Assembly scaffold NifEN: A structural and functional homolog of the nitrogenase catalytic component.

NifEN is a biosynthetic scaffold for the cofactor of Mo-nitrogenase (designated the M-cluster). Previous studies have revealed the sequence and structural homology between NifEN and NifDK, the catalytic component of nitrogenase. However, direct proof for the functional homology between the two proteins has remained elusive. Here we show that, upon maturation of a cofactor precursor (designated the L-cluster) on NifEN, the cluster species extracted from NifEN is spectroscopically equivalent and functionally interchangeable with the native M-cluster extracted from NifDK. Both extracted clusters display nearly indistinguishable EPR features, X-ray absorption spectroscopy/extended X-ray absorption fine structure (XAS/EXAFS) spectra and reconstitution activities, firmly establishing the M-cluster-bound NifEN (designated NifEN(M)) as the only protein other than NifDK to house the unique nitrogenase cofactor. Iron chelation experiments demonstrate a relocation of the cluster from the surface to its binding site within NifEN(M) upon maturation, which parallels the insertion of M-cluster into an analogous binding site in NifDK, whereas metal analyses suggest an asymmetric conformation of NifEN(M) with an M-cluster in one αß-half and an empty cluster-binding site in the other αß-half, which led to the proposal of a stepwise assembly mechanism of the M-cluster in the two αß-dimers of NifEN. Perhaps most importantly, NifEN(M) displays comparable ATP-independent substrate-reducing profiles to those of NifDK, which establishes the M-cluster-bound αß-dimer of NifEN(M) as a structural and functional mimic of one catalytic αß-half of NifDK while suggesting the potential of this protein as a useful tool for further investigations of the mechanistic details of nitrogenase.

Identification and characterization of functional homologs of nitrogenase cofactor biosynthesis protein NifB from methanogens.

Nitrogenase biosynthesis protein NifB catalyzes the radical S-adenosyl-L-methionine (SAM)-dependent insertion of carbide into the M cluster, the cofactor of the molybdenum nitrogenase from Azotobacter vinelandii. Here, we report the identification and characterization of two naturally "truncated" homologs of NifB from Methanosarcina acetivorans (NifB(Ma)) and Methanobacterium thermoautotrophicum (NifB(Mt)), which contain a SAM-binding domain at the N terminus but lack a domain toward the C terminus that shares homology with NifX, an accessory protein in M cluster biosynthesis. NifB(Ma) and NifB(Mt) are monomeric proteins containing a SAM-binding [Fe4S4] cluster (designated the SAM cluster) and a [Fe4S4]-like cluster pair (designated the K cluster) that can be processed into an [Fe8S9] precursor to the M cluster (designated the L cluster). Further, the K clusters in NifB(Ma) and NifB(Mt) can be converted to L clusters upon addition of SAM, which corresponds to their ability to heterologously donate L clusters to the biosynthetic machinery of A. vinelandii for further maturation into the M clusters. Perhaps even more excitingly, NifB(Ma) and NifB(Mt) can catalyze the removal of methyl group from SAM and the abstraction of hydrogen from this methyl group by 5'-deoxyadenosyl radical that initiates the radical-based incorporation of methyl-derived carbide into the M cluster. The successful identification of NifB(Ma) and NifB(Mt) as functional homologs of NifB not only enabled classification of a new subset of radical SAM methyltransferases that specialize in complex metallocluster assembly, but also provided a new tool for further characterization of the distinctive, NifB-catalyzed methyl transfer and conversion to an iron-bound carbide.

Uncoupling binding of substrate CO from turnover by vanadium nitrogenase.

Biocatalysis by nitrogenase, particularly the reduction of N2 and CO by this enzyme, has tremendous significance in environment- and energy-related areas. Elucidation of the detailed mechanism of nitrogenase has been hampered by the inability to trap substrates or intermediates in a well-defined state. Here, we report the capture of substrate CO on the resting-state vanadium-nitrogenase in a catalytically competent conformation. The close resemblance of this active CO-bound conformation to the recently described structure of CO-inhibited molybdenum-nitrogenase points to the mechanistic relevance of sulfur displacement to the activation of iron sites in the cofactor for CO binding. Moreover, the ability of vanadium-nitrogenase to bind substrate in the resting-state uncouples substrate binding from subsequent turnover, providing a platform for generation of defined intermediate(s) of both CO and N2 reduction.

Assembly of nitrogenase MoFe protein.

