Anaerobic agarose electrofocusing was not amendable to study all molybdoproteins. Anaerobic multiphasic gel electrophoresis identified seven distinct (\u2799)Mo-zones. Five of the (\u2799)Mo-labeled species are molybdoproteins; membrane fragments, MoFe protein, CO(,2) reductase, Mo binding-storage protein, and a 30 KD molybdoprotein. The MoFe protein, CO(,2) reductase, and Mo binding-storage protein exhibited electrophoretic heterogeneity because of complex formation, denaturation and amount of Mo bound to the protein, respectively. The other two (\u2799)Mo-species were identified as free Mo-co and MoO(,4)(\u27=). Cells grown with NH(,3) as a nitrogen source accumulated (\u2799)Mo primarily in the membrane, free Mo-co and MoO(,4)(\u27=). Mo accumulation by clostridial cells during the derepression of the nitrogenase system increased 1 1/2 hours before nitrogenase activity was detected. The increase in Mo accumulation by the cells is a result of Mo being incorporated into membrane fragments, free Mo-co, Mo binding-storage protein, 30 KD molybdoprotein and CO(,2) reductase. Mo incorporation into the MoFe protein was detected one hour after the onset of metal uptake. Kinetics of Mo accumulation into the Mo-species during the derepression of nitrogenase suggests Mo incorporation occurs in the following sequence: (a) membrane fragments and MoO(,4)(\u27=), (b) free Mo-co, (c) Mo binding-storage protein and 30 KD molybdoprotein, (d) CO(,2) reductase, and (e) the MoFe protein. Clostridial cells grown in the presence of limiting amounts of Mo become Mo-deficient as a result of growth. A MoO(,4) supplement added to Mo-deficient cells rapidly accumulates within the cells to levels five times that found in steady-state nitrogen-fixing cells. The Mo accumulated by the Mo-deficient cells is rapidly incorporated into preformed demolybdoproteins in the absence of de novo protein synthesis. The increase in Mo accumulation by Mo-deficient cells was a result of an increase in all molybdoproteins except the MoFe protein. The intracellular level of all Mo-species except nitrogenase is influenced by the availability of Mo