Nitrogenase MoFe Protein Research Articles

Detection of nitrogenase in individual cells of a natural population of trichodesmium using immunocytochemical methods for fluorescent cells

The intercellular distribution of nitrogenase was detected in a natural population of Trichodesmium spp. collected from Shitaba Bay, Uwajima, Ehime Prefecture, Japan. Polyclonal antibodies raised against the Fe-protein and the MoFe-protein (α-subunit) of nitrogenase were used as probes. Visualization of the antibody binding to the nitrogenase was performed using horseradish peroxidase-conjugated secondary antibody and the chromogenic substrate, 3,3′-diaminobenzidine tetrahydrochloride, to avoid the masking effects caused by the bright autofluorescence emitted by the Trichodesmium cells. Nitrogenase proteins were detected in 66 to 81% of the trichomes. More than 77% of the cells were immunostained in individual nitrogenase-containing trichome. The data show natural populations of Trichodesmium spp. do not developed heterocyst-equivalent cells.

A substrate channel in the nitrogenase MoFe protein

Nitrogenase catalyzes the six electron/six proton reduction of N2 to two ammonia molecules at a complex organometallocluster called "FeMo cofactor." This cofactor is buried within the alpha-subunit of the MoFe protein, with no obvious access for substrates. Examination of high-resolution X-ray crystal structures of MoFe proteins from several organisms has revealed the existence of a water-filled channel that extends from the solvent-exposed surface to a specific face of FeMo cofactor. This channel could provide a pathway for substrate and product access to the active site. In the present work, we examine this possibility by substituting four different amino acids that line the channel with other residues and analyze the impact of these substitutions on substrate reduction kinetic parameters. Each of the MoFe protein variants was purified and kinetic parameters were established for the reduction of the substrates N2, acetylene, azide, and propyne. For each MoFe protein, V (max) values for the different substrates were found to be nearly unchanged when compared with the values for the wild-type MoFe protein, indicating that electron delivery to the active site is not compromised by the various substitutions. In contrast, the K(m) values for these substrates were found to increase significantly (up to 22-fold) in some of the MoFe protein variants compared with the wild-type MoFe protein values. Given that each of the amino acids that were substituted is remote from the active site, these results are consistent with the water-filled channel functioning as a substrate channel in the MoFe protein.

VTVH-MCD Study of the ΔnifBΔnifZ 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.

Climbing Nitrogenase: Toward a Mechanism of Enzymatic Nitrogen Fixation

"Nitrogen fixation", the reduction of dinitrogen (N2) to two ammonia (NH3) molecules, by the Mo-dependent nitrogenase is essential for all life. Despite four decades of research, a daunting number of unanswered questions about the mechanism of nitrogenase activity make it the "Everest of enzymes". This Account describes our efforts to climb one "face" of this mountain by meeting two interdependent challenges central to determining the mechanism of biological N2 reduction. The first challenge is to determine the reaction pathway: the composition and structure of each of the substrate-derived moieties bound to the catalytic FeMo cofactor (FeMo-co) of the molybdenum-iron (MoFe) protein of nitrogenase. To overcome this challenge, it is necessary to discriminate between the two classes of potential reaction pathways: (1) a "distal" (D) pathway, in which H atoms add sequentially at a single N or (2) an "alternating" (A) pathway, in which H atoms add alternately to the two N atoms of N2. Second, it is necessary to characterize the dynamics of conversion among intermediates within the accepted Lowe-Thorneley kinetic scheme for N2 reduction. That goal requires an experimental determination of the number of electrons and protons delivered to the MoFe protein as well as their "inventory", a partition into those residing on each of the reaction components and released as H2 or NH3. The principal obstacle to this "climb" has been the inability to generate N2 reduction intermediates for characterization. A combination of genetic, biochemical, and spectroscopic approaches recently overcame this obstacle. These experiments identified one of the four-iron Fe-S faces of the active-site FeMo-co as the specific site of reactivity, indicated that the side chain of residue alpha70V controls access to this face, and supported the involvement of the side chain of residue alpha195H in proton delivery. We can now freeze-quench trap N2 reduction pathway intermediates and use electron-nuclear double resonance (ENDOR) and electron spin-echo envelope modulation (ESEEM) spectroscopies to characterize them. However, even successful trapping of a N2 reduction intermediate occurs without synchronous electron delivery to the MoFe protein. As a result, the number of electrons and protons, n, delivered to MoFe during its formation is unknown. To determine n and the electron inventory, we initially employed ENDOR spectroscopy to analyze the substrate moiety bound to the FeMo-co and 57Fe within the cofactor. Difficulties in using that approach led us to devise a robust kinetic protocol for determining n of a trapped intermediate. This Account describes strategies that we have formulated to bring this "face" of the nitrogenase mechanism into view and afford approaches to its climb. Although the summit remains distant, we look forward to continued progress in the ascent.

