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PubMed1991

Involvement of GroEL in nif gene regulation and nitrogenase assembly.

Several approaches were used to study the role of GroEL, the prototype chaperonin, in the nitrogen fixation (nif) system. An Escherichia coli groEL mutant transformed with the Klebsiella pneumoniae nif gene cluster accumulated very low to nondetectable levels of nitrogenase components compared with the isogenic wild-type strain or the mutant cotransformed with the wild-type groE operon. In K. pneumoniae, overexpression of the E. coli groE operon markedly accelerated the rate of appearance of the MoFe protein and its constituent polypeptides after the start of derepression. The groEL mutation in E. coli decreased NifA-dependent beta-galactosidase expression from the nifH promoter but did not affect the constitutive expression of nifA from the tet promoter of ntr-controlled expression from the nifLA promoter. The possibility that GroEL is required for the correct folding of NifA was supported by coimmunoprecipitation of NifA with anti-GroEL antibodies. Kinetic analyses of nitrogenase assembly in 35S pulse-chased K. pneumoniae pointed to the existence of high-molecular-weight intermediates in MoFe protein assembly and demonstrated the transient binding of newly synthesized NifH and NifDK to GroEL. Overall, these results indicate that GroEL fulfills both regulatory and structural functions in the nif system.

Bacterial Proteins↗
PubMed1994

Reciprocal light-dark transcriptional control of nif and rbc expression and light-dependent posttranslational control of nitrogenase activity in Synechococcus sp. strain RF-1.

Synechococcus sp. strain RF-1 exhibits a circadian rhythm of N2 fixation when cells are grown under a light-dark cycle, with nitrogenase activity observed only during the dark period. This dark-dependent activity correlated with nif gene transcription in strain RF-1. By using antibodies against dinitrogenase reductase (the Fe protein of the nitrogenase complex), it was found that there was a distinct shift in the mobility of this protein on sodium dodecyl sulfate gels during the light-dark cycle. The Fe protein was present only when cells were incubated in the dark. Upon illumination, there was a conversion of all Fe protein to a modified form, after which it rapidly disappeared from extracts. These studies indicated that all nitrogenase activity present during the dark cycle resulted from de novo synthesis of nitrogenase. Upon entering the light phase, cells appeared to quickly degrade the modified form of Fe protein, perhaps as a result of activating or inducing a protease. By contrast, transcription of the rbcL gene, which encodes the catalytic subunit of the key enzyme of CO2 fixation (a light-dependent process), was enhanced in the light.

Bacterial Proteins↗
PubMed1994

Nucleotide and divalent cation specificity of in vitro iron-molybdenum cofactor synthesis.

The nucleotide and divalent cation requirements of the in vitro iron-molybdenum cofactor (FeMo-co) synthesis system have been compared with those of substrate reduction by nitrogenase. The FeMo-co synthesis system specifically requires ATP, whereas both 1,N6-etheno-ATP and 2'-deoxy-ATP function in place of ATP in substrate reduction (M. F. Weston, S. Kotake, and L. C. Davis, Arch. Biochem. Biophys. 225:809-817, 1983). Mn2+, Ca2+, and Fe2+ substitute for Mg2+ to various extents in in vitro FeMo-co synthesis, whereas Ca2+ is ineffective in substrate reduction by nitrogenase. The observed differences in the nucleotide and divalent cation specificities suggest a role(s) for the nucleotide and divalent cation in in vitro FeMo-co synthesis that is distinct from their role(s) in substrate reduction.

Adenosine Triphosphate↗
PubMed1998

Circadian rhythm of nitrogenase gene expression in the diazotrophic filamentous nonheterocystous cyanobacterium Trichodesmium sp. strain IMS 101.

