The Requirement of Reductant for in Vitro Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase

Ronda M Allen,Ranjini Chatterjee,Paul W Ludden,Vinod K Shah

doi:10.1074/jbc.271.8.4256

Ronda M Allen, Ranjini Chatterjee + Show 2 more

Open Access

https://doi.org/10.1074/jbc.271.8.4256

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Abstract

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.

Highlights

Nitrogenase is a two-component metalloprotein that catalyzes the conversion of dinitrogen to ammonium [1]
Requirement of Reductant for in Vitro FeMo-co Synthesis— The initial approach employed to address the requirement of reductant for FeMo-co biosynthesis involved preparing two cell-free extracts of A. vinelandii strain UW45 in parallel, one that contained 1.7 mM dependent on the addition of reductant (DTH) and the other that lacked DTH
When added to the FeMo-co synthesis assays from which DTH was excluded during the preincubation phase, both extracts exhibited similar FeMo-co synthesis activities (Table I, compare lines b and d)

Summary

EXPERIMENTAL PROCEDURES

Materials—AG1-X8 and Sephadex G-25 were Pharmacia Biotech Inc. products. Sodium dithionite (Na2S2O4, DTH) was purchased from Fluka. In Vitro FeMo-co Synthesis Assay—9-ml serum vials were flushed with purified argon and rinsed with anoxic 25 mM Tris-HCl (pH 7.4) that contained 1.7 mM DTH. 5 ␮l of the purified NifB-co contained approximately 8.5 nmol of DTH, which is not sufficient to support significant FeMo-co synthesis in assays that contained indigo carmine-oxidized cell-free extract (described below, see Table III). The supernatant was discarded, and the pellet was resuspended in 2 ml of 25 mM Tris-HCl (pH 7.4) that had been sparged with N2 and degassed on a gassing manifold This suspension, which is free of added reductant, was used as a source of FeMo-co. Activation of Apodinitrogenase by FeMo-co (FeMo-co Insertion Assay)—The FeMo-co insertion assays were performed as described previously [5] except that the extract of A. vinelandii strain UW45 was free of DTH and had been oxidized with indigo carmine. NADH and dithiothreitol have midpoint potentials (EoЈ) of Ϫ320 and Ϫ330 mV, respectively [20]

RESULTS AND DISCUSSION

Second addition following preincubation

Activityb nmol