Whole-cell immunocytochemical detection of nitrogenase in cyanobacteria: improved protocol for highly fluorescent cells

An improved immunocytochemical method was developed to detect nitrogenase in sin- gle cells of cyanobacteria using a unicellular diazotrophic strain (Gloeothece sp. 68DGA) and a non- diazotrophic strain (Synechocystis sp. PCC6714) as model organisms. Polyclonal antibodies raised against the Fe-protein and the MoFe-protein (α-subunit) of nitrogenase were used as probes. The antigenicity of nitrogenase was maintained for at least 6 mo when the cells were preserved in chilled methanol (-30°C) after paraformaldehyde fixation (3%). The cells were permeabilized for antibody penetration and non-specific binding was prevented by incubation in phosphate-buffered saline con- taining dimethyl sulfoxide (10%) and normal rabbit serum (15%). Antibody binding was visualized by a horseradish peroxidase-conjugated secondary antibody and the chromogenic substrate 3-3'- diaminobenzidine tetrachloride because of the difficulty in discriminating between fluorescence from additive fluorochrome and natural autofluorescence from cyanobacteria. Almost all cells (>97%) were immunostained when they were grown diazotrophically and expressed nitrogenase (acetylene reduction) activity. Non-specific staining of both the diazotrophic strains grown with combined nitro- gen and the non-diazotrophic strain was negligible. Our protocol was able to detect nitrogenase in unicellular and filamentous non-heterocystous strains without modification.

Structural basis for VO2+-inhibition of nitrogenase activity: (B) pH-sensitive inner-sphere rearrangements in the 1H-environment of the metal coordination site of the nitrogenase Fe–protein identified by ENDOR spectroscopy

The nitrogenase Fe-protein is the specific ATP-activated electron donor to the active site-containing nitrogenase MoFe-protein. It has been previously demonstrated that different VO(2+)-nucleotide coordination environments exist for the Fe-protein that depend on pH and are distinguishable by EPR spectroscopy. After having studied the nitrogenase 31P and 23Na superhyperfine structure for this system by electron nuclear double resonance (ENDOR) spectroscopy (Petersen et al. 2008 in J Biol Inorg Chem. doi:10.1007/s00775-008-0360-0), we here report on the 1H-interactions with the nucleotide-bound metal center after substitution of the natural diamagnetic metal Mg2+ with paramagnetic oxo-vanadium(IV). ENDOR spectra show a number of resonances arising from interactions of the VO2+ ion with protons. In the presence of reduced Fe-protein and VO2+ ADP, at least three sets of nonexchangeable protons are detected. At low pH the superhyperfine couplings of most of these are consistent with proton interactions originating from the nucleotide. There is no indication of 1H-resonances that exchange in D2O at neutral pH and could be assigned to inner-sphere hydroxyl coordination. Exchangeable hydroxyl protons in the inner coordination sphere with reduced Fe-protein are only found in the low pH form; based on their hyperfine tensor components these have been assigned to an axially coordinated hydroxyl water molecule. The pH-dependent alterations of the proton couplings that exchange in D2O suggest that they are partially caused by a rearrangement in the local hydroxyl coordination environment of the metal center. These rearrangements especially affect the apical metal position, where an axially coordinated water present at low pH is absent at neutral pH. Oxidation of the Fe-protein induced substantial changes in the electron-nucleus interactions. This indicates that the oxidation state of the iron-sulfur cluster has an important effect on the metal coordination environment at the nucleotide binding site of the Fe-protein. The distinct VO(2+)-nucleotide coordination structures with ADP and ATP and the redox state of the [4Fe-4S] cluster imply that VO2+ has a critical influence on the switch regions of the regulatory protein, and, taken together, this provides a plausible explanation for the inhibitory action of VO2+.

