Molecular insights into nitrogenase FeMoco insertion: TRP-444 of MoFe protein alpha-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.     

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.     

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.     

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 α-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.     

Alkyne substrate interaction within the nitrogenase MoFe protein

Nitrogenase catalyzes the biological reduction of N(2) to ammonia (nitrogen fixation), as well as the two-electron reduction of the non-physiological alkyne substrate acetylene (HC triple bond 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 alpha-70(Val) 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 alpha-70(Val) by alanine results in an increased capacity for the reduction of the larger alkyne propyne (HC triple bond C-CH(3)), 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 alpha-70(Val) residue was substituted by glycine and this MoFe protein variant shows an increased capacity for reduction of the terminal alkyne, 1-butyne (HC triple bond C-CH(2)-CH(3)). This protein shows no detectable reduction of the internal alkyne 2-butyne (H(3)C-C triple bond C-CH(3)). In contrast, substitution of the nearby alpha-191(Gln) residue by alanine, in combination with the alpha-70(Ala) 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.     

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 alphaH195Q, alphaH195N, and alphaQ191K variant MoFe proteins (where His at position 195 in the alpha subunit is replaced by Gln/Asn or Gln at position alpha-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 1 s of initiating turnover under N(2) with the alphaH195Q and alphaH195N variants, but not with the alphaQ191K 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, alphaH195Q or alphaH195N 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 alphaQ191K 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 (57)Fe M?ssbauer spectroscopy, which clearly showed FeMo-factor reduction without P cluster oxidation. Because the wild-type, alphaH195Q and alphaH195N MoFe proteins can bind N(2), but alphaQ195K cannot, these results suggest that P cluster oxidation occurs only under high electron flux as required for N(2) reduction.     

NifI inhibits nitrogenase by competing with Fe protein for binding to the MoFe protein

Reduction of substrate by nitrogenase requires direct electron transfer from the Fe protein to the MoFe protein. Inhibition of nitrogenase activity in Methanococcus maripaludis occurs when the regulatory protein NifI_1,2 binds the MoFe protein. This inhibition is relieved by 2-oxoglutarate. Here we present evidence that NifI_1,2 binding prevents association of the two nitrogenase components. Increasing amounts of Fe protein competed with NifI_1,2, decreasing its inhibitory effect. NifI_1,2 prevented the co-purification of MoFe protein with a mutant form of the Fe protein that forms a stable complex with the MoFe protein, and NifI_1,2 was unable to bind to an -stabilized Fe protein:MoFe protein complex. NifI_1,2 inhibited ATP- and MoFe protein-dependent oxidation of the Fe protein, and 2OG relieved this inhibition. These results support a model where NifI_1,2 competes with the Fe protein for binding to MoFe protein and prevents electron transfer.     

Functional expression of a fusion-dimeric MoFe protein of nitrogenase in Azotobacter vinelandii

The MoFe protein component of the complex metalloenzyme nitrogenase is an alpha2beta2 tetramer encoded by the nifD and the nifK genes. In nitrogen fixing organisms, the alpha and beta subunits are translated as separate polypeptides and then assembled into tetrameric MoFe protein complex that includes two types of metal centers, the P cluster and the FeMo cofactor. In Azotobacter vinelandii, the NifEN complex, the site for biosynthesis of the FeMo cofactor, is an alpha2beta2 tetramer that is structurally similar to the MoFe protein and encoded as two separate polypeptides by the nifE and the nifN genes. In Anabaena variabilis it was shown that a NifE-N fusion protein encoded by translationally fused nifE and nifN genes can support biological nitrogen fixation. The structural similarity between the MoFe protein and the NifEN complex prompted us to test whether the MoFe protein could also be functional when synthesized as a single protein encoded by nifD-K translational fusion. Here we report that the NifD-K fusion protein encoded by nifD-K translational fusion in A. vinelandii is a large protein (as determined by Western blot analysis) and is capable of supporting biological nitrogen fixation. These results imply that the MoFe protein is flexible in that it can accommodate major structural changes and remain functional.     