Recent studies suggested that the daily cycle of nitrogen fixation activity in the marine filamentous nonheterocystous cyanobacterium Trichodesmium sp. is controlled by a circadian rhythm. In this study, we evaluated the rhythm of nitrogen fixation in Trichodesmium sp. strain IMS 101 by using the three criteria for an endogenous rhythm. Nitrogenase transcript abundance oscillated with a period of approximately 24 h, and the cycle was maintained even under constant light conditions. The cyclic pattern of transcript abundance was maintained when the culture was grown at 24 and 28.5 degrees C, although the period was slightly longer (26 h) at the higher temperature. The cycle of gene expression could be entrained with light-dark cues. Results of inhibitor experiments indicated that transcript abundance was regulated primarily by transcription initiation, rather than by degradation. The circadian rhythm, the first conclusively demonstrated endogenous rhythm in a filamentous cyanobacterium, was also reflected in nitrogenase MoFe protein abundance and patterns of Fe protein posttranslational modification-demodification.

Bacterial Proteins↗
PubMed1999

Requirement of NifX and other nif proteins for in vitro biosynthesis of the iron-molybdenum cofactor of nitrogenase.

The iron-molybdenum cofactor (FeMo-co) of nitrogenase contains molybdenum, iron, sulfur, and homocitrate in a ratio of 1:7:9:1. In vitro synthesis of FeMo-co has been established, and the reaction requires an ATP-regenerating system, dithionite, molybdate, homocitrate, and at least NifB-co (the metabolic product of NifB), NifNE, and dinitrogenase reductase (NifH). The typical in vitro FeMo-co synthesis reaction involves mixing extracts from two different mutant strains of Azotobacter vinelandii defective in the biosynthesis of cofactor or an extract of a mutant strain complemented with the purified missing component. Surprisingly, the in vitro synthesis of FeMo-co with only purified components failed to generate significant FeMo-co, suggesting the requirement for one or more other components. Complementation of these assays with extracts of various mutant strains demonstrated that NifX has a role in synthesis of FeMo-co. In vitro synthesis of FeMo-co with purified components is stimulated approximately threefold by purified NifX. Complementation of these assays with extracts of A. vinelandii DJ42. 48 (DeltanifENX DeltavnfE) results in a 12- to 15-fold stimulation of in vitro FeMo-co synthesis activity. These data also demonstrate that apart from the NifX some other component(s) is required for the cofactor synthesis. The in vitro synthesis of FeMo-co with purified components has allowed the detection, purification, and identification of an additional component(s) required for the synthesis of cofactor.

Azotobacter vinelandii↗
PubMed2001

Properties of a thermostable nitrate reductase from the hyperthermophilic archaeon Pyrobaculum aerophilum.

The nitrate reductase of the hyperthermophilic archaeon Pyrobaculum aerophilum was purified 137-fold from the cytoplasmic membrane. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the enzyme complex consists of three subunits with apparent molecular weights of 130,000, 52,000, and 32,000. The enzyme contained molybdenum (0.8-mol/mol complex), iron (15.4-mol/mol complex) and cytochrome b (0.49-mol/mol complex) as cofactors. The P. aerophilum nitrate reductase distinguishes itself from nitrate reductases of mesophilic bacteria and archaea by its very high specific activity using reduced benzyl viologen as the electron donor (V(max) with nitrate, 1,162 s(-1) (326 U/mg); V(max) with chlorate, 1,348 s(-1) (378 U/mg) [assayed at 75 degrees C]). The K(m) values for nitrate and chlorate were 58 and 140 microM, respectively. Azide was a competitive inhibitor and cyanide was a noncompetitive inhibitor of the nitrate reductase activity. The temperature optimum for activity was > 95 degrees C. When incubated at 100 degrees C, the purified nitrate reductase had a half-life of 1.5 h. This study constitutes the first description of a nitrate reductase from a hyperthermophilic archaeon.

Enzyme Stability↗
PubMed1980

Catalysis of exchange of terminal phosphate groups of ATP and ADP by purified nitrogenase proteins.