Biosynthesis of MoFe protein and, particularly, that of its associated P-cluster and FeMoco has raised a significant amount of interest because of the biological importance and chemical exclusiveness of these unique clusters. Following a brief introduction to the properties of Azotobacter vinelandii MoFe protein, this chapter will focus on the recent progress toward understanding the assembly mechanism of MoFe protein, with an emphasis on studies that provide important structural or spectroscopic insights into this process.

Purification of nitrogenase proteins.

Nitrogenase is one of the most complex enzymes known to date. The extensively studied molybdenum nitrogenase consists of two protein components and three metal centers that are critical for nitrogenase activity. The inherent complexity of this enzyme system, which is further compounded by the sensitivity of the metal clusters toward oxygen, makes the large-scale purification of fully active nitrogenase proteins a formidable task. This chapter highlights several methods that have been developed for the purification of nitrogenase proteins over the past few decades. Techniques used include weak anion exchange chromatography, size exclusion chromatography, and immobilized metal affinity chromatography. These methods can be selectively applied to nitrogenase variants and other related proteins.

Protocols for cofactor isolation of nitrogenase.

The iron-molybdenum cofactor (FeMoco) of the nitrogenase MoFe protein has remained a focal point in the field of bioinorganic chemistry for decades. This unique metal cluster has long been regarded as the actual site of dinitrogen reduction, and it is structurally complex and chemically unprecedented. A detailed characterization of the isolated FeMoco is crucial for elucidating the physiochemical properties of this biologically important cofactor. Such a study requires an effective technique to extract FeMoco intact, and in high yield, from the MoFe protein. A method involving the acid treatment of the MoFe protein and the subsequent extraction of FeMoco into an organic solvent was developed over 30 years ago and has been improved upon ever since. FeMoco isolated by this strategy is catalytically active and spectrally interesting, which provides a useful platform for future structure-function analyses of this unique cofactor. A general working protocol for FeMoco isolation is described in this chapter, along with some of the major modifications reported in the past years.

Variable-temperature, variable-field magnetic circular dichroism spectroscopic study of NifEN-bound precursor and "FeMoco".

NifEN plays a key role in the biosynthesis of the iron-molybdenum cofactor (FeMoco) of nitrogenase. A scaffold protein that hosts the conversion of a FeMoco precursor to a mature cofactor, NifEN can assume three conformations during the process of FeMoco maturation. One, designated ΔnifB NifEN, contains only two permanent [Fe(4)S(4)]-like clusters. The second, designated NifEN(Precursor), contains the permanent clusters and a precursor form of FeMoco. The third, designated NifEN("FeMoco"), contains the permanent [Fe(4)S(4)]-like clusters and a fully complemented, "FeMoco"-like structure. Here, we report a variable-temperature, variable-field magnetic circular dichroism spectroscopic investigation of the electronic structure of the metal clusters in the three forms of dithionite-reduced NifEN. Our data indicate that the permanent [Fe(4)S(4)]-like clusters are structurally and electronically conserved in all three NifEN species and exhibit spectral features of classic [Fe(4)S(4)](+) clusters; however, they are present in a mixed spin state with a small contribution from the S > ½ spin state. Our results also suggest that both the precursor and "FeMoco" have a conserved Fe/S electronic structure that is similar to the electronic structure of FeMoco in the MoFe protein, and that the "FeMoco" in NifEN("FeMoco") exists, predominantly, in an S = 3/2 spin state with spectral parameters identical to those of FeMoco in the MoFe protein. These observations provide strong support to the outcome of our previous EPR and X-ray absorption spectroscopy/extended X-ray absorption fine structure analysis of the three NifEN species while providing significant new insights into the unique electronic properties of the precursor and "FeMoco" in NifEN.

Characterization of isolated nitrogenase FeVco.

The cofactors of the Mo- and V-nitrogenases (i.e., FeMoco and FeVco) are homologous metal centers with distinct catalytic properties. So far, there has been only one report on the isolation of FeVco from Azotobacter chroococcum. However, this isolated FeVco species did not carry the full substrate-reducing capacity, as it is unable to restore the N(2)-reducing ability of the cofactor-deficient MoFe protein. Here, we report the isolation and characterization of a fully active species of FeVco from A. vinelandii. Our metal and activity analyses show that FeVco has been extracted intact, carrying with it the characteristic capacity to reduce C(2)H(2) to C(2)H(6) and, perhaps even more importantly, the ability to reduce N(2) to NH(3). Moreover, our EPR and XAS/EXAFS investigations indicate that FeVco is similar to, yet distinct from FeMoco in electronic properties and structural topology, which could account for the differences in the reactivity of the two cofactors. The outcome of this study not only permits the proposal of the first EXAFS-based structural model of the isolated FeVco but also lays a foundation for future catalytic and structural investigations of this unique metallocluster.