Mechanistic features and structure of the nitrogenase alpha-Gln195 MoFe protein

EPR signals observed under CO and C(2)H(2) during nitrogenase turnover were investigated for the alpha-Gln(195) MoFe protein, an altered form for which the alpha-His(195) residue has been substituted by glutamine. Under CO, samples show S = 1/2 hi- and lo-CO EPR signals identical to those recognized for the wild-type protein, whereas the S = 3/2 signals generated under high CO/high flux conditions differ. Previous work has revealed that the EPR spectrum generated under C(2)H(2) exhibits a signal (S(EPR1)) originating from the FeMo-cofactor having two or more bound C(2)H(2) adducts and a second signal (S(EPR2)) arising from a radical species [S?rlie, M., Christiansen, J., Dean, D. R., and Hales, B. J. (1999) J. Am. Chem. Soc. 121, 9457-9458]. Pressure-dependent studies show that the intensity of these signals has a sigmoidal dependency at low pressures and maximized at 0.1 atm C(2)H(2) with a subsequent decrease in steady-state intensity at higher pressures. Analogous signals are not recognized for the wild-type MoFe protein. Analysis of the principal g-factors of S(EPR2) suggests that it either represents an unusual metal cluster or is a carboxylate centered radical possibly originating from homocitrate. Both S(EPR1) and S(EPR2) exhibit similar relaxation properties that are atypical for S = 1/2 signals originating from Fe-S clusters or radicals and indicate a coupled relaxation pathway. The alpha-Gln(195) MoFe protein also exhibits these signals when incubated under turnover conditions in the presence of C(2)H(4). Under these conditions, additional inflections in the g 4-6 region assigned to ground-state transitions of an S = 3/2 spin system are also recognized and assigned to turnover states of the MoFe protein without C(2)H(4) bound. The structure of alpha-Gln(195) was crystallographically determined and found to be virtually identical to that of the wild-type MoFe protein except for replacement of an NuH-S hydrogen bond interaction between FeMo-cofactor and the imidazole side chain of alpha-His(195) by an analogous interaction involving Gln.     

Long-range interactions between the Fe protein binding sites of the MoFe protein of nitrogenase

We report the properties and reactivity of the catalytically active heterologous nitrogenase formed between the Fe protein from Clostridium pasteurianum (Cp2) and the MoFe protein from Klebsiella pneumoniae (Kp1). Under turnover conditions, in the presence of MgATP, a stable 2:1 (Cp2)2Kp1 electron transfer complex is formed, in which the [4Fe-4S]+ centre of Cp2 is protected from chelation by alpha,alpha'-bipyridyl. However, the two Fe protein-binding sites on Kp1 are not equivalent, since a 1:1 Cp2.Kp1 complex was isolated by gel filtration. The non-equivalence of the Fe protein binding sites was also indicated by the inhibition pattern of Klebsiella nitrogenase by Cp2. The EPR spectrum of the isolated 1:1 Cp2.Kp1 complex showed an S=1/2 signal characteristic of dithionite-reduced Cp2 and signals with g values of 4.27, 3.73, 2.01 and 4.32, 3.63, 2.00 characteristic of the high- and low-pH forms of the FeMoco centre of Kp1, respectively. The unoccupied binding site of Kp1 of the isolated 1:1 Cp2Kp1 complex was shown to be catalytically fully functional in combination with Kp2. In contrast to homologous nitrogenases, which require MgATP for detectable rates of electron transfer from the Fe protein, stopped-flow kinetic studies revealed that electron transfer from Cp2 to Kp1 occurred in the absence of MgATP with a rate constant of 0.065 s(-1). Subsequently, a slower transient decrease and restoration of absorption in the electronic spectrum in the 500-700 nm region was observed. These changes corresponded with those in the intensity of the S=3/2 EPR signal of the FeMoco centres of Kp1 and were consistent with the transient reduction of the FeMoco centre of Kp1 to an EPR-silent form, followed by restoration of the signal at longer reaction times. These changes were not associated with catalysis since no evolution of H2 was detectable.     

The biosynthesis of nitrogenase MoFe protein polypeptides in free-living cultures of Rhizobium japonicum

The biosynthesis of the constituent polypeptides of nitrogenase component I (Rj 1) in free-living cultures of Rhizobium japonicum (strain 110) was investigated under different growth conditions. Cells were pulse-labelled and the proteins analysed by one and two-dimensional gel electrophoresis. The positions of the constituent Rj 1 polypeptides were identified by co-electrophoresis with purified Rj 1 isolated from bacteroids of soybean nodules, and by comparison with an immunoprecipitate from a culture induced for nitrogenase. The synthesis of the proteins preceded any detectable enzyme activity and increased with time, reaching a maximum after 3 days. At this time, between 6 and 8% of the total sodium dodecyl sulfate-soluble protein synthesized was Rj 1. Exposure to air led to a dramatic decrease in the rate of Rj 1 synthesis, with almost complete regression after 20 min. In the presence of KNO3, there was no nitrogenase activity, but the proteins were present in similar amounts (7%) as the control culture. When mannitol and glycerol were used as the sole carbon sources, the amount of Rj 1 synthesized was extremely low.     