ATP-driven Reduction by Dark-operative Protochlorophyllide Oxidoreductase from Chlorobium tepidum Mechanistically Resembles Nitrogenase Catalysis

During chlorophyll and bacteriochlorophyll biosynthesis in gymnosperms, algae, and photosynthetic bacteria, dark-operative protochlorophyllide oxidoreductase (DPOR) reduces ring D of aromatic protochlorophyllide stereospecifically to produce chlorophyllide. We describe the heterologous overproduction of DPOR subunits BchN, BchB, and BchL from Chlorobium tepidum in Escherichia coli allowing their purification to apparent homogeneity. The catalytic activity was found to be 3.15 nmol min(-1) mg(-1) with K(m) values of 6.1 microm for protochlorophyllide, 13.5 microm for ATP, and 52.7 microm for the reductant dithionite. To identify residues important in DPOR function, 21 enzyme variants were generated by site-directed mutagenesis and investigated for their metal content, spectroscopic features, and catalytic activity. Two cysteine residues (Cys(97) and Cys(131)) of homodimeric BchL(2) are found to coordinate an intersubunit [4Fe-4S] cluster, essential for low potential electron transfer to (BchNB)(2) as part of the reduction of the protochlorophyllide substrate. Similarly, Lys(10) and Leu(126) are crucial to ATP-driven electron transfer from BchL(2). The activation energy of DPOR electron transfer is 22.2 kJ mol(-1) indicating a requirement for 4 ATP per catalytic cycle. At the amino acid level, BchL is 33% identical to the nitrogenase subunit NifH allowing a first tentative structural model to be proposed. In (BchNB)(2), we find that four cysteine residues, three from BchN (Cys(21), Cys(46), and Cys(103)) and one from BchB (Cys(94)), coordinate a second inter-subunit [4Fe-4S] cluster required for catalysis. No evidence for any type of molybdenum-containing cofactor was found, indicating that the DPOR subunit BchN clearly differs from the homologous nitrogenase subunit NifD. Based on the available data we propose an enzymatic mechanism of DPOR.

Intercellular localization of nitrogenase in a non-heterocystous cyanobacterium (cyanophyte), Trichodesmium sp. NIBB1067

The nitrogenase activities of cyanobacteria of the genus Trichodesmium are strictly light-dependent, although they do not develop heterocysts. Recently, the development of heterocyst-equivalent cells (diazocytes) was suggested in Trichodesmium spp. However, no cells with a similar appearance to diazocytes could be found in Trichodesmium sp. NIBB1067. An immunocytochemical analysis of nitrogenase in individual cells was performed using polyclonal antibodies generated from DNA fragments of genes encoding the Fe-protein and the α-and the β-subunit of the MoFe-protein of nitrogenase of Trichodesmium sp. NIBB1067. Visualization of the antibody binding was carried out using a horseradish peroxidase-conjugated secondary antibody and a chromogenic substrate, 3,3′-diaminobenzidine, to avoid the masking effects caused by the bright autofluorescence emitted by Trichodesmium cells. Nitrogenase proteins were detected in almost all the cells (always higher than 95% of total cells) grown under nitrogen-limited conditions. These results indicate that Trichodesmium sp. NIBB1067 does not differentiate heterocyst-equivalent cells.

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.

Conformations generated during turnover of the Azotobacter vinelandii nitrogenase MoFe protein and their relationship to physiological function

Various S=3/2 EPR signals elicited from wild-type and variant Azotobacter vinelandii nitrogenase MoFe proteins appear to reflect different conformations assumed by the FeMo-cofactor with different protonation states. To determine whether these presumed changes in protonation and conformation reflect catalytic capacity, the responses (particularly to changes in electron flux) of the αH195Q, αH195N, and αQ191K variant MoFe proteins (where His at position 195 in the α subunit is replaced by Gln/Asn or Gln at position α-191 by Lys), which have strikingly different substrate-reduction properties, were studied by stopped-flow or rapid-freeze techniques. Rapid-freeze EPR at low electron flux (at 3-fold molar excess of wild-type Fe protein) elicited two transient FeMo-cofactor-based EPR signals within 1s of initiating turnover under N2 with the αH195Q and αH195N variants, but not with the αQ191K variant. No EPR signals attributable to P cluster oxidation were observed for any of the variants under these conditions. Furthermore, during turnover at low electron flux with the wild-type, αH195Q or αH195N MoFe protein, the longer-time 430-nm absorbance increase, which likely reflects P cluster oxidation, was also not observed (by stopped-flow spectrophotometry); it did, however, occur for all three MoFe proteins under higher electron flux. No 430-nm absorbance increase occurred with the αQ191K variant, not even at higher electron flux. This putative lack of involvement of the P cluster in electron transfer at low electron flux was confirmed by rapid-freeze 57Fe Mössbauer spectroscopy, which clearly showed FeMo-factor reduction without P cluster oxidation. Because the wild-type, αH195Q and αH195N MoFe proteins can bind N2, but αQ195K cannot, these results suggest that P cluster oxidation occurs only under high electron flux as required for N2 reduction.