A crude Azotobacter nitrogenase complex contained a highly active adenylate kinase which caused rapid equilibration of AMP, ADP, and ATP. Purified molybdenum-iron protein preparations also contained measurable adenylate kinase activity which could be removed by adsorption and elution from hydroxylapatite. Independent of adenylate kinase, the highly purified molybdenum-iron protein from both Klebsiella pneumoniae and Azotobacter chroococcum catalyzed the exchange of [32P]orthophosphate with the terminal phosphate of ATP or ADP. The exchange labeling of ATP was stimulated by ferricyanide ion due to the inhibition of ATP hydrolysis linked to substrate reductions which cannot occur in the absence of reductant. This exchange reaction is responsible for reported ATP synthesis by crude nitrogenase preparations. Binding of ATP labeled with 32P in the terminal phosphate group was measured directly with concentrated solutions of the molybdenum-iron nitrogenase protein from K. pneumoniae by the column gel filtration method. The protein was saturated with ATP at a calculated ratio of 4.0 +/- 0.3 mol ATP/mol protein; half-saturation of 220 microM protein occurred at 600 +/- 100 microM. The interactions between adenine nucleotides and the molybdenum-iron protein suggest the involvement of the nucleotides in a role distinct from the established reactions with the iron protein of the nitrogenase complex. A dual role for ATP in the reduction of dinitrogen by isolated nitrogenase proteins would be consistent with the existence of binding sites for ATP on both the iron and molybdenum-iron proteins.

Adenosine Diphosphate↗
PubMed1995

Nitrogenase structure and function: a biochemical-genetic perspective.

Biological nitrogen fixation is catalyzed by nitrogenase, an enzyme composed of two component proteins called the Fe protein and the MoFe protein. During catalysis, electrons are delivered one at a time from the Fe protein to the MoFe protein in a process involving component-protein association and dissociation and hydrolysis of at least two MgATP for each electron transfer. The Fe protein contains the sites for MgATP binding and hydrolysis, whereas the site for substrate binding and reduction is located on the MoFe protein. Among the important aspects of nitrogenase enzymology discussed here are (a) the structures of the metal centers that participate in electron transfer, (b) the organization of the metalloclusters within the polypeptides and their contributions to substrate binding and electron transfer, (c) the nature of the dynamic interactions between the two component proteins that lead to nucleotide hydrolysis and intermolecular electron transfer, (d) the mechanism by which the multiple electrons necessary for substrate reduction are distributed within the MoFe protein, (e) the nature of the intramolecular electron path within the MoFe protein, and (f) where and how substrate and various inhibitors become bound to the substrate-reduction site. This chapter summarizes biochemical-genetic strategies used to address these questions and discussed them in the context of the recently proposed three-dimensional models for both the Fe protein and MoFe protein from Azotobacter vinelandii.

Metalloproteins↗
PubMed1994

Biosynthesis of the iron-molybdenum cofactor of nitrogenase.

The iron-molybdenum cofactor (FeMo-co) of nitrogenase is a unique molybdenum-containing prosthetic group that has been proposed to form an integral part of the active site of dinitrogenase. In Klebsiella pneumoniae, at least six nif (nitrogen fixation) gene products are required for the biosynthesis of FeMo-co, including NIFB, NIFNE, NIFH, NIFQ, and NIFV. An in vitro system for the synthesis of FeMo-co, which requires MgATP, molybdate, homocitrate, and at least the products of nifN, E, B, and H, has provided an enzymatic assay for the purification of many of the gene products required for FeMo-co biosynthesis. Although the structure of the cofactor has been solved recently, much about the biosynthetic pathway remains unknown. This article reviews what is known about the various components required for FeMo-co biosynthesis.

Genes, Bacterial↗
PubMed1999

MgATP-independent hydrogen evolution catalysed by nitrogenase: an explanation for the missing electron(s) in the MgADP-AlF4 transition-state complex.

