RESUMEN
The NifS and NifU nitrogen fixation-specific gene products are required for the full activation of both the Fe-protein and MoFe-protein of nitrogenase from Azotobacter vinelandii. Because the two nitrogenase component proteins both require the assembly of [Fe-S]-containing clusters for their activation, it has been suggested that NifS and NifU could have complementary functions in the mobilization of sulfur and iron necessary for nitrogenase-specific [Fe-S] cluster assembly. The NifS protein has been shown to have cysteine desulfurase activity and can be used to supply sulfide for the in vitro catalytic formation of [Fe-S] clusters. The NifU protein was previously purified and shown to be a homodimer with a [2Fe-2S] cluster in each subunit. In the present work, primary sequence comparisons, amino acid substitution experiments, and optical and resonance Raman spectroscopic characterization of recombinantly produced NifU and NifU fragments are used to show that NifU has a modular structure. One module is contained in approximately the N-terminal third of NifU and is shown to provide a labile rubredoxin-like ferric-binding site. Cysteine residues Cys35, Cys62, and Cys106 are necessary for binding iron in the rubredoxin-like mode and visible extinction coefficients indicate that up to one ferric ion can be bound per NifU monomer. The second module is contained in approximately the C-terminal half of NifU and provides the [2Fe-2S] cluster-binding site. Cysteine residues Cys137, Cys139, Cys172, and Cys175 provide ligands to the [2Fe-2S] cluster. The cysteines involved in ligating the mononuclear Fe in the rubredoxin-like site and those that provide the [2Fe-2S] cluster ligands are all required for the full physiological function of NifU. The only two other cysteines contained within NifU, Cys272 and Cys275, are not necessary for iron binding at either site, nor are they required for the full physiological function of NifU. The results provide the basis for a model where iron bound in labile rubredoxin-like sites within NifU is used for [Fe-S] cluster formation. The [2Fe-2S] clusters contained within NifU are proposed to have a redox function involving the release of Fe from bacterioferritin and/or the release of Fe or an [Fe-S] cluster precursor from the rubredoxin-like binding site.
Asunto(s)
Proteínas Bacterianas/química , Genes Bacterianos , Hierro/metabolismo , Secuencia de Aminoácidos , Azotobacter vinelandii/genética , Azotobacter vinelandii/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Cisteína/química , ADN/química , ADN/genética , Electroforesis en Gel de Poliacrilamida , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fijación del Nitrógeno/genética , Plásmidos/química , Plásmidos/genética , Unión Proteica/genética , Espectrofotometría Ultravioleta , Espectrometría RamanRESUMEN
The NifS and NifU proteins from Azotobacter vinelandii are required for the full activation of nitrogenase. NifS is a homodimeric cysteine desulfurase that supplies the inorganic sulfide necessary for formation of the Fe-S clusters contained within the nitrogenase component proteins. NifU has been suggested to complement NifS either by mobilizing the Fe necessary for nitrogenase Fe-S cluster formation or by providing an intermediate Fe-S cluster assembly site. As isolated, the homodimeric NifU protein contains one [2Fe-2S](2+, +) cluster per subunit, which is referred to as the permanent cluster. In this report, we show that NifU is able to interact with NifS and that a second, transient [2Fe-2S] cluster can be assembled within NifU in vitro when incubated in the presence of ferric ion, L-cysteine, and catalytic amounts of NifS. Approximately one transient [2Fe-2S] cluster is assembled per homodimer. The transient [2Fe-2S] cluster species is labile and rapidly released on reduction. We propose that transient [2Fe-2S] cluster units are formed on NifU and then released to supply the inorganic iron and sulfur necessary for maturation of the nitrogenase component proteins. The role of the permanent [2Fe-2S] clusters contained within NifU is not yet known, but they could have a redox function involving either the formation or release of transient [2Fe-2S] cluster units assembled on NifU. Because homologs to both NifU and NifS, respectively designated IscU and IscS, are found in non-nitrogen fixing organisms, it is possible that the function of NifU proposed here could represent a general mechanism for the maturation of Fe-S cluster-containing proteins.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Hierro-Azufre/química , Sustitución de Aminoácidos , Azotobacter vinelandii/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Hierro/química , Sustancias Macromoleculares , Oxidación-Reducción , Espectrofotometría Ultravioleta , Espectrometría RamanRESUMEN
A genetic strategy was developed for the isolation of a mutant strain of Azotobacter vinelandii that exhibits in vivo nitrogenase activity resistant to inhibition by acetylene. Examination of the kinetic features of the altered nitrogenase MoFe protein produced by this strain, which has serine substituted for the alpha-subunit Gly(69) residue, is consistent with other studies that indicate the MoFe protein normally contains at least two acetylene binding/reduction sites. The first of these is a high affinity site and is the one primarily accessed during typical acetylene reduction assays. Results of the present work indicate that this acetylene binding/reduction site is not directly relevant to the mechanism of nitrogen reduction because it can be eliminated or severely altered without significantly affecting nitrogen reduction. Elimination of this site also results in the manifestation of a low affinity acetylene-binding site to which both acetylene and nitrogen are able to bind with approximately the same affinity. In contrast to the normal enzyme, nitrogen and acetylene binding to the altered MoFe protein are mutually competitive. The location of the alpha-Ser(69) substitution is interpreted to indicate that the 4Fe-4S face of the FeMo cofactor capped by the alpha-subunit Val(70) residue is the most likely region within FeMo cofactor to which acetylene binds with high affinity.