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.

Alkyne substrate interaction within the nitrogenase MoFe protein

Nitrogenase catalyzes the biological reduction of N2 to ammonia (nitrogen fixation), as well as the two-electron reduction of the non-physiological alkyne substrate acetylene (HC≡CH). A complex metallo-organic species called FeMo-cofactor provides the site of substrate reduction within the MoFe protein, but exactly where and how substrates interact with FeMo-cofactor remains unknown. Recent results have shown that the MoFe protein α-70Val residue, whose side chain approaches one Fe–S face of FeMo-cofactor, plays a significant role in defining substrate access to the active site. For example, substitution of α-70Val by alanine results in an increased capacity for the reduction of the larger alkyne propyne (HC≡C–CH3), whereas, substitution by isoleucine at this position nearly eliminates the capacity for the reduction of acetylene. These and complementary spectroscopic studies led us to propose that binding of short chain alkynes occurs with side-on binding to Fe atom 6 within FeMo-cofactor. In the present work, the α-70Val residue was substituted by glycine and this MoFe protein variant shows an increased capacity for reduction of the terminal alkyne, 1-butyne (HC≡C–CH2–CH3). This protein shows no detectable reduction of the internal alkyne 2-butyne (H3C–C≡C–CH3). In contrast, substitution of the nearby α-191Gln residue by alanine, in combination with the α-70Ala substitution, does result in significant reduction of 2-butyne, with the exclusive product being 2-cis-butene. These results indicate that the reduction of alkynes by nitrogenases involves side-on binding of the alkyne to Fe6 within FeMo-cofactor, and that a terminal acidic proton is not required for reduction. The successful design of amino acid substitutions that permit the targeted accommodation of an alkyne that otherwise is not a nitrogenase substrate provides evidence to support the current model for alkyne interaction within the nitrogenase MoFe protein.

Molecular insights into nitrogenase FeMoco insertion – The role of His 274 and His 451 of MoFe protein α 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 α274 and His α451 of Azotobacter vinelandii MoFe protein in this process. Our results from combined metal, activity, EPR, stability and insertion analyses show that mutations of His α274 and/or His α451, 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.

Variable-Temperature, Variable-Field Magnetic Circular Dichroism Spectroscopic Study of the Metal Clusters in the ΔnifB and ΔnifH MoFe Proteins of Nitrogenase from Azotobacter vinelandii

Deletion of nifB results in the formation of a variant nitrogenase MoFe protein (DeltanifB MoFe protein) that appears to contain two normal [8Fe-7S] P clusters. This protein can be reactivated to form the holo MoFe protein upon addition of isolated FeMo cofactor. In contrast, deletion of nifH results in a variant protein (DeltanifH MoFe protein) that appears to contain FeS clusters different from the normal P cluster, presumably representing precursors of the normal P cluster. The DeltanifH MoFe protein is not reconstituted to the holo MoFe protein with isolated FeMo cofactor. The EPR and EXAFS spectroscopic properties of FeS clusters in the DeltanifH MoFe protein clearly differ from those of the normal P cluster found in the DeltanifB MoFe protein and suggest the presence of [4Fe-4S]-like clusters. To further characterize the metal cluster structures in the DeltanifH MoFe protein, a variable-temperature, variable-field magnetic circular dichroism (VTVH-MCD) spectroscopic study has been undertaken on both the DeltanifB MoFe protein and the DeltanifH MoFe protein in both the dithionite-reduced and oxidized states. This study clearly shows that each half of the dithionite-reduced DeltanifH MoFe protein contains a [4Fe-4S]+ cluster paired with a diamagnetic [4Fe-4S]-like cluster. Upon oxidation, the VTVH-MCD spectrum of the DeltanifH MoFe protein reveals a paramagnetic, albeit EPR-silent system, suggesting an integer spin state. These results suggest that the DeltanifH MoFe protein contains a pair of neighboring, unusual [4Fe-4S]-like clusters, which are paramagnetic in their oxidized state.

