Purification and characterization of the alternative nitrogenase from the photosynthetic bacterium Rhodospirillum rubrum

Abstract

The alternative nitrogenase from a nifH mutant of the photosynthetic bacterium Rhodospirillum rubrum has been purified and characterized. The dinitrogenase protein (ANF1) contains three subunits in an apparent ␣ 2 ␤ 2 ␥ 2 structure and contains Fe but no Mo or V. A factor capable of activating apo-dinitrogenase (lacking the FeMo cofactor) from Azotobacter vinelandii was extracted from the alternative dinitrogenase protein with N-methylformamide. The electron paramagnetic resonance (EPR) signal of the dinitrogenase protein is not characteristic of the EPR signals of molybdenum-or vanadium-containing dinitrogenases. The alternative dinitrogenase reductase (ANF2) was purified as an ␣ 2 dimer containing an Fe 4 S 4 cluster and exhibited an EPR spectrum characteristic of dinitrogenase reductases. The enzyme complex reduces protons to H 2 very well but reduces N 2 to ammonium poorly. Acetylene is reduced to a mixture of ethylene and ethane.

In Vitro Synthesis of the Iron-Molybdenum Cofactor and Maturation of the nif-encoded Apodinitrogenase

“…The Specificity for Molybdenum of the in Vitro FeMo-co Synthesis System-Cofactor structures of the three nitrogenases are proposed to be essentially similar with vanadium and iron atoms replacing the molybdenum atom in FeV-co and FeFe-co, respectively (2,21,40). The requirement of the nifB and nifV gene products for the biosynthesis of all three cofactors suggests that certain steps in the biosynthesis of FeMo-co are shared in the biosynthetic pathways of all three cofactors.…”

Maturation of Nitrogenase: a Biochemical Puzzle

“…Some organisms, like Azotobacter vinelandii, possess all three types of nitrogenases (6,13,56). Other organisms, like Rhodobacter capsulatus and Rhodospirillum rubrum, carry the Mo-containing and Fe-only nitrogenases (17,73), while some organisms, like Anabaena variabilis, carry the Mo-and V-containing nitrogenases (76). Finally, Klebsiella pneumoniae possesses only the Mo-containing nitrogenase (2).…”

Characterization of Diazotrophs Containing Mo-Independent Nitrogenases, Isolated from Diverse Natural Environments

“…Over the years it has become clear that Mo-independent nitrogenases are present in a diverse group of diazotrophic microorganisms which include Clostridium pasteurianum (55), Rhodobacter capsulatus (46,47), Anabaena variabilis (30,50), Rhodospirillum rubrum (11,32), Heliobacterium gestii (31), Azospirillum brasilense (9), Azotobacter salinestris (34), Azotobacter paspali (34), Azomonas macrocytogenes (34), Rhodopseudomonas palustris (39), and Methanosarcina acetivorans (19). Loveless et al (35) demonstrated that diazotrophs with Moindependent nitrogenases were easily isolated from aquatic environments using Mo-deficient, nitrogen-free media under aerobic conditions.…”