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Journal of Bacteriology, May 1999, p. 2797-2801, Vol. 181, No. 9
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Requirement of NifX and Other nif Proteins for In Vitro Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase

Vinod K. Shah,1,2 Priya Rangaraj,1,2 Ranjini Chatterjee,1,2 Ronda M. Allen,1,2 Jon T. Roll,2,3 Gary P. Roberts,2,3 and Paul W. Ludden1,2,*

Departments of Biochemistry1 and Bacteriology3 and Center for the Study of Nitrogen Fixation,2 College of Agricultural and Life Sciences, University of Wisconsin---Madison, Madison, Wisconsin 53706

Received 26 October 1998/Accepted 22 February 1999

The iron-molybdenum cofactor (FeMo-co) of nitrogenase contains molybdenum, iron, sulfur, and homocitrate in a ratio of 1:7:9:1. In vitro synthesis of FeMo-co has been established, and the reaction requires an ATP-regenerating system, dithionite, molybdate, homocitrate, and at least NifB-co (the metabolic product of NifB), NifNE, and dinitrogenase reductase (NifH). The typical in vitro FeMo-co synthesis reaction involves mixing extracts from two different mutant strains of Azotobacter vinelandii defective in the biosynthesis of cofactor or an extract of a mutant strain complemented with the purified missing component. Surprisingly, the in vitro synthesis of FeMo-co with only purified components failed to generate significant FeMo-co, suggesting the requirement for one or more other components. Complementation of these assays with extracts of various mutant strains demonstrated that NifX has a role in synthesis of FeMo-co. In vitro synthesis of FeMo-co with purified components is stimulated approximately threefold by purified NifX. Complementation of these assays with extracts of A. vinelandii DJ42.48 (Delta nifENX Delta vnfE) results in a 12- to 15-fold stimulation of in vitro FeMo-co synthesis activity. These data also demonstrate that apart from the NifX some other component(s) is required for the cofactor synthesis. The in vitro synthesis of FeMo-co with purified components has allowed the detection, purification, and identification of an additional component(s) required for the synthesis of cofactor.

* Corresponding author. Mailing address: Department of Biochemistry, 433 Babcock Dr., Madison, WI 53706. Phone: (608) 262-6859. Fax: (608) 262-3453. E-mail: ludden{at}biochem.wisc.edu.

Journal of Bacteriology, May 1999, p. 2797-2801, Vol. 181, No. 9
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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