When the MoFe (Kp1) and Fe (Kp2) component proteins of Klebsiella pneumoniae nitrogenase are incubated with MgADP and AlF4(-) in the presence of dithionite as a reducing agent, a stable putative transition-state complex is produced [Yousafzai and Eady (1997) Biochem. J. 326, 637-640]. Surprisingly, the EPR signal associated with reduced Kp2 is not detectable, but Kp1 retains the S=3/2 EPR signal arising from the dithionite reduced state of the MoFe cofactor centre of the protein. This is consistent with the [Fe4S4] centre of the Fe protein in the complex being oxidized, and similar observations have been made with the complex of Azotobacter vinelandii [Spee, Arendsen, Wassink, Marritt, Hagen and Haaker (1998) FEBS Lett. 432, 55-58]. No satisfactory explanation for the fate of the electrons lost by Kp2 has been forthcoming. However, we report here that during the preparation of the MgADP-AlF4 K. pneumoniae complex under argon, H2 was evolved in amounts corresponding to one half of the FeMoco content of the Kp1 (FeMoco is the likely catalytic site of nitrogenase with a composition Mo:Fe7:S9:homocitrate). This is surprising, since activity is observed during incubation in the absence of MgATP, normally regarded as being essential for nitrogenase function, and in the presence of MgADP, a strong competitive inhibitor of nitrogenase. The formation of H2 by nitrogenase in the absence of AlF4(-) was also observed in reaction mixtures containing MgADP but not MgATP. The reaction showed saturation kinetics when Kp1 was titrated with increasing amounts of Kp2 and, at saturation, the amount of H2 formed was stoichiometric with the FeMoco content of Kp1. The dependence of the rate of formation of H2 on [MgADP] was inconsistent with the activity arising from MgATP contamination. We conclude that MgATP is not obligatory for H+ reduction by nitrogenase since MgADP supports a very low rate of hydrogen evolution.

Adenosine Diphosphate↗
PubMed1992

Nitrogenase-catalyzed ethane production and CO-sensitive hydrogen evolution from MoFe proteins having amino acid substitutions in an alpha-subunit FeMo cofactor-binding domain.

Unlike wild type, certain Mo-dependent nitrogenases, which are expressed in non-N2-fixing mutant strains of Azotobacter vinelandii and have single amino acid substitutions within a region of the MoFe protein alpha-subunit proposed to encompass an FeMo cofactor-binding domain, are able to catalyze the reduction of acetylene by both two and four electrons to yield ethylene and ethane, respectively (Scott, D. J., May, H. D., Newton, W. E., Brigle, K. E., and Dean, D. R. (1990) Nature 343, 188-190). Although the V-dependent nitrogenase is also able to catalyze the reduction of acetylene to the same two- and four-electron products (Dilworth, M. J., Eady, R. R., Robson, R. L., and Miller, R. W. (1987) Nature 327, 167-168), we find that ethane formation from acetylene catalyzed by the altered Mo-dependent nitrogenases occurs by a different mechanism, which is distinguished by: (i) an increased sensitivity to CO; (ii) the absence of a lag; and (iii) no temperature dependence of product distribution among ethylene and ethane during acetylene reduction. An altered MoFe protein, which was purified from one such mutant strain having the alpha-subunit glutaminyl 191 residue substituted by lysyl, exhibited both a changed S = 3/2 EPR spectrum and changes in the distribution of electrons to various products when compared to wild type. Also, unlike wild type, this altered MoFe protein catalyzed proton reduction that is inhibited by carbon monoxide (CO). Because proton reduction catalyzed by a nitrogenase that has a FeMo cofactor with citrate rather than homocitrate as its organic constituent (Liang, J., Madden, M., Shah, V. K., and Burris, R. H. (1990) Biochemistry 29, 8577-8581) is also inhibited by CO, the possibility arose that changes in the polypeptide environment of FeMo cofactor might have caused a rearrangement in its molecular structure or composition. However, this possibility was ruled out by biochemical reconstitution studies (using FeMo cofactor isolated from both the wild-type and altered MoFe proteins), which were monitored by EPR spectroscopy and resulting catalytic activity.

