Klebsiella pneumoniae nitrogenase: pre-steady-state absorbance changes show that redox changes occur in the MoFe protein that depend on substrate and component protein ratio; a role for P-centres in reducing dinitrogen?
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ABSTRACT:
SUBMITTER: Lowe DJ
PROVIDER: S-EPMC1134273 | biostudies-other | 1993 May
REPOSITORIES: biostudies-other
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Project description: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.
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Project description:Our previous investigation of substrates reduction catalyzed by nitrogenase suggested that ?-Ile423 of MoFe protein possibly functions as an electron transfer gate to Mo site of active center-"FeMoco". Amino acid residue ?-Lys424 connects directly to ?-Ile423, and they are located in the same ?-helix (?423-431). In the present study, function of ?-Lys424 was investigated by replacing it with Arg (alkaline, like Lys), Gln (neutral), Glu (acidic), and Ala (neutral) through site-directed mutagenesis and homologous recombination. The mutants were, respectively, termed 424R, 424Q, 424E, and 424A. Studies of diazotrophic cell growth, cytological, and enzymatic properties indicated that none of the substitutions altered the secondary structure of MoFe protein, or normal expression of nifA, nifL, and nifD. Substitution of alkaline amino acid (i.e., 424R) maintained acetylene (C2H2) and proton (H+) reduction activities at normal levels similar to that of wild-type (WT), because its FeMoco content did not reduce. In contrast, substitution of acidic or neutral amino acid (i.e., 424Q, 424E, 424A) impaired the catalytic activity of nitrogenase to varying degrees. Combination of MoFe protein structural simulation and the results of a series of experiments, the function of ?-Lys424 in ensuring insertion of FeMoco to MoFe protein was further confirmed, and the contribution of ?-Lys424 in maintaining low potential of the microenvironment causing efficient catalytic activity of nitrogenase was demonstrated.
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Project description:The engineering of transgenic organisms with the ability to fix nitrogen is an attractive possibility. However, oxygen sensitivity of nitrogenase, mainly conferred by the reductase component (NifH)2 , is an imminent problem. Nitrogenase-like enzymes involved in coenzyme F430 and chlorophyll biosynthesis utilize the highly homologous reductases (CfbC)2 and (ChlL)2 , respectively. Chimeric protein-protein interactions of these reductases with the catalytic component of nitrogenase (MoFe protein) did not support nitrogenase activity. Nucleotide-dependent association and dissociation of these complexes was investigated, but (CfbC)2 and wild-type (ChlL)2 showed no modulation of the binding affinity. By contrast, the interaction between the (ChlL)2 mutant Y127S and the MoFe protein was markedly increased in the presence of ATP (or ATP analogues) and reduced in the ADP state. Upon formation of the octameric (ChlL)2 MoFe(ChlL)2 complex, the ATPase activity of this variant is triggered, as seen in the homologous nitrogenase system. Thus, the described reductase(s) might be an attractive tool for further elucidation of the diverse functions of (NifH)2 and the rational design of a more robust reductase.
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Project description:A molybdenum L-edge X-ray absorption spectroscopy (XAS) study is presented for native and oxidized MoFe protein of nitrogenase as well as Mo-Fe model compounds. Recently collected data on MoFe protein (in oxidized and reduced forms) is compared to previously published Mo XAS data on the isolated FeMo cofactor in NMF solution and put in context of the recent Mo K-edge XAS study, which showed a MoIII assignment for the molybdenum atom in FeMoco. The L3-edge data are interpreted within a simple ligand-field model, from which a time-dependent density functional theory (TDDFT) approach is proposed as a way to provide further insights into the analysis of the molybdenum L3-edges. The calculated results reproduce well the relative spectral trends that are observed experimentally. Ultimately, these results give further support for the MoIII assignment in protein-bound FeMoco, as well as isolated FeMoco.
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Project description:Another iron in the fire: X-ray anomalous diffraction studies on the nitrogenase MoFe protein show the presence of a mononuclear iron site, designated as Fe16, which was previously identified as either Ca(2+) or Mg(2+). The position of the absorption edge indicates that this site is in the oxidation state +2. The high sequence conservation of the residues coordinated to Fe16 emphasizes the potential importance of the site in nitrogenase.
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Project description: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.
| S-EPMC2819195 | biostudies-literature
Project description:The reduction of dinitrogen to ammonia catalyzed by nitrogenase involves a complex series of events, including ATP hydrolysis, electron transfer, and activation of metal clusters for N2 reduction. Early evidence shows that an essential part of the mechanism involves transducing information between the nitrogenase component proteins through conformational dynamics. Here, millisecond time-resolved hydrogen-deuterium exchange mass spectrometry was used to unravel peptide-level protein motion on the time scale of catalysis of Mo-dependent nitrogenase from Azotobacter vinelandii. Normal mode analysis calculations complemented this data, providing insights into the specific signal transduction pathways that relay information across protein interfaces at distances spanning 100 Å. Together, these results show that conformational changes induced by protein docking are rapidly transduced to the active site, suggesting a specific mechanism for activating the metal cofactor in the enzyme active site.