Molecular Insights into Nitrogenase FeMoco Insertion: TRP-444 OF MoFe PROTEIN α-SUBUNIT LOCKS FeMoco IN ITS BINDING SITE

Biosynthesis of the FeMo cofactor (FeMoco) of nitrogenase MoFe protein is arguably one of the most complex processes in metalloprotein biochemistry. Here we investigate the role of a MoFe protein residue (Trp-alpha444) in the final step of FeMoco assembly, which involves the insertion of FeMoco into its binding site. Mutations of this aromatic residue to small uncharged ones result in significantly decreased levels of FeMoco insertion/retention and drastically reduced activities of MoFe proteins, suggesting that Trp-alpha444 may lock the FeMoco tightly in its binding site through the sterically restricting effect of its bulky, aromatic side chain. Additionally, these mutations cause partial conversion of the P-cluster to a more open conformation, indicating a potential connection between FeMoco insertion and P-cluster assembly. Our results provide some of the initial molecular insights into the FeMoco insertion process and, moreover, have useful implications for the overall scheme of nitrogenase assembly.

Structural insights into a protein-bound iron-molybdenum cofactor precursor

The iron-molybdenum cofactor (FeMoco) of the nitrogenase MoFe protein is a highly complex metallocluster that provides the catalytically essential site for biological nitrogen fixation. FeMoco is assembled outside the MoFe protein in a stepwise process requiring several components, including NifB-co, an iron- and sulfur-containing FeMoco precursor, and NifEN, an intermediary assembly protein on which NifB-co is presumably converted to FeMoco. Through the comparison of Azotobacter vinelandii strains expressing the NifEN protein in the presence or absence of the nifB gene, the structure of a NifEN-bound FeMoco precursor has been analyzed by x-ray absorption spectroscopy. The results provide physical evidence to support a mechanism for FeMoco biosynthesis. The NifEN-bound precursor is found to be a molybdenum-free analog of FeMoco and not one of the more commonly suggested cluster types based on a standard [4Fe-4S] architecture. A facile scheme by which FeMoco and alternative, non-molybdenum-containing nitrogenase cofactors are constructed from this common precursor is presented that has important implications for the biosynthesis and biomimetic chemical synthesis of FeMoco.

Identification of Contaminations Hiding Beneath the alpha- and beta-Subunits of Partially Purified Nitrogenase MoFe Protein on the Sodium Dodecyl Sulfate Gel

Abstract: To identify the unknown proteins that would contaminate the α- and β-subunits of nitrogenase MoFe protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the partially purified MoFe protein (Av1) preparation was obtained from Azotobacter vinelandii Lipmann OP by chromatography on DEAE-cellulose (DE52) and Sephacryl S-200 columns and analyzed by PAGE and matrixassisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The Av1 preparation was shown to have two main bands at the position of the α- and β-subunits of crystalline Av1 on the SDS gel. However, on the anoxic native PAGE, in addition to the Av1 band, the preparation was shown to have three other main bands that migrated slower than Av1. Of these three main bands, the protein with the fastest migration was identified as bacterioferritin elsewhere. The proteins on the other two bands, termed Upper and Middle, were suggested to be two different homopolymers with the same apparent subunit electrophoretic mobilities as the α- and β-subunits of Av1, respectively. By analysis of MALDI-TOF mass spectrometry, the Upper was identified as GroEL, which belongs to the heat shock protein 60 family, and the Middle was identified as glucose-6-phosphate isomerase (PGI). In our preparation, anoxic native electrophoresis indicated that GroEL was composed of 14 identical subunits and that PGI was composed of 10 identical subunits. This is the first report of PGI, with so many subunits. The contaminating proteins in the Av1 preparation, mainly GroEL and PGI, could be totally or partially removed from Av1 if the shoulders and center of the elution peak were collected separately from the Sephacryl S-200 column and the center fraction was purified further by Q-Sepharose developed with an NaCl concentration gradient. Thus, Av1 with more than 90% purity was obtained. Obviously, this modified method is useful for the purification of mutant MoFe proteins with a high purity. (Managing editor: Wei WANG)

The Interstitial Atom of the Nitrogenase FeMo-Cofactor: ENDOR and ESEEM Evidence That it is Not a Nitrogen

X-ray crystallographic study of the nitrogenase MoFe protein revealed electron density from an atom (denoted X) inside the active-site metal cluster, the [MoFe7S9:homocitrate] FeMo-cofactor. The electron density associated with X is consistent with a single N, O, or C atom. We now have tested whether X is an N or not by comparing the Q-band ENDOR and ESEEM signals from resting-state (S = 3/2) MoFe protein and NMF-extracted FeMo-co from bacteria grown with either 14N or 15N as the exclusive N source. All of the 14N or 15N signals associated with the protein are lost upon extraction of the FeMo-co. We interpret this as strong evidence that X is not an N.