Dual functions of NifEN: insights into the evolution and mechanism of nitrogenase.

Nitrogenase catalyzes the nucleotide-dependent conversion of dinitrogen to ammonia at the iron-molybdenum cofactor (FeMoco) center of its molybdenum-iron (MoFe) protein component. Biosynthesis of FeMoco is arguably one of the most complex processes in the field of bioinorganic chemistry, which involves the participation of a number of nif (nitrogen fixing) gene products. One key player in this process, NifEN (encoded by nifE and nifN), is homologous to the MoFe protein with regard to both the primary sequences and the types of the metal centers. Recently, an all-iron precursor has been identified on NifEN, which closely resembles the Fe/S core structure of the mature cofactor. Such a precursor-bound form of NifEN has not only served as an excellent platform for the investigation of FeMoco assembly, but also facilitated the examination of the capacity of NifEN as a catalytic homolog of MoFe protein. This perspective will focus on the recent advances toward elucidating the dual functions of NifEN in nitrogenase assembly and catalysis, and the insights afforded by these advances into the evolution and mechanism of nitrogenase.

Insertion of heterometals into the NifEN-associated iron-molybdenum cofactor precursor.

The cofactors of Mo-, V-, Fe-dependent nitrogenases are believed to be highly homologous in structure despite the different types of heterometals (Mo, V, and Fe) they contain. Previously, a precursor form of the FeMo cofactor (FeMoco) was captured on NifEN, a scaffold protein for FeMoco biosynthesis. This all-Fe precursor closely resembles the Fe/S core structure of the FeMoco and, therefore, could reasonably serve as a precursor for all nitrogenase cofactors. Here, we report the heterologous incorporation of V and Fe into the NifEN-associated FeMoco precursor. EPR and activity analyses indicate that V and Fe can be inserted at much reduced efficiencies compared with Mo, and incorporation of both V and Fe is enhanced in the presence of homocitrate. Further, native polyacrylamide gel electrophoresis experiments suggest that NifEN undergoes a significant conformational rearrangement upon metal insertion, which allows the subsequent NifEN-MoFe protein interactions and the transfer of the cofactor between the two proteins. The combined outcome of these in vitro studies leads to the proposal of a selective mechanism that is utilized in vivo to maintain the specificity of heterometals in nitrogenase cofactors, which is likely accomplished through the redox regulation of metal mobilization by different Fe proteins (encoded by nifH, vnfH, and anfH, respectively), as well as the differential interactions between these Fe proteins and their respective scaffold proteins (NifEN and VnfEN) in the Mo-, V-, and Fe-dependent nitrogenase systems.

Stepwise formation of P-cluster in nitrogenase MoFe protein.

The P-cluster of nitrogenase is one of the most complex biological metallocenters known to date. Despite the recent advances in the chemical synthesis of P-cluster topologs, the biosynthetic mechanism of P-cluster has not been well defined. Here, we present a combined biochemical, electron paramagnetic resonance, and X-ray absorption spectroscopy/extended X-ray absorption fine-structure investigation of the maturation process of P-clusters in DeltanifH molybdenum-iron (MoFe) protein. Our data indicate that the previously identified, [Fe(4)S(4)]-like cluster pairs in DeltanifH MoFe protein are indeed the precursors to P-clusters, which can be reductively coupled into the mature [Fe(8)S(7)] structures in the presence of Fe protein, MgATP, and dithionite. Moreover, our observation of a biphasic maturation pattern of P-clusters in DeltanifH MoFe protein provides dynamic proof for the previously hypothesized, stepwise assembly mechanism of the two P-clusters in the alpha(2)beta(2)-tetrameric MoFe protein, i.e., one P-cluster is formed in one alphabeta dimer before the other in the second alphabeta dimer.

Catalytic activities of NifEN: implications for nitrogenase evolution and mechanism.

