nifU Gene Product from Azotobacter vinelandii Is a Homodimer That Contains Two Identical [2Fe-2S] Clusters

Abstract

The nifU gene product is required for the full activation of the metalloenzyme nitrogenase, the catalytic component of biological nitrogen fixation. In the present work, a hybrid plasmid that contains the Azotobacter vinelandii nifU gene was constructed and used to hyperexpress the NIFU protein in Escherichia coli. Recombinant NIFU was purified to homogeneity and was found to be a homodimer of 33-kDa subunits with approximately two Fe atoms per subunit. The combination of UV/visible absorption, variable-temperature magnetic circular dichroism, EPR, and resonance Raman spectroscopies shows the presence of a [2Fe-2S]2+,+ center (Em = -254 mV) with complete cysteinyl coordination in each subunit. The electronic, magnetic, and vibrational properties of the [2Fe-2S]2+,+ center do not conform to those established for any of the spectroscopically distinct types of 2Fe ferredoxins. These distinctive properties appear to be a consequence of a novel arrangement of coordinating cysteinyl residues in NIFU, and the residues likely to be involved in cluster coordination are discussed in light of primary sequence comparisons to other putative [2Fe-2S] proteins. The observed physicochemical properties of NIFU and its constituent [2Fe-2S] cluster also provide insight into the role of this protein in nitrogenase metallocluster biosynthesis.

Iron-Sulfur Cluster Assembly

“…This strain is not further impaired in diazotrophic growth, so it seems unlikely that Nif IscA significantly contributes to nitrogenase maturation under the conditions used in the present work. 1 Another possibility is that, as previously suggested, the isc-encoded system could make some very minor contributions to nitrogenase maturation, but this possibility has not been tested because deletion of the A. vinelandii isc operon is lethal (5). Yet another possibility is that an A. vinelandii gene, which we designate nfuA, that encodes a product bearing primary sequence homology to the C-terminal domain of NifU could contribute to nitrogenase-specific Fe-S cluster biogenesis in the absence of intact NifU function.…”

“…The Azotobacter vinelandii NifU protein is an ϳ60-kDa homodimer proposed to provide a molecular scaffold for formation of [Fe-S] clusters or Fe-S cluster precursors required for full activation of the nitrogenase catalytic components (1,2). Primary sequence comparisons among NifU homologs indicate that it is a modular protein having three distinct domains (3,4).…”

Contributions of the LPPVK Motif of the Iron-Sulfur Template Protein IscU to Interactions with the Hsc66-Hsc20 Chaperone System

“…Effect of the V102E Substitution on IscU Interactions with Hsc66 and Hsc20 -In addition to IscU, diazotrophpic organisms contain a second iron-sulfur template protein, NifU, that functions in the assembly of the nitrogenase protein (28,29). The N-terminal regions of NifU proteins display sequence homology to IscU proteins, but there are no known chaperones associated with iron-sulfur cluster assembly specific to the nitrogen fixation machinery (5,12).…”

Subcellular compartmentalization of human Nfu, an iron–sulfur cluster scaffold protein, and its ability to assemble a [4Fe–4S] cluster

“…Enzymes involved in Fe-S cluster assembly were first identified in the nif (nitrogen fixation) operon in Azotobacter vinelandii (2). Genetic and biochemical studies have shown that two gene products, NifS and NifU, are essential for assembly of the Fe-S clusters in nitrogenase (3)(4)(5)(6). Subsequent studies in a wide variety of prokaryotes resulted in the identification of the isc (iron-sulfur cluster assembly) operon (7,8), which encodes the translational regulator IscR (9), cysteine desulfurase IscS (7), scaffold proteins IscU (10) and IscA (11,12), chaperone proteins HscA and HscB (13), and ferredoxin Fdx.…”