Asunto(s)
Acetileno/farmacología , Azotobacter vinelandii/enzimología , Inhibidores Enzimáticos/farmacología , Nitrogenasa/química , Acetileno/metabolismo , Sitios de Unión , Nitrogenasa/antagonistas & inhibidores , Nitrogenasa/aislamiento & purificación , Fenotipo , Relación Estructura-ActividadRESUMEN
An enzyme having the same L-cysteine desulfurization activity previously described for the NifS protein was purified from a strain of Azotobacter vinelandii deleted for the nifS gene. This protein was designated IscS to indicate its proposed role in iron-sulfur cluster assembly. Like NifS, IscS is a pyridoxal-phosphate containing homodimer. Information gained from microsequencing of oligopeptides obtained by tryptic digestion of purified IscS was used to design a strategy for isolation and DNA sequence analysis of a 7,886-base pair A. vinelandii genomic segment that includes the iscS gene. The iscS gene is contained within a gene cluster that includes homologs to nifU and another gene contained within the major nif cluster of A. vinelandii previously designated orf6. These genes have been designated iscU and iscA, respectively. Information available from complete genome sequences of Escherichia coli and Hemophilus influenzae reveals that they also encode iscSUA gene clusters. A wide conservation of iscSUA genes in nature and evidence that NifU and NifS participate in the mobilization of iron and sulfur for nitrogenase-specific iron-sulfur cluster formation suggest that the products of the iscSUA genes could play a general role in the formation or repair of iron-sulfur clusters. The proposal that IscS is involved in mobilization of sulfur for iron-sulfur cluster formation in A. vinelandii is supported by the presence of a cysE-like homolog in another gene cluster located immediately upstream from the one containing the iscSUA genes. O-Acetylserine synthase is the product of the cysE gene, and it catalyzes the rate-limiting step in cysteine biosynthesis. A similar cysE-like gene is also located within the nif gene cluster of A. vinelandii. The likely role of such cysE-like gene products is to increase the cysteine pool needed for iron-sulfur cluster formation. Another feature of the iscSUA gene cluster region from A. vinelandii is that E. coli genes previously designated as hscB, hscA, and fdx are located immediately downstream from, and are probably co-transcribed with, the iscSUA genes. The hscB, hscA, and fdx genes are also located adjacent to the iscSUA genes in both E. coli and H. influenzae. The E. coli hscA and hscB gene products have previously been shown to bear primary sequence identity when respectively compared with the dnaK and dnaJ gene products and have been proposed to be members of a heat-shock-cognate molecular chaperone system of unknown function. The close proximity and apparent co-expression of iscSUA and hscBA in A. vinelandii indicate that the proposed chaperone function of the hscBA gene products could be related to the maturation of iron-sulfur cluster-containing proteins. Attempts to place non-polar insertion mutations within either A. vinelandii iscS or hscA revealed that such mutations could not be stably maintained in the absence of the corresponding wild-type allele. These results reveal a very strong selective pressure against the maintenance of A. vinelandii iscS or hscA knock-out mutations and suggest that such mutations are either lethal or highly deleterious. In contrast to iscS or hscA, a strain having a polar insertion mutation within the cysE-like gene was readily isolated and could be stably maintained. These results show that the cysE-like gene located upstream from iscS is not essential for cell growth and that the cysE-like gene and the iscSUA-hscBA-fdx genes are contained within separate transcription units.
