Functional NifD-K fusion protein in Azotobacter vinelandii is a homodimeric complex equivalent to the native heterotetrameric MoFe protein Surobhi Lahiri, Lakshmi Pulakat, Nara Gavini * Department of Biological Sciences, Mississippi State University, Mississippi State, MS 39762, USA Received 31 August 2005 Available online 26 September 2005 Abstract The MoFe protein of the complex metalloenzyme nitrogenase folds as a heterotetramer containing two copies each of the homologous a and b subunits, encoded by the nifD and the nifK genes respectively. Recently, the functional expression of a fusion NifD-K protein of nitrogenase was demonstrated in Azotobacter vinelandii, strongly implying that the MoFe protein is flexible as it could accommodate major structural changes, yet remain functional [M.H. Suh, L. Pulakat, N. Gavini, J. Biol. Chem. 278 (2003) 5353–5360]. This finding led us to further explore the type of interaction between the fused MoFe protein units. We aimed to determine whether an interaction exists between the two fusion MoFe proteins to form a homodimer that is equivalent to native heterotetrameric MoFe protein. Using the Bacteriomatch Two-Hybrid System, translationally fused constructs of NifD-K (fusion) with the full-length kCI of the pBT bait vector and also NifD-K (fusion) with the N-terminal a-RNAP of the pTRG target vector were made. To compare the extent of interaction between the fused NifD-K proteins to that of the b–b interactions in the native MoFe protein, we proceeded to generate translationally fused constructs of NifK with the a-RNAP of the pTRG vector and kCI protein of the pBT vector. The strength of the interaction between the proteins in study was determined by measuring the b-galactosidase activity and extent of ampicillin resistance of the colonies expressing these proteins. This analysis demonstrated that direct protein–protein interaction exists between NifD-K fusion proteins, sug- gesting that they exist as homodimers. As the interaction takes place at the b-interfaces of the NifD-K fusion proteins, we propose that these homodimers of NifD-K fusion protein may function in a similar manner as that of the heterotetrameric native MoFe protein. The observation that the extent of protein–protein interaction between the b-subunits of the native MoFe protein in BacterioMatch Two- Hybrid System is comparable to the extent of protein–protein interaction observed between the NifD-K fusion proteins in the same sys- tem further supports this idea. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Nitrogenase; NifD-K fusion protein; Protein–protein interaction; Bacterial two-hybrid The abundant but inert N 2 in the atmosphere is convert- ed to the metabolically useful NH 3 by the metalloenzyme nitrogenase. Although nitrogen fixation is a property of a phylogenetically diverse set of bacteria and cyanobacteria, in general, the sequences, structures, and functional prop- erties of the nitrogenase are highly conserved between dif- ferent organisms [1–3]. The diazotroph Azotobacter vinelandii possesses three classes of nitrogenases [4,5]; these differ in part, by the heterometal atom contained in the ac- tive site metal cluster (Mo, V or Fe) [6–8]. The Mo-depen- dent nitrogenases are the best studied and most widely distributed [7–9]. Much of the understanding of the struc- tural properties of the Mo-dependent nitrogenase was ob- tained through studies on the crystallographic structures of both component proteins, the Fe protein and the MoFe protein of nitrogenase and their metal clusters from A. vin- elandii, Klebsiella pneumoniae, and Clostridium pasteuria- num [10–16]. The Fe protein has a molecular weight of about 60 kDa and is a dimer of identical subunits encoded by the nifH gene. Both subunits are bridged by one 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.09.105 * Corresponding author. Fax: +1 662 325 7939. E-mail address: gavini@biology.msstate.edu (N. Gavini). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 337 (2005) 677–684 BBRC