Analysis of steady state Fe and MoFe protein interactions during nitrogenase catalysis

Steady state kinetic measurements are reported for nitrogenase from Azotobacter vinelandii (Av) and Clostridium pasteurianum (Cp) under a variety of conditions, using dithionite as reductant. The specific activities of Av1 and Cp1 are determined as functions of Av2:Av1 and Cp2:Cp1, respectively, at component protein ratios from 0.4 to 50, and conform to a simple hyperbolic rate law for the interaction of Av2 with Av1 and Cp2 with Cp1. The specific activities of Av2 and Cp2 are also measured as a function of increasing Av1 and Cp1 concentrations, producing 'MoFe inhibition' at large MoFe:Fe ratios. When the rate of product formation under MoFe inhibited conditions is re-plotted as increasing Av2:Av1 or Cp2:Cp1 ratios, sigmoidal kinetic behavior is observed, suggesting that the rate constants in the Thorneley and Lowe (T&L) model are more dependent upon the oxidation level of MoFe protein than previously suspected [R.N.F. Thorneley, D.J. Lowe, Biochem. J. 224 (1984) 887-894], at least when applied to Av and Cp. Calculation of Hill coefficients gave values of 1.7-1.9, suggesting a highly cooperative Fe-MoFe protein interaction in both Av and Cp nitrogenase catalysis. The T&L model lacks analytical solutions [R.N.F. Thorneley, D.J. Lowe, Biochem. J. 215 (1983) 393-404], so the ease of its application to experimental data is limited. To facilitate the study of steady state kinetic data for H(2) evolution, analytical equations are derived from a different mechanism for nitrogenase activity, similar to that of Bergersen and Turner [Biochem. J. 131 (1973) 61-75]. This alternative cooperative model assumes that two Fe proteins interact with one MoFe protein active site. The derived rate laws for this mechanism were fitted to the observed sigmoidal behavior for low Fe:MoFe ratios (<0.4), as well as to the commonly observed hyperbolic behavior for high values of Fe:MoFe for both Av and Cp.     

Stoichiometry and spectral properties of the MoFe cofactor and noncofactor redox centers in the MoFe protein of nitrogenase from Azotobacter vinelandii

Reductive EPR and optical titrations of oxidized MoFe protein using reduced methyl viologen as reductant were used to quantitate the stoichiometry of the various spectroscopically and electrochemically distinct redox centers in the oxidized MoFe protein. Three centers were found to correlate with the EPR signal development (MoFe cofactor centers), and three centers were found to be independent of the EPR signal (P clusters) but to demonstrate distinct optical and kinetic properties. Oxidative EPR and optical titrations of reduced MoFe protein are reported which support the presence of three P-cluster centers. The optical titrations show a distinct change in kinetic behavior between the MoFe cofactor and P-cluster centers. Controlled potential coulometry demonstrates that incremental oxidation of reduced protein by methylene blue, thionine, or indigodisulfonate occurs specifically at three P-cluster sites. Subsequent oxidation by methylene blue and thionine (but not indigodisulfonate) causes the EPR signal to disappear. Three P-cluster sites, two EPR sites, and one presently uncharacterized site are suggested by the results of this study.     

Crystallographic analysis of the MoFe protein of nitrogenase from a nifV mutant of Klebsiella pneumoniae identifies citrate as a ligand to the molybdenum of iron molybdenum cofactor (FeMoco)

The x-ray crystal structure of NifV(-) Klebsiella pneumoniae nitrogenase MoFe protein (NifV(-) Kp1) has been determined and refined to a resolution of 1.9 A. This is the first structure for a nitrogenase MoFe protein with an altered cofactor. Moreover, it is the first direct evidence that the organic acid citrate is not just present, but replaces homocitrate as a ligand to the molybdenum atom of the iron molybdenum cofactor (FeMoco). Subsequent refinement of the structure revealed that the citrate was present at reduced occupancy.     