Variant MoFe proteins of Azotobacter vinelandii: effects of carbon monoxide on electron paramagnetic resonance spectra generated during enzyme turnover

The resting state of wild-type nitrogenase MoFe protein exhibits an S=3/2 electron paramagnetic resonance (EPR) signal originating from the FeMo cofactor, the enzyme's active site. When nitrogenase turns over under CO, this signal disappears and one (sometimes two) of three new EPR signals, which also arise from the FeMo cofactor, appears, depending on the CO concentration. The appearance and properties of these CO-inducible EPR signals, which were also generated with variant MoFe proteins (alphaR96Q, alphaR96K, alphaQ191K, alphaR359K, alphaR96K/alphaR359K, alphaR277C, alphaR277H, and DeltanifV) that are impacted around the FeMo cofactor, have been investigated. No new CO-induced EPR signals arise from any variant, suggesting that no new CO-binding sites are produced by the substitutions. All variant proteins, except alphaR277H, produce the lo-CO signal; all, except alphaQ191K, produce the hi(5)-CO signal; but only two (alphaR96Q and DeltanifV) exhibit the hi-CO signal. FeMo cofactor's environment clearly dictates which CO-induced EPR signals are generated; however, none of these EPR signals correlate with CO inhibition of H(2) evolution observed with some of these variants. CO inhibition of H(2) evolution is, therefore, due to CO binding to a different site(s) from those responsible for the CO-induced EPR signals. Some resting-state variants have overlapping S=3/2 EPR signals, whose intensities simultaneously decrease under turnover conditions, indicating that all FeMo cofactor conformations are catalytically active. Moreover, these variants produce a similar number of hi(5)-CO signals after turnover under CO to the number of resting-state S=3/2 signals. The FeMo cofactor associated with the hi(5)-CO signal likely contains two bridging CO molecules.

The composition and distribution of metal clusters in the MoFe protein from a nifZ deletion strain (DJ 194) of Azotobacter vinelandii

Through the anaerobic chromatography on the columns of DEAE 52, Q-Sepharose and Sephacryl S-200, a nitrogenase MoFe protein (ΔnifZ Av1) was obtained from a nifZ deleted mutant of Azotobacter vinelandii (stain DJ194). The results of Western blotting after anoxic native electrophoresis and SDS-PAGE showed that ΔnifZ Av1 was similar to wild type MoFe protein (OP Av1) at the electrophoretic mobility, molecular weight and subunit composition. Furthermore, ΔnifZ Av1 was also similar to OP Av1 at the molybdenum content, EPR signal (g ≈ 4.3, 3.65 and 2.01), and the molar extinction coefficient (Δe) of circular dichroism (CD) at 660 nm region. All of these indicated that, besides having the same α2β2 composition as OP Av1, the ΔnifZ Av1 also contained equal amount of reductive FeMoco in the spin state of S=3/2 to OP Av1. However, the iron content and substrate (C2H2, H+ and N2)-reduction activity of ΔnifZ Av1 were 74% and 46%–50% of those of OP Av1, respectively. Furthermore, the Δe at around 450 nm, which reflects P-cluster in Av1, was obviously lower than that of OP Av1. It suggested that the difference between ΔnifZ Av1 and OP Av1 resulted from P-cluster rather than FeMoco, and from the half number of P-cluster in ΔnifZ Av1, but the composition or redox state of P-cluster in ΔnifZ Av1 were not changed. Thus it could propose that ΔnifZ Av1 is composed of two different αβ subunit pairs. One is a FeMoco- and P-cluster-containing pair, and the other is a P-cluster-deficient but FeMoco-containing pair. Since the deletion of nifZ gene leads to the deficiency of only one of two P-clusters in a α2β2 tetramer, the assembly of P-cluster may not simply depend on one gene product, and so a possible mechanism of NifZ is supposed here.