| S-EPMC10657360 | biostudies-literature
Project description:The X-ray crystal structure of the nitrogenase MoFe protein from Clostridium pasteurianum (Cp1) has been determined at 1.08 Å resolution by multiwavelength anomalous diffraction phasing. Cp1 and the ortholog from Azotobacter vinelandii (Av1) represent two distinct families of nitrogenases, differing primarily by a long insertion in the α-subunit and a deletion in the β-subunit of Cp1 relative to Av1. Comparison of these two MoFe protein structures at atomic resolution reveals conserved structural arrangements that are significant to the function of nitrogenase. The FeMo cofactors defining the active sites of the MoFe protein are essentially identical between the two proteins. The surrounding environment is also highly conserved, suggesting that this structural arrangement is crucial for nitrogen reduction. The P clusters are likewise similar, although the surrounding protein and solvent environment is less conserved relative to that of the FeMo cofactor. The P cluster and FeMo cofactor in Av1 and Cp1 are connected through a conserved water tunnel surrounded by similar secondary-structure elements. The long α-subunit insertion loop occludes the presumed Fe protein docking surface on Cp1 with few contacts to the remainder of the protein. This makes it plausible that this loop is repositioned to open up the Fe protein docking surface for complex formation.
| S-EPMC4321486 | biostudies-literature
Project description:Proton uptake accompanies the reduction of all known substrates by nitrogenase. As a consequence, a higher pH should limit the availability of protons as a substrate essential for turnover, thereby increasing the proportion of more highly reduced forms of the enzyme for further study. The utility of the high-pH approach would appear to be problematic in view of the observation reported by Pham and Burgess [(1993) Biochemistry 32, 13725-13731] that the MoFe-protein undergoes irreversible protein denaturation above pH 8.65. In contrast, we found by both enzyme activity and crystallographic analyses that the MoFe-protein is stable when incubated at pH 9.5. We did observe, however, that at higher pHs and under turnover conditions, the MoFe-protein is slowly inactivated. While a normal, albeit low, level of substrate reduction occurs under these conditions, the MoFe-protein undergoes a complex transformation; initially, the enzyme is reversibly inhibited for substrate reduction at pH 9.5, yet in a second, slower process, the MoFe-protein becomes irreversibly inactivated as measured by substrate reduction activity at the optimal pH of 7.8. The final inactivated MoFe-protein has an increased hydrodynamic radius compared to that of the native MoFe-protein, yet it has a full complement of iron and molybdenum. Significantly, the modified MoFe-protein retains the ability to specifically interact with its nitrogenase partner, the Fe-protein, as judged by the support of ATP hydrolysis and by formation of a tight complex with the Fe-protein in the presence of ATP and aluminum fluoride. The turnover-dependent inactivation coupled to conformational change suggests a mechanism-based transformation that may provide a new probe of nitrogenase catalysis.
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Project description:Nitrogenase reduces dinitrogen (N2) by six electrons and six protons at an active-site metallocluster called FeMo cofactor, to yield two ammonia molecules. Insights into the mechanism of substrate reduction by nitrogenase have come from recent successes in trapping and characterizing intermediates generated during the reduction of protons as well as nitrogenous and alkyne substrates by MoFe proteins with amino acid substitutions. Here, we describe an intermediate generated at a high concentration during reduction of the natural nitrogenase substrate, N2, by wild-type MoFe protein, providing evidence that it contains N2 bound to the active-site FeMo cofactor. When MoFe protein was frozen at 77 K during steady-state turnover with N2, the S = 3/2 EPR signal (g = [4.3, 3.64, 2.00]) arising from the resting state of FeMo cofactor was observed to convert to a rhombic, S = 1/2, signal (g = [2.08, 1.99, 1.97]). The intensity of the N2-dependent EPR signal increased with increasing N2 partial pressure, reaching a maximum intensity of approximately 20% of that of the original FeMo cofactor signal at > or = 0.2 atm N2. An almost complete loss of resting FeMo cofactor signal in this sample implies that the remainder of the enzyme has been reduced to an EPR-silent intermediate state. The N2-dependent EPR signal intensity also varied with the ratio of Fe protein to MoFe protein (electron flux through nitrogenase), with the maximum signal intensity observed with a ratio of 2:1 (1:1 Fe protein:FeMo cofactor) or higher. The pH optimum for the signal was 7.1. The N2-dependent EPR signal intensity exhibited a linear dependence on the square root of the EPR microwave power in contrast to the nonlinear response of signal intensity observed for hydrazine-, diazene-, and methyldiazene-trapped states. 15N ENDOR spectroscopic analysis of MoFe protein captured during turnover with 15N2 revealed a 15N nuclear spin coupled to the FeMo cofactor with a hyperfine tensor A = [0.9, 1.4, 0.45] MHz establishing that an N2-derived species was trapped on the FeMo cofactor. The observation of a single type of 15N-coupled nucleus from the field dependence, along with the absence of an associated exchangeable 1H ENDOR signal, is consistent with an N2 molecule bound end-on to the FeMo cofactor.
| S-EPMC2814451 | biostudies-literature