Mapping the interaction of the [2Fe-2S] Clostridium pasteurianum ferredoxin with nitrogenase MoFe protein

The [2Fe-2S] ferredoxin from Clostridium pasteurianum had previously been shown to interact specifically with the nitrogenase MoFe protein, and electrostatic forces were found to be important contributors to the interaction. This phenomenon has now been analyzed in detail by using ferredoxin variants in which charge inversions or cancellations were introduced on all charged residues. The mutated forms of the ferredoxin were covalently cross-linked to the MoFe protein. The reaction products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their nitrogenase activity was measured. The latter displayed a consistent inverse correlation with the amount of cross-linked MoFe protein. The data allowed an unambiguous identification of the ferredoxin residues (glutamates 31, 34, 38, 39, 84, 85) that are involved in the interaction with the MoFe protein. Furthermore, whereas the wild-type ferredoxin yielded approximately equal amounts of cross-linked products with the alpha and beta subunits of the MoFe protein, some of its molecular variants displayed a differential decrease of reactivity towards one or the other of these subunits. The positions on the ferredoxin molecule of the residues interacting with the MoFe protein were determined using the recently elucidated crystal structure of the homologous [2Fe-2S] ferredoxin from Aquifex aeolicus.     

A novel S = 3/2 EPR signal associated with native Fe-proteins of nitrogenase

In addition to their g = 1.94 EPR signal, nitrogenase Fe-proteins from Azotobacter vinelandii, Azotobacter chroococcum and Klebsiella pneumoniae exhibit a weak EPR signal with g approximately equal to 5. Temperature dependence of the signal was consistent with an S = 3/2 system with negative zero-field splitting, D = -5 +/- 0.7 cm-1. The ms = +/- 3/2 ground state doublet gives rise to a transition with geff = 5.90 and the transition within the excited ms = +/- 1/2 doublet has a split geff = 4.8, 3.4. Quantitation gave 0.6 to 0.8 spin . mol-1 which summed with the spin intensity of the S = 1/2 g = 1.94 line to roughly 1 spin/mol. MgATP and MgADP decreased the intensity of the S = 3/2 signal with no concomitant changes in intensity of the S = 1/2 signal.     

Quantitative EPR of an S = 7/2 system in thionine-oxidized MoFe proteins of nitrogenase. A redefinition of the P-cluster concept

Thionine-oxidized nitrogenase MoFe proteins from Azotobacter vinelandii. Azotobacter chroococcum and Klebsiella pneumoniae exhibit excited-state EPR signals with g = 10.4, 5.8 and 5.5 with a maximal amplitude in the temperature range of 20-50 K. The magnitude of these effective g values, combined with the temperature dependence of the peak area at g = 10.4 from 12 K to 86 K, are consistent with an S = 7/2 system with spin Hamiltonian parameters D = -3.7 +/- 0.7 cm-1, [E] = 0.16 +/- 0.01 cm-1 and g = 2.00. This interpretation predicts nine additional effective g values some of which have been detected as broad features of low intensity at g approximately 10, approximately 2.5 and approximately 1.8. The S = 7/2 EPR is ascribed to the multi-iron exchange-coupled entities known as the P clusters. Quantification relative to the S = 3/2 EPR signal from dithionite-reduced MoFe protein indicates a stoichiometry of one P cluster per FeMo cofactor. Two possible interpretations for these observations, together with data from the literature, are proposed. In the first model there are two P clusters per tetrameric MoFe protein. Each P cluster encompasses approximately 8Fe ions and releases a total of three electrons on oxidation with excess thionine. In the second model the conventional view of four P clusters, each containing approximately 4Fe, is retained. This alternative requires that following one-electron oxidation, the P clusters factorize into two populations, Pa and Pb, only one of which is further oxidized with thionine resulting in the S = 7/2 system. Both models require eight-electron oxidation of tetrameric MoFe protein to reach the S = 7/2 state.     

Structure-function relationships in the alpha subunit of Klebsiella pneumoniae nitrogenase MoFe protein from analysis of nifD mutants.