Amino Acids↗
PubMed1992

FeMo cofactor synthesis by a nifH mutant with altered MgATP reactivity.

We have characterized a Nif- mutant of Azotobacter vinelandii, designated UW91 (Shah, V. K., Davis, L. C., Gordon, J. K., Orme-Johnson, W. H., and Brill, W. J. (1973) Biochim. Biophys. Acta 292, 246-255). The specific Fe protein mutation giving rise to the Nif- phenotype was shown by DNA sequencing and site-directed mutagenesis to be the substitution of a conserved alanine at position 157 by a serine. The UW91 Fe protein was purified and shown to have a normal [4Fe-4S] cluster and normal MgATP binding activity. The substitution of alanine 157 by serine, however, prevents the MgATP-induced conformational change that occurs for the wild-type Fe protein, prevents MgATP hydrolysis, and prevents productive electron transfer to the MoFe protein. The UW91 Fe protein does bind to the MoFe protein to give a normal cross-linking pattern; however, it does not compete very successfully with wild-type Fe protein in an activity assay. The UW91 MoFe protein was also purified and characterized and shown to be indistinguishable from the wild-type protein. Thus, the substitution of Fe protein residue alanine 157 by serine does not change the Fe protein's ability to function in FeMo cofactor biosynthesis or insertion. This demonstrates that these events do not require the MgATP-induced conformational change, MgATP hydrolysis, or productive electron transfer to the MoFe protein.

Acetylene↗
PubMed1992

Electrophoretic studies on the assembly of the nitrogenase molybdenum-iron protein from the Klebsiella pneumoniae nifD and nifK gene products.

The electrophoretic properties of the molybdenum-iron (MoFe) protein component of nitrogenase and an iron-molybdenum cofactor (FeMoco)-reactivatable apoMoFe protein from Klebsiella pneumoniae were examined under anaerobic ([O2] < 5 ppm), nondenaturing conditions. In wild type K. pneumoniae extracts, two immunoreactive species migrating more slowly than purified MoFe protein were detected using anti-MoFe protein antibodies. The uppermost species comigrates with the apoMoFe protein produced by a K. pneumoniae mutant unable to synthesize FeMoco (UN106) and by Escherichia coli harboring the plasmids pVL222+pVL15 (nifHDKTYUSWZM+A). In vitro FeMoco titration of the UN106 and pVL222+pVL15 extracts increases the electrophoretic mobility of the apoMoFe protein to that of purified MoFe protein in a two-step process giving rise to a species of intermediate mobility between the apo- and holoMoFe proteins. Two-dimensional gel electrophoresis showed that a 20-kDa peptide is associated with the apoMoFe protein and with the intermediate species, but not with the holoMoFe protein. N-terminal sequencing identified this associated peptide as the nifY gene product, which we propose is acting as a temporary enforcer of the apoMoFe protein structure required for cofactor binding that is released upon FeMoco activation. This FeMoco-induced mobility shift was used to characterize the mutant apoMoFe proteins produced in E. coli as a result of deleting the various nitrogen fixation (nif) genes from the plasmid pVL222. E. coli extracts bearing plasmids deleted in nifH, nifS, nifTYUM, or nifWZM exhibit less than 10% of the apoMoFe protein activity of derepressed UN106 and contain an immunoreactive species whose electrophoretic mobility is increased upon addition of FeMoco from that of apoMoFe protein to that of holoMoFe protein in a single step. Anaerobic nondenaturing gel electrophoresis of 55Fe-labeled E. coli extracts followed by autoradiography showed that these inactive apoMoFe species do not contain iron, indicating that the P-clusters are absent. We therefore propose that NifH, S, U, W, Z, and M are all involved, to varying degrees, in P-cluster assembly. In addition, the presence of the P-clusters does appear to be necessary for the two-step FeMoco activation of the apoMoFe protein to occur.

Amino Acid Sequence↗