Structure of the molybdate/tungstate binding protein mop from Sporomusa ovata

BACKGROUND: Transport of molybdenum into bacteria involves a high-affinity ABC transporter system whose expression is controlled by a repressor protein called ModE. While molybdate transport is tightly coupled to utilization in some bacteria, other organisms have molybdenum storage proteins. One class of putative molybdate storage proteins is characterized by a sequence consisting of about 70 amino acids (Mop). A tandem repeat of Mop sequences also constitutes the molybdate binding domain of ModE. RESULTS: We have determined the crystal structure of the 7 kDa Mop protein from the methanol-utilizing anaerobic eubacterium Sporomusa ovata grown in the presence of molybdate and tungstate. The protein occurs as highly symmetric hexamers binding eight oxyanions. Each peptide assumes a so-called OB fold, which has previously also been observed in ModE. There are two types of oxyanion binding sites in Mo at the interface between two or three peptides. All oxyanion binding sites were found to be occupied by WO(4) rather than MoO(4). CONCLUSIONS: The biological function of proteins containing only Mop sequences is unknown, but they have been implicated in molybdate homeostasis and molybdopterin cofactor biosynthesis. While there are few indications that the S. ovata Mop binds pterin, the structure suggests that only the type-1 oxyanion binding sites would be sufficiently accessible to bind a cofactor. The observed occupation of the oxyanion binding sites by WO(4) indicates that Mop might also be involved in controlling intracellular tungstate levels.     

EFFECT OF TUNGSTATE ON THE UPTAKE AND FUNCTION OF MOLYBDATE IN AZOTOBACTER AGILIS.

Bulen, William A. (Charles F. Kettering Foundation, Yellow Springs, Ohio). Effect of tungstate on the uptake and function of molybdate in Azotobacter agilis. J. Bacteriol. 82:130-134. 1961.-The reported competitive inhibition of molybdate by tungstate was investigated in an effort to elucidate molybdenum functions associated with nitrogen fixation by Azotobacter agilis (A. vinelandii). Growth, respiration, and N(2) (15)-incorporation experiments with normal and molybdenum-deficient cells indicated that tungstate inhibits the uptake of molybdate but does not compete with the metabolically functional molybdenum of cells metabolizing N(2). Neither a molybdenum requirement nor a tungstate inhibition was observed with cells metabolizing urea.     

无机钼酸在棕色固氮菌(Azotobacter vinelandii)体内的累积和转化

醋酸铵培养的棕色固氮菌(Azotobacter vinelandii),经超声击碎高速离心制备粗提取液、DEAE-纤维素柱层析表明,体内~(99)MoFe蛋白合成受到阻遏,在0.15 M NaGl洗脱分部中,除~(99)Mo储存蛋白峰外,还存在一个无机~(99)MoO_4~=组分。醋酸铵培养的棕色固氮菌经去阻遏后,在体内固氮活性出现的同时,可观察到原先菌体内累积的~(99)Mo储存蛋白峰降低,无机~(99)MoO_4~=的组分几乎消失以及~(99)MoFe蛋白合成。若去阻遏过程存在氯霉素,则菌体不显示固氮活性,(99)MoFe蛋白不再合成,储存蛋白和无机铝酸组分中~(99)Mo的转移停止。     

Purification of a NifEN protein complex that contains bound molybdenum and a FeMo-Co precursor from an Azotobacter vinelandii DeltanifHDK strain

The NifEN protein complex serves as a molecular scaffold where some of the steps for the assembly of the iron-molybdenum cofactor (FeMo-co) of nitrogenase take place. A His-tagged version of the NifEN complex has been previously purified and shown to carry two identical [4Fe-4S] clusters of unknown function and a [Fe-S]-containing FeMo-co precursor. We have improved the purification of the his-NifEN protein from a DeltanifHDK strain of Azotobacter vinelandii and have found that the amounts of iron and molybdenum within NifEN were significantly higher than those reported previously. In an in vitro FeMo-co synthesis system with purified components, the NifEN protein served as a source of both molybdenum and a [Fe-S]-containing FeMo-co precursor, showing significant FeMo-co synthesis activity in the absence of externally added molybdate. Thus, the NifEN scaffold protein, purified from DeltanifHDK background, contained the Nif-Bco-derived Fe-S cluster and molybdenum, although these FeMo-co constituents were present at different levels within the protein complex.     

Mutational analysis of genes of the mod locus involved in molybdenum transport, homeostasis, and processing in Azotobacter vinelandii.