NifEN is a key player in the biosynthesis of nitrogenase MoFe protein. It not only shares a considerable degree of sequence homology with the MoFe protein, but also contains clusters that are homologous to those found in the MoFe protein. Here we present an investigation of the catalytic activities of NifEN. Our data show that NifEN is catalytically competent in acetylene (C(2)H(2)) and azide (N(3)(-)) reduction, yet unable to reduce dinitrogen (N(2)) or evolve hydrogen (H(2)). Upon turnover, C(2)H(2) gives rise to an additional S = 1/2 signal, whereas N(3)(-) perturbs the signal originating from the NifEN-associated FeMoco homolog. Combined biochemical and spectroscopic studies reveal that N(3)(-) can act as either an inhibitor or an activator for the binding and/or reduction of C(2)H(2), while carbon monoxide (CO) is a potent inhibitor for the binding and/or reduction of both N(3)(-) and C(2)H(2). Taken together, our results suggest that NifEN is a catalytic homolog of MoFe protein; however, it is only a "skeleton" version of the MoFe protein, as its associated clusters are simpler in structure and less versatile in function, which, in turn, may account for its narrower range of substrates and lower activities of substrate reduction. The resemblance of NifEN to MoFe protein in catalysis points to a plausible, sequential appearance of the two proteins in nitrogenase evolution. More importantly, the discrepancy between the two systems may provide useful insights into nitrogenase mechanism and allow reconstruction of a fully functional nitrogenase from the "skeleton" enzyme, NifEN.

Optimization of FeMoco maturation on NifEN.

Mo-nitrogenase catalyzes the reduction of dinitrogen to ammonia at the cofactor (i.e., FeMoco) site of its MoFe protein component. Biosynthesis of FeMoco involves NifEN, a scaffold protein that hosts the maturation of a precursor to a mature FeMoco before it is delivered to the target location in the MoFe protein. Previously, we have shown that the NifEN-bound precursor could be converted in vitro to a fully complemented "FeMoco" in the presence of 2 mM dithionite. However, such a conversion was incomplete, and Mo was only loosely associated with the NifEN-bound "FeMoco". Here we report the optimized maturation of the NifEN-associated precursor in 20 mM dithionite. Activity analyses show that upon the optimal conversion of precursor to "FeMoco", NifEN is capable of activating a FeMoco-deficient form of MoFe protein to the same extent as the isolated FeMoco. Furthermore, EPR and XAS/EXAFS analyses reveal the presence of a tightly organized Mo site in NifEN-bound "FeMoco", which allows the observation of a FeMoco-like S = 3/2 EPR signal and the modeling of a NifEN-bound "FeMoco" that adopts a conformation very similar to that of the MoFe protein-associated FeMoco. The sensitivity of FeMoco maturation to dithionite concentration suggests an essential role of redox chemistry in this process, and the optimal potential of dithionite solution could serve as a guideline for future identification of in vivo electron donors for FeMoco maturation.

VTVH-MCD study of the Delta nifB Delta nifZ MoFe protein from Azotobacter vinelandii.

NifZ is a member of a series of proteins associated with the maturation of the nitrogenase MoFe protein. An MCD spectroscopic study was undertaken on the Delta nifB Delta nifZ MoFe protein generated in the absence of both NifZ and NifB (deletion of NifB generates an apo-MoFe protein lacking the FeMo cofactor). Results presented here show that, in the absence of NifZ, only one of the two P-clusters of the MoFe protein is matured to the ultimate [8Fe-7S] structure. The other P-cluster site in the protein contains a [4Fe-4S] cluster pair, representing a P-cluster precursor that is electronically identical to the analogous clusters observed in the Delta nifH MoFe protein. These results suggest that the MoFe protein is synthesized in a stepwise fashion where NifZ is specifically required for the formation of the second P-cluster.

Assembly of nitrogenase MoFe protein.

Assembly of nitrogenase MoFe protein is arguably one of the most complex processes in the field of bioinorganic chemistry, requiring, at least, the participation of nifS, nifU, nifB, nifE, nifN, nifV, nifQ, nifZ, nifH, nifD, and nifK gene products. Previous genetic studies have identified factors involved in MoFe protein assembly; however, the exact functions of these factors and the precise sequence of events during the process have remained unclear until the recent characterization of a number of assembly-related intermediates that provided significant insights into this biosynthetic "black box". This review summarizes the recent advances in elucidation of the mechanism of FeMoco biosynthesis in four aspects: (1) the ex situ assembly of FeMoco on NifEN, (2) the incorporation of FeMoco into MoFe protein, (3) the in situ assembly of P-cluster on MoFe protein, and (4) the stepwise assembly of MoFe protein.

P-cluster maturation on nitrogenase MoFe protein.