Asunto(s)
Azotobacter vinelandii/genética , Proteínas Bacterianas/genética , Liasas de Carbono-Azufre/genética , Proteínas de Escherichia coli , Ferredoxinas/genética , Proteínas HSP70 de Choque Térmico/genética , Proteínas Hierro-Azufre/genética , Familia de Multigenes , Secuencia de Aminoácidos , Proteínas Bacterianas/aislamiento & purificación , Liasas de Carbono-Azufre/química , Escherichia coli/genética , Genes Bacterianos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Homología de Secuencia de AminoácidoRESUMEN
The nifS gene product (NIFS) is a pyridoxal phosphate binding enzyme that catalyzes the desulfurization of L-cysteine to yield L-alanine and sulfur. In Azotobacter vinelandii this activity is required for the full activation of the nitrogenase component proteins. Because the nitrogenase component proteins, Fe protein and MoFe protein, both contain metalloclusters which are required for their respective activities, it is suggested that NIFS participates in the biosynthesis of the nitrogenase metalloclusters by providing the inorganic sulfur required for Fe-S core formation [Zheng, L., White, R. H., Cash, V. L. Jack, R. F., & Dean, D. R. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2754-2758]. In the present study the mechanism for the desulfurization of L-cysteine catalyzed by NIFS was determined in the following ways. First, the substrate analogs, L-allylglycine and vinylglycine, were shown to irreversibly inactivate NIFS by formation of a gamma-methylcystathionyl or cystathionyl residue, respectively, through nucleophilic attack by an active site cysteinyl residue on the corresponding analog-pyridoxal phosphate adduct. Second, this reactive cysteinyl residue, which is required for L-cysteine desulfurization activity, was identified as Cys325 by the specific alkylation of that residue and by site-directed mutagenesis experiments. Third, the formation of an enzyme-bound cysteinyl persulfide was identified as an intermediate in the NIFS-catalyzed reaction. Fourth, evidence was obtained for an enamine intermediate in the formation of L-alanine.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Proteínas Bacterianas/metabolismo , Cisteína/metabolismo , Azufre/metabolismo , Alilglicina/farmacología , Azotobacter vinelandii/enzimología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Secuencia de Bases , Catálisis , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Naftalenosulfonatos/metabolismo , Nitrogenasa/genética , Nitrogenasa/metabolismo , Fosfato de Piridoxal/metabolismoRESUMEN
Biological nitrogen fixation is catalyzed by nitrogenase, a complex metalloenzyme composed of two separately purifiable component proteins encoded by the structural genes nifH, nifD, and nifK. Deletion of the Azotobacter vinelandii nifS gene lowers the activities of both nitrogenase component proteins. Because both nitrogenase component proteins have metallocluster prosthetic groups that are composed of iron- and sulfur-containing cores, this result indicated that the nifS gene product could be involved in the mobilization of the iron or sulfur required for metallocluster formation. In the present work, it is shown that NIFS is a pyridoxal phosphate-containing homodimer that catalyzes the formation of L-alanine and elemental sulfur by using L-cysteine as substrate. NIFS activity is extremely sensitive to thiol-specific alkylating reagents, which indicates the participation of a cysteinyl thiolate at the active site. Based on these results we propose that an enzyme-bound cysteinyl persulfide that requires the release of the sulfur from the substrate L-cysteine for its formation ultimately provides the inorganic sulfide required for nitrogenase metallocluster formation. The recent discovery of nifS-like genes in non-nitrogen-fixing organisms also raises the possibility that the reaction catalyzed by NIFS represents a universal mechanism that involves pyridoxal phosphate chemistry, in the mobilization of the sulfur required for metallocluster formation.
Asunto(s)
Azotobacter vinelandii/enzimología , Azotobacter vinelandii/genética , Proteínas Bacterianas/metabolismo , Liasas de Carbono-Azufre , Genes Bacterianos , Liasas/metabolismo , Familia de Multigenes , Fijación del Nitrógeno/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Cromatografía por Intercambio Iónico , Clonación Molecular , Cisteína/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Escherichia coli/genética , Etilmaleimida/farmacología , Cinética , Datos de Secuencia Molecular , Peso Molecular , Homología de Secuencia de Aminoácido , EspectrofotometríaRESUMEN
Azotobacter vinelandii ferredoxin I is a small protein that contains one [4Fe-4S] cluster and one [3Fe-4S] cluster. Recently the x-ray crystal structure has been redetermined and the fdxA gene, which encodes the protein, has been cloned and sequenced. Here we report the site-directed mutation of Cys-20, which is a ligand of the [4Fe-4S] cluster in the native protein, to alanine and the characterization of the protein product by x-ray crystallographic and spectroscopic methods. The data show that the mutant protein again contains one [4Fe-4S] cluster and one [3Fe-4S] cluster. The new [4Fe-4S] cluster obtains its fourth ligand from Cys-24, a free cysteine in the native structure. The formation of this [4Fe-4S] cluster drives rearrangement of the protein structure.