Crude extracts of wild-type, nitrogenase-derepressed Klebsiella pneumoniae fractionated by nondenaturing gel electrophoresis contain, in addition to the major form of the MoFe protein, two minor variants of lower electrophoretic mobility. Of seven Nif- mutants of K. pneumoniae with nonpolar point mutations in nifD (encoding the alpha subunit of Kp1), three exhibit a wild-type-like electrophoretic pattern, whereas in the remaining four, the slowest-migrating form becomes the predominant species. Amino acid substitutions in mutants of the first type are located in the N terminus of NifD and include Gly-85 to Arg (UN1661), Glu-121 to Lys (UN1649), and Gly-161 to Asp (UN1683). Mutations of the second type are Gly-186 to Asp (UN1648), Gly-195 to Glu (UN1680), Ser-443 to Pro (UN1793), and Gly-455 to Asp (UN1650). Six of the mutated residues show interspecies conservation, three are close to conserved cysteines, and two are located next to conserved histidines. Based on evidence pointing to the possibility that the lowest-mobility form lacks the iron-molybdenum cofactor, these results provide insights into the functional significance of specific sites in the alpha subunit of the MoFe protein.     

The inactive MoFe protein (NifB-Kp1) of the nitrogenase from nifB mutants of Klebsiella pneumoniae. Its interaction with FeMo-cofactor and the properties of the active MoFe protein formed.

The inactive MoFe protein (NifB-Kp1) of nitrogenase from nifB mutants of Klebsiella pneumoniae may be activated by addition of the iron-molybdenum cofactor (FeMoco) extracted from active MoFe protein (Kp1). However, when apparently saturated with FeMoco, our preparations of NifB-Kp1 yielded activated protein, Kp1-asm, with a specific activity that was at best only 40% of that expected. This was not due to degradation of Kp1-asm, NifB-Kp1 or FeMoco during the activation reaction. Nor could activation be enhanced by addition of other nif-gene products or other proteins. Whereas fully active Kp1 contains 2 FeMoco/molecule, apparent saturation of our NifB-Kp1 preparations required the binding of only 0.4-0.65 FeMoco/molecule. By using chromatography Kp1-asm could be largely resolved from NifB-Kp1 that had not been activated. However, we were unable to isolate fully active MoFe protein (i.e. Kp1-asm containing 2 FeMoco/molecule) from solutions of NifB-Kp1 activated with FeMoco. The maximum activity/ng-atom of total Mo obtained for our purified Kp1-asm was approximately half the maximum activity for FeMoco. Since all NifB-Kp1 preparations contained some Mo, we suggest that FeMoco activated only those NifB-Kp1 molecules already containing one atom of (non-FeMoco) Mo, thus forming Kp1-asm with 2 Mo but only 1 FeMoco/molecule. Kp1-asm was identical with normal Kp1 in terms of its Mr, stability, e.p.r. signals, pattern of substrate reductions, CO inhibition and ATP/2e ratio. In addition, for preparations of differing specific activity, there was a constant and identical relationship between the e.p.r. signal intensity (from FeMoco) and the activity of both Kp1 and Kp1-asm. Assuming the above hypothesis on the structure of Kp1-asm, these data demonstrate that the two FeMoco sites in wild-type Kp1 operate independently.     

Low-temperature magnetic-circular-dichroism spectroscopy of the iron-molybdenum cofactor and the complementary cofactor-less MoFe protein of Klebsiella pneumoniae nitrogenase

The major metal clusters of the MoFe protein, Kpl , of Klebsiella pneumoniae nitrogenase were characterized separately by low-temperature magnetic-circular-dichroism spectroscopy. The spectra and magnetization curves of the extracted iron-molybdenum cofactor, FeMoco , and of 'P' clusters in NifB - Kpl , the inactive, FeMoco -less, MoFo protein from an nifB mutant, were measured and compared with those of the holoprotein. (When FeMoco and NifB - Kpl are combined, active Kpl is formed.) Reduced NifB - Kpl had a spectrum with a weak, paramagnetic, component superimposed on a diamagnetic background. The paramagnetic component was assigned to a contaminating, e.p.r.-active, species. Thionine-oxidized NifB - Kpl had a spectrum and magnetization properties very similar to those of thionine-oxidized Kpl , demonstrating that the 'P' clusters are not significantly affected by the absence of the FeMoco clusters. The spectra of reduced isolated FeMoco had similar magnetization curves but sharper features and higher intensities than those of this centre in dithionite-reduced Kpl . Furthermore, a shoulder near 580 nm in the Kpl spectrum was absent from that of FeMoco . This may be due to the loss of a ligand or to a change in symmetry of the FeMoco cluster on extraction.