Biosynthesis of nitrogenase P-cluster has attracted considerable attention because it is biologically important and chemically unprecedented. Previous studies suggest that P-cluster is formed from a precursor consisting of paired [4Fe-4S]-like clusters and that P-cluster is assembled stepwise on MoFe protein, i.e., one cluster is assembled before the other. Here, we specifically tackle the assembly of the second P-cluster by combined biochemical and spectroscopic approaches. By using a P-cluster maturation assay that is based on purified components, we show that the maturation of the second P-cluster requires the concerted action of NifZ, Fe protein, and MgATP and that the action of NifZ is required before that of Fe protein/MgATP, suggesting that NifZ may act as a chaperone that facilitates the subsequent action of Fe protein/MgATP. Furthermore, we provide spectroscopic evidence that the [4Fe-4S] cluster-like fragments can be converted to P-clusters, thereby firmly establishing the physiological relevance of the previously identified P-cluster precursor.

Conformational differences between Azotobacter vinelandii nitrogenase MoFe proteins as studied by small-angle X-ray scattering.

The nitrogenase MoFe protein is a heterotetramer containing two unique high-nuclearity metalloclusters, FeMoco and the P-cluster. FeMoco is assembled outside the MoFe protein, whereas the P-cluster is assembled directly on the MoFe protein polypeptides. MoFe proteins isolated from different genetic backgrounds have been analyzed using biochemical and spectroscopic techniques in attempting to elucidate the pathway of P-cluster biosynthesis. The DeltanifH MoFe protein is less stable than other MoFe proteins and has been shown by extended X-ray absorption fine structure studies to contain a variant P-cluster that most likely exists as two separate [Fe4S4]-like clusters instead of the subunit-bridging [Fe8S7] cluster found in the wild-type and DeltanifB forms of the MoFe protein [Corbett, M. C., et al. (2004) J. Biol. Chem. 279, 28276-28282]. Here, a combination of small-angle X-ray scattering and Fe chelation studies is used to show that there is a correlation between the state of the P-cluster and the conformation of the MoFe protein. The DeltanifH MoFe protein is found to be larger than the wild-type or DeltanifB MoFe proteins, an increase in size that can be modeled well by an opening of the subunit interface consistent with P-cluster fragmentation and solvent exposure. Importantly, this opening would allow for the insertion of P-cluster precursors into a region of the MoFe protein that is buried in the wild-type conformation. Thus, DeltanifH MoFe protein could represent an early intermediate in MoFe protein biosynthesis where the P-cluster precursors have been inserted, but P-cluster condensation and tetramer stabilization have yet to occur.

Molecular insights into nitrogenase FeMoco insertion--the role of His 274 and His 451 of MoFe protein alpha subunit.

The final step of FeMo cofactor (FeMoco) assembly involves the insertion of FeMoco into its binding site in the molybdenum-iron (MoFe) protein of nitrogenase. Here we examine the role of His alpha274 and His alpha451 of Azotobacter vinelandii MoFe protein in this process. Our results from combined metal, activity, EPR, stability and insertion analyses show that mutations of His alpha274 and/or His alpha451, two of the histidines that belong to a so-called His triad, to small uncharged Ala specifically reduce the accumulation of FeMoco in MoFe protein. This observation indicates that the enrichment of histidines at the His triad is important for FeMoco insertion and that the His triad potentially serves as an intermediate docking point for FeMoco through transitory ligand coordination and/or electrostatic interaction.

Molecular insights into nitrogenase FeMo cofactor insertion: the role of His 362 of the MoFe protein alpha subunit in FeMo cofactor incorporation.

The assembly of the complex iron-molybdenum cofactor (FeMoco) of nitrogenase molybdenum-iron (MoFe) protein has served as one of the central topics in the field of bioinorganic chemistry for decades. Here we examine the role of a MoFe protein residue (His alpha362) in FeMoco insertion, the final step of FeMoco biosynthesis where FeMoco is incorporated into its binding site in the MoFe protein. Our data from combined metal, activity and electron paramagnetic resonance analyses show that mutations of His alpha362 to small uncharged Ala or negatively charged Asp result in significantly reduced FeMoco accumulation in MoFe protein, indicating that His alpha362 plays a key role in the process of FeMoco insertion. Given the strategic location of His alpha362 at the entry point of the FeMoco insertion funnel, this residue may serve as one of the initial docking points for FeMoco insertion through transient ligand coordination and/or electrostatic interaction.