Cloning and Mutational Analysis of the γ Gene fromAzotobacter vinelandii Defines a New Family of Proteins Capable of Metallocluster Binding and Protein Stabilization

Abstract

Dinitrogenase is a heterotetrameric (␣ 2 ␤ 2 ) enzyme that catalyzes the reduction of dinitrogen to ammonium and contains the iron-molybdenum cofactor (FeMo-co) at its active site. Certain Azotobacter vinelandii mutant strains unable to synthesize FeMo-co accumulate an apo form of dinitrogenase (lacking FeMo-co), with a subunit composition ␣ 2 ␤ 2 ␥ 2 , which can be activated in vitro by the addition of FeMo-co. The ␥ protein is able to bind FeMo-co or apodinitrogenase independently, leading to the suggestion that it facilitates FeMo-co insertion into the apoenzyme. In this work, the non-nif gene encoding the ␥ subunit (nafY) has been cloned, sequenced, and found to encode a NifY-like protein. This finding, together with a wealth of knowledge on the biochemistry of proteins involved in FeMo-co and FeV-co biosyntheses, allows us to define a new family of iron and molybdenum (or vanadium) cluster-binding proteins that includes NifY, NifX, VnfX, and now ␥. In vitro FeMo-co insertion experiments presented in this work demonstrate that ␥ stabilizes apodinitrogenase in the conformation required to be fully activable by the cofactor. Supporting this conclusion, we show that strains containing mutations in both nafY and nifX are severely affected in diazotrophic growth and extractable dinitrogenase activity when cultured under conditions that are likely to occur in natural environments. This finding reveals the physiological importance of the apodinitrogenase-stabilizing role of which both proteins are capable. The relationship between the metal cluster binding capabilities of this new family of proteins and the ability of some of them to stabilize an apoenzyme is still an open matter.Nitrogenase catalyzes the reduction of nitrogen gas to ammonium, in an ATP-and reductant-dependent reaction. It is one of the best characterized metalloenzymes and is an excellent model for elucidating metalloprotein assembly. Nitrogenase is composed of two oxygen-labile metalloproteins: dinitrogenase and dinitrogenase reductase (1, 2). Dinitrogenase (also termed component I or molybdenum-iron protein) is a 240-kDa ␣ 2 ␤ 2 tetramer of the nifD and nifK gene products (3). Each ␣␤ nitrogenase dimer contains an iron-molybdenum cofactor (FeMo-co) 1 and a P cluster (3, 4). Dinitrogenase reductase (also termed component II or iron protein) is a 60-kDa ␣ 2 dimer of the nifH gene product which contains a single 4Fe-4S center coordinated between the two subunits (5). NifH has at least three roles in the nitrogenase enzyme system (6): first, it serves as electron donor to nitrogenase; second, it participates in the biosynthesis of FeMo-co; and third, it is required for maturation of apodinitrogenase to a FeMo-co-activable form.

Nitrogenase reactivity with P-cluster variants

“…This observation, in combination with results from quantitative analysis of the SDS͞PAGE and N-terminal amino acid sequencing (data not shown), indicates a subunit composition of ␣ 2 ␤ 2 for ⌬nifH Av1Ј and ␣␤ 2 k for ⌬nifB Av1 V (Table 2). Additional small subunits, encoded by nafY and vnfG, have been reported to be associated with various species of Av1 and Av1 V , respectively (23,27,45,46). However, no additional subunits are detectable in ⌬nifH Av1Ј or ⌬nifB Av1 V based on SDS͞PAGE (Fig.…”

Accumulation of 99Mo-containing Iron-Molybdenum Cofactor Precursors of Nitrogenase on NifNE, NifH, and NifX ofAzotobacter vinelandii

“…Homer et al (30) have shown that NafY binds FeMo-co specifically and have postulated a role for NafY in the cofactor insertion process. However, Rubio et al (22) have shown that nafY mutants exhibit a nif phenotype only under stress conditions. One interpretation of their data is that NafY is required for the stability of apodinitrogenase in an open conformation that is favorable to the insertion of FeMo-co.…”

The NifX Protein is Involved in the Final Stages of FeMo-cofactor Transport to the MoFe Protein

“…al in 2002 [25] . The A. vinelandii NafY protein (nitrogenase accessory factor Y, also known as γ  , was reported to be a molecular chaperone that assisted in maintaining the apodinitrogenase in a conformation that facilitated the insertion of the FeMoco by acting as a metalloinsertase [25] .…”

“…The A. vinelandii NafY protein (nitrogenase accessory factor Y, also known as γ  , was reported to be a molecular chaperone that assisted in maintaining the apodinitrogenase in a conformation that facilitated the insertion of the FeMoco by acting as a metalloinsertase [25] . Mutational analysis of the strains containing mutations in both nafY and nifX showed that these were severely affected in diazotrophic growth, indicating their physiological importance in the stabilization of the apodinitrogenase [25] . Subsequently, the 3-dimensional structure of the NafY protein from A. vinelandii revealed two distinct domains of this protein:…”

“…This application proved immensely useful in recognizing NifH, NifEN and NifV as some of the key players in the FeMoco biosynthetic pathway and helped in the purification of an apo-MoFe protein [13][14][15][16][17] . As understood from research conducted thus far, a stepwise overview of the events involved in the biosynthesis and assembly of the FeMoco includes: (i) the formation of an Fe/S core of the FeMoco (designated NifB-co) by NifB [18,19] (ii) transfer of the NifB-co to the α 2 β 2 tetrameric scaffold NifEN protein [20,21] (iii) maturation of the FeMoco and apo-MoFe protein by the combined action of the Fe protein and MgATP [17,22,23] and further processing of the NifB-co on NifEN by an unknown mechanism, forming the completed FeMoco (iv) the final transfer of the fully formed FeMoco to a nafYencoded protein, called γ  which is a FeMoco carrier that aids in the insertion of the FeMoco into the apoMoFe [24,25] . In addition to these major events, a few other important steps are also involved in the entire process.The NifU and NifS proteins participate in the initial Fe/S mobilization [26,27] , the NifQ protein is involved in an early stage of the metal core formation and found to be essential in Mo-limiting conditions [28] , the NifV protein contributes the homocitrate component of the FeMoco [29] and the NifX protein probably functions as an 'escort' protein that delivers the FeMoco or its precursors from one assembly site to another or plays a role in specifying the organic acid moiety of FeMoco [30,31] .…”