Nitrogenase iron-molybdenum cofactor binding site: Protein conformational changes associated with cofactor binding

doi:10.1016/S0040-4020(97)00710-2

Tetrahedron

Volume 53, Issue 35, 1 September 1997, Pages 11971-11984

https://doi.org/10.1016/S0040-4020(97)00710-2 Get rights and content

Abstract

Formation of active nitrogenase MoFe-protein requires the assembly and insertion of a unique molybdenum containing Fe:S cluster, the FeMoco. The protein acceptor is held in an open conformation by the binding of a small molecular weight protein, γ. Using selective alkylation of the protein cysteines, conformational changes associated with γ binding and cofactor insertion were evaluated and a model for the change presented. Reversible oxygen damage to the precursor is identified and evaluated in terms of the chemistry of dithionite used in the buffer as an oxygen scavenger.

Active nitrogenase MoFe-protein requires the assembly and insertion of an unusual molybdenum containing Fe:S cluster, the FeMoco.

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Sequential and differential interaction of assembly factors during nitrogenase MoFe protein maturation
2018, Journal of Biological Chemistry
Citation Excerpt :
In contrast to Strep-tagged MoFe protein produced by a ΔnifH strain, two different proteins co-purify with Strep-tagged MoFe protein produced by a ΔnifB strain (Fig. 5). These proteins, NafY and NifY, share primary structure similarity to each other and have been variously proposed to act as “molecular props” that assist FeMo-cofactor insertion or as FeMo-cofactor–trafficking proteins (33–39). These possibilities are not mutually exclusive, and indeed NafY has already been shown to co-purify with apo-MoFe protein produced by a NifB-deficient strain (33, 35).
Nitrogenases reduce atmospheric nitrogen, yielding the basic inorganic molecule ammonia. The nitrogenase MoFe protein contains two cofactors, a [7Fe-9S-Mo-C-homocitrate] active-site species, designated FeMo-cofactor, and a [8Fe-7S] electron-transfer mediator called P-cluster. Both cofactors are essential for molybdenum-dependent nitrogenase catalysis in the nitrogen-fixing bacterium Azotobacter vinelandii. We show here that three proteins, NafH, NifW, and NifZ, copurify with MoFe protein produced by an A. vinelandii strain deficient in both FeMo-cofactor formation and P-cluster maturation. In contrast, two different proteins, NifY and NafY, copurified with MoFe protein deficient only in FeMo-cofactor formation. We refer to proteins associated with immature MoFe protein in the following as “assembly factors.” Copurifications of such assembly factors with MoFe protein produced in different genetic backgrounds revealed their sequential and differential interactions with MoFe protein during the maturation process. We found that these interactions occur in the order NafH, NifW, NifZ, and NafY/NifY. Interactions of NafH, NifW, and NifZ with immature forms of MoFe protein preceded completion of P-cluster maturation, whereas interaction of NafY/NifY preceded FeMo-cofactor insertion. Because each assembly factor could independently bind an immature form of MoFe protein, we propose that subpopulations of MoFe protein–assembly factor complexes represent MoFe protein captured at different stages of a sequential maturation process. This suggestion was supported by separate isolation of three such complexes, MoFe protein–NafY, MoFe protein–NifY, and MoFe protein–NifW. We conclude that factors involved in MoFe protein maturation sequentially bind and dissociate in a dynamic process involving several MoFe protein conformational states.
A sterile α-motif domain in NafY targets apo-NifDK for iron-molybdenum cofactor delivery via a tethered domain
2011, Journal of Biological Chemistry
Citation Excerpt :
Because the surface of n-NafY displays a large negatively charged region (supplemental Fig. S3), it is possible that n-NafY would bind between the positively charged insertion funnel and the disordered region of apo-NifDK (NifD residues 381–407) that would act as a lid (29). Selective alkylation studies have shown that NafY-stabilized apo-NifDK has the side chain of α-Cys275 exposed to solvent (α-Cys275 serves as ligand to FeMo-co) (28). Fourth, it could also be possible that n-NafY increases the KD of the FeMo-co·apo-NifDK complex.
NafY participates in the final steps of nitrogenase maturation, having a dual role as iron-molybdenum cofactor (FeMo-co) carrier and as chaperone to the FeMo-co-deficient apo-NifDK (apo-dinitrogenase). NafY contains an N-terminal domain of unknown function (n-NafY) and a C-terminal domain (core-NafY) necessary for FeMo-co binding. We show here that n-NafY and core-NafY have very weak interactions in intact NafY. The NMR structure of n-NafY reveals that it belongs to the sterile α-motif (SAM) family of domains, which are frequently involved in protein-protein interactions. The presence of a SAM domain in NafY was unexpected and could not be inferred from its amino acid sequence. Although SAM domains are very commonly found in eukaryotic proteins, they have rarely been identified in prokaryotes. The n-NafY SAM domain binds apo-NifDK. As opposed to full-length NafY, n-NafY impaired FeMo-co insertion when present in molar excess relative to FeMo-co and apo-NifDK. The implications of these observations are discussed to offer a plausible mechanism of FeMo-co insertion. NafY domain structure, molecular tumbling, and interdomain motion, as well as NafY interaction with apo-NifDK are consistent with the function of NafY in FeMo-co delivery to apo-NifDK.