Asunto(s)
Azotobacter/genética , Ferredoxinas/genética , Genes Bacterianos , Mutación , Azotobacter/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Hierro/análisis , Modelos Moleculares , Datos de Secuencia Molecular , Fijación del Nitrógeno/genética , Conformación Proteica , Espectrofotometría , Azufre/análisis , Difracción de Rayos XRESUMEN
Azotobacter vinelandii genes contained within the major nif-cluster and designated orf6, nifU, nifS, nifV, orf7, orf8, nifW, nifZ, nifM, and orf9 are organized into at least two overlapping transcriptional units. Nitrogenase derepressed crude extracts of Azotobacter vinelandii mutant strains having individual deletions located within nifU, nifS, nifV, nifW, nifZ, or nifM were examined for nitrogenase component protein activities. The results of these experiments indicated that, in A. vinelandii, the nifU, nifS and nifM gene products are required for the full activation or the catalytic stability of the nitrogenase Fe protein. Deletion of the nifV gene resulted in lower MoFe protein activity, probably resulting from the accumulation of an altered FeMo-cofactor. The nifW and nifZ gene products were required for the full activation or catalytic stability of the MoFe protein. Deletion of nifZ alone or nifM alone did not appear to affect FeMo-cofactor biosynthesis. However, deletion of both nifZ and nifM eleminated either FeMo-cofactor biosynthesis or the insertion of FeMo-cofactor into the apo-MoFe protein. Other genes contained within the nifUSVWZM gene cluster (orf6, orf7, orf8, and orf9) were not required for Mo-dependent diazotrophic growth.
Asunto(s)
Azotobacter/genética , Genes Bacterianos , Familia de Multigenes , Fijación del Nitrógeno/genética , Nitrogenasa/metabolismo , Azotobacter/enzimología , Azotobacter/crecimiento & desarrollo , Secuencia de Bases , Mapeo Cromosómico , Electroforesis en Gel Bidimensional , Cinética , Datos de Secuencia Molecular , Molibdoferredoxina/biosíntesis , Molibdoferredoxina/metabolismo , Mutación , Nitrogenasa/genética , Transcripción GenéticaRESUMEN
Serine substitutions for the five conserved cysteins (residues 38, 85, 97, 132, and 184) have been made in the Azotobacter vinelandii nitrogenase Fe-protein by site-specific mutagenesis. At least moderate levels of enzyme activity (greater than 10% of wild type enzyme) were found for enzymes with serine substitutions at residues 38, 85, and 184; whereas, no activity was detected for enzymes with serines at residues 97 and 132. This is consistent with cysteines 97 and 132 being the four ligands to the Fe:S cluster (two ligands from each of the two identical subunits). Although previous chemical modification studies had implicated these residues as ligands, the earlier results did not portend the new finding that of all the conserved cysteines only these 2 residues are required for a second function of the Fe-protein. Namely, if either cysteine 97 or 132 is replaced, it appears that a functional Fe:S cluster cannot be incorporated into the apo-Fe-protein. The consequence is that these altered Fe-proteins cannot participate either in substrate reduction or in the biosynthesis of FeMo-cofactor, a metallocofactor of the MoFe-protein. These results implicate the Fe:S center of Fe-protein in the biosynthesis mechanism as either a redox partner or Fe:S donor. Additional results suggest that the posttranslational modification of Fe-protein by nifM product is not the insertion of the Fe:S center.
Asunto(s)
Azotobacter/enzimología , Cisteína , Proteínas Hierro-Azufre/metabolismo , Metaloproteínas/metabolismo , Nitrogenasa/metabolismo , Oxidorreductasas , Adenosina Trifosfato/metabolismo , Sitios de Unión , Fenómenos Químicos , Química , Cinética , Sustancias Macromoleculares , Molibdoferredoxina/biosíntesis , Mutación , Nitrogenasa/genética , Serina , Relación Estructura-ActividadRESUMEN
Determination of a 28,793-base-pair DNA sequence of a region from the Azotobacter vinelandii genome that includes and flanks the nitrogenase structural gene region was completed. This information was used to revise the previously proposed organization of the major nif cluster. The major nif cluster from A. vinelandii encodes 15 nif-specific genes whose products bear significant structural identity to the corresponding nif-specific gene products from Klebsiella pneumoniae. These genes include nifH, nifD, nifK, nifT, nifY, nifE, nifN, nifX, nifU, nifS, nifV, nifW, nifZ, nifM, and nifF. Although there are significant spatial differences, the identified A. vinelandii nif-specific genes have the same sequential arrangement as the corresponding nif-specific genes from K. pneumoniae. Twelve other potential genes whose expression could be subject to nif-specific regulation were also found interspersed among the identified nif-specific genes. These potential genes do not encode products that are structurally related to the identified nif-specific gene products. Eleven potential nif-specific promoters were identified within the major nif cluster, and nine of these are preceded by an appropriate upstream activator sequence. A + T-rich regions were identified between 8 of the 11 proposed nif promoter sequences and their upstream activator sequences. Site-directed deletion-and-insertion mutagenesis was used to establish a genetic map of the major nif cluster.