Incorporation of Molybdenum into the Iron-Molybdenum Cofactor of Nitrogenase

Ronda M Allen,Jon T Roll,Priya Rangaraj,Vinod K Shah,Gary P Roberts,Paul W Ludden

doi:10.1074/jbc.274.22.15869

Ronda M Allen, Jon T Roll + Show 4 more

Open Access

https://doi.org/10.1074/jbc.274.22.15869

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Abstract

The biosynthesis of the iron-molybdenum cofactor (FeMo-co) of dinitrogenase was investigated using 99Mo to follow the incorporation of Mo into precursors. 99Mo label accumulates on dinitrogenase only when all known components of the FeMo-co synthesis system, NifH, NifNE, NifB-cofactor, homocitrate, MgATP, and reductant, are present. Furthermore, 99Mo label accumulates only on the gamma protein, which has been shown to serve as a chaperone/insertase for the maturation of apodinitrogenase when all known components are present. It appears that only completed FeMo-co can accumulate on the gamma protein. Very little FeMo-co synthesis was observed when all known components are used in purified forms, indicating that additional factors are required for optimal FeMo-co synthesis. 99Mo did not accumulate on NifNE under any conditions tested, suggesting that Mo enters the pathway at some other step, although it remains possible that a Mo-containing precursor of FeMo-co that is not sufficiently stable to persist during gel electrophoresis occurs but is not observed. 99Mo accumulates on several unidentified species, which may be the additional components required for FeMo-co synthesis. The molybdenum storage protein was observed and the accumulation of 99Mo on this protein required nucleotide.

Highlights

The iron-molybdenum cofactor (FeMo-co)1 of dinitrogenase (Fig. 1) constitutes the active site of the nif-encoded, molybdenum-containing dinitrogenase protein in Azotobacter vinelandii and other nitrogen-fixing organisms [1,2,3]
99Mo Is Detected on the Molybdenum Storage Protein—When 99Mo-labeled molybdate is supplied to extracts capable of FeMo-co synthesis, two proteins become prominently labeled (Fig. 2, lane 2)
molybdenum storage protein (Mo-Sto) is not co-regulated with nitrogenase, and the protein purified as an ␣2␤2 tetramer of 21 and 24 kDa subunits with approximately 15 Mo atoms/ tetramer, the Mo content of the protein decreased during purification

Summary

Introduction

The iron-molybdenum cofactor (FeMo-co)1 of dinitrogenase (Fig. 1) constitutes the active site of the nif-encoded, molybdenum-containing dinitrogenase protein in Azotobacter vinelandii and other nitrogen-fixing organisms [1,2,3]. Two hundred ␮l of the appropriate A. vinelandii cell-free extract, 10 ␮l of a solution containing an excess of NifB-co as Fe and S donor for FeMo-co [16], and 0.1 mg of purified dinitrogenase reductase (10 ␮l) were added to the reaction mixtures.

Results

Conclusion