Comparison of iron-molybdenum cofactor-deficient nitrogenase MoFe proteins by x-ray absorption spectroscopy. Implications for P-cluster biosynthesis
2004, Journal of Biological Chemistry
Citation Excerpt :
This result helps define the role of the Fe protein (NifH) in MoFe biosynthesis. It is presumed that the Fe protein is involved in forming the open conformation of the MoFe protein that accepts preassembled FeMoco (67). The protein-protein interaction that leads to this conformational change could be driven by, or could drive, the condensation of two [Fe4S4] centers in “apoMoFe protein” to form the P-cluster.
Nitrogenase, the enzyme system responsible for biological nitrogen fixation, is believed to utilize two unique metalloclusters in catalysis. There is considerable interest in understanding how these metalloclusters are assembled in vivo. It has been presumed that immature iron-molybdenum cofactor-deficient nitrogenase MoFe proteins contain the P-cluster, although no biosynthetic pathway for the assembly of this complex cluster has been identified as yet. Through the comparison by iron K-edge x-ray absorption edge and extended fine structure analyses of cofactor-deficient MoFe proteins resulting from nifH and nifB deletion strains of Azotobacter vinelandii, a novel [Fe-S] cluster is identified in the ΔnifH MoFe protein. The iron-iron scattering displayed by the ΔnifH MoFe protein is more similar to that of a standard [Fe₄S₄]-containing protein than that of the ΔnifB MoFe protein, which is shown to contain a “normal” P-cluster. The iron-sulfur scattering of the ΔnifH MoFe protein, however, indicates differences in its cluster from an [Fe₄S₄](Cys)₄ site that may be consistent with the presence of either oxygenic or nitrogenic ligation. Based on these results, models for the [Fe-S] center in the ΔnifH MoFe protein are constructed, the most likely of which consist of two separate [Fe₄S₄] sites, each with some non-cysteinyl coordination. This type of model suggests that the P-cluster is formed by the condensation of two [Fe₄S₄] fragments, possibly concomitant with Fe protein (NifH)-induced conformational change.
Purification and Characterization of NafY (Apodinitrogenase γ Subunit) from Azotobacter vinelandii
2004, Journal of Biological Chemistry
The formation of an active dinitrogenase requires the synthesis and the insertion of the iron-molybdenum cofactor (FeMo-co) into a presynthesized apodinitrogenase. In Azotobacter vinelandii, NafY (also known as γ protein) has been proposed to be a FeMo-co insertase because of its ability to bind FeMo-co and apodinitrogenase. Here we report the purification and biochemical characterization of NafY and reach the following conclusions. First, NafY is a 26-kDa monomeric protein that binds one molecule of FeMo-co with very high affinity (K_d ≈ 60 nm); second, the NafY-FeMo-co complex exhibits a S = 3/2 EPR signal with features similar to the signals for extracted FeMo-co and the M center of dinitrogenase; third, site-directed mutagenesis of nafY indicates that the His¹²¹ residue of NafY is involved in cofactor binding; and fourth, NafY binding to apodinitrogenase or to FeMo-co does not require the presence of any additional protein. In addition, we have obtained evidence that suggests the ability of NafY to bind NifB-co, an FeS cluster of unknown structure that is a biosynthetic precursor to FeMo-co.
Structure, Function, and Biosynthesis of the Metallosulfur Clusters in Nitrogenases
1999, Advances in Inorganic Chemistry
This chapter focuses on the structure, biosynthesis, and function of the metallosulfur clusters found in nitrogenases. The chapter focuses on the recent advances and attempts to describe the present level of understanding. Molybdenum nitrogenase has been the subject of intensive study for many more decades, but much less work has been done on the vanadium and iron-only nitrogenases. The chapter discusses the properties of Mo nitrogenase and outlines what is known of the other two enzymes. The solutions of the structures of the Fe protein, the MoFe proteins, and the putative transition-state complex of these two proteins with adenosine diphosphate (ADP) and AlF^4- represent major advances in our understanding of nitrogenase, but still many questions remain. A great deal has been learned about the biosynthesis of nitrogenases, but the process is understood only in broad outline. The detailed roles of the individual gene products require much further investigation that may once more indicate fresh approaches to some of the problems identified.
Biosynthesis of Nitrogenase Cofactors
2020, Chemical Reviews

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