DNA sequencing of the region upstream from the Azotobacter vinelandii operon (modEABC) that contains genes for the molybdenum transport system revealed an open reading frame (modG) encoding a hypothetical 14-kDa protein. It consists of a tandem repeat of an approximately 65-amino-acid sequence that is homologous to Mop, a 7-kDa molybdopterin-binding protein of Clostridium pasteurianum. The tandem repeat is similar to the C-terminal half of the product of modE. The effects of mutations in the mod genes provide evidence for distinct high- and low-affinity Mo transport systems and for the involvement of the products of modE and modG in the processing of molybdate. modA, modB, and modC, which encode the component proteins of the high-affinity Mo transporter, are required for 99Mo accumulation and for the nitrate reductase activity of cells growing in medium with less than 10 microM Mo. The exchange of accumulated 99Mo with nonradioactive Mo depends on the presence of modA, which encodes the periplasmic molybdate-binding protein. 99Mo also exchanges with tungstate but not with vanadate or sulfate. modA, modB, and modC mutants exhibit nitrate reductase activity and 99Mo accumulation only when grown in more than 10 microM Mo, indicating that A. vinelandii also has a low-affinity Mo uptake system. The low-affinity system is not expressed in a modE mutant that synthesizes the high-affinity Mo transporter constitutively or in a spontaneous tungstate-tolerant mutant. Like the wild type, modG mutants only show nitrate reductase activity when grown in > 10 nM Mo. However, a modE modG double mutant exhibits maximal nitrate reductase activity at a 100-fold lower Mo concentration. This indicates that the products of both genes affect the supply of Mo but are not essential for nitrate reductase cofactor synthesis. However, nitrogenase-dependent growth in the presence or absence of Mo is severely impaired in the double mutant, indicating that the products of modE and modG may be involved in the early steps of nitrogenase cofactor biosynthesis in A. vinelandii.     

Expression of an alternative nitrogen fixation system in Azotobacter vinelandii.

Nitrogenase activities were determined from maximum acetylene reduction rates for mutant strains of Azotobacter vinelandii which are unable to fix N2 in the presence of molybdenum (Nif-) but undergo phenotypic reversal to Nif+ under conditions of Mo deficiency. The system responsible for N2 fixation under these conditions is thought to be an alternative N2 fixation system (Bishop et al., Proc. Natl. Acad. Sci. U.S.A. 77:7342-7346, 1980). Phenotypic reversal of Nif- strains to Nif+ strains was also observed in N-free medium without Mo but with either V or Re. Two protein patterns were found on two-dimensional gels of proteins from the extracts of wild-type cells cultured in N-free medium without Mo and with or without V or Re. The expression of each protein pattern in the wild-type strain of A. vinelandii seemed to depend upon the physiological state of the N2-fixing culture. Electron paramagnetic resonance experiments were conducted on whole cells of A. vinelandii grown under conditions of Mo deprivation in the absence of fixed N. No g = 3.65 signal (an electron paramagnetic resonance signal characteristic of the Mo-containing component of nitrogenase) was detectable in these cells, regardless of whether V or Re was present during growth of these cells, These results are discussed from the perspective that the well-known effect of V on N2 fixation by A. vinelandii may involve an alternative N2 fixation system.     

Activation of vanadium nitrogenase expression in Azotobacter vinelandii DJ54 revertant in the presence of molybdenum

Azotobacter vinelandii carries three different and genetically distinct nitrogenase systems on its chromosome. Expression of all three nitrogenases is repressed by high concentrations of fixed nitrogen. Expression of individual nitrogenase systems is under the control of specific metal availability. We have isolated a novel type of A. vinelandii DJ54 revertant, designated A. vinelandii BG54, which carries a defined deletion in the nifH gene and is capable of diazotrophic growth in the presence of molybdenum. Inactivation of nifDK has no effect on growth of this mutant strain in nitrogen-free medium suggesting that products of the nif system are not involved in supporting diazotrophic growth of A. vinelandii BG54. Similar to the wild type, A. vinelandii BG54 is also sensitive to 1 mM tungsten. Tn5-B21 mutagenesis to inactivate the genes specific to individual systems revealed that the structural genes for vnf nitrogenase are required for diazotrophic growth of A. vinelandii BG54. Analysis of promoter activity of different nif systems revealed that the vnf promoter is activated in A. vinelandii BG54 in the presence of molybdenum. Based on these data we conclude that A. vinelandii BG54 strain utilizes vnf nitrogenase proteins to support its diazotrophic growth.     

Tungsten transport protein A (WtpA) in Pyrococcus furiosus: the first member of a new class of tungstate and molybdate transporters

A novel tungstate and molybdate binding protein has been discovered from the hyperthermophilic archaeon Pyrococcus furiosus. This tungstate transport protein A (WtpA) is part of a new ABC transporter system selective for tungstate and molybdate. WtpA has very low sequence similarity with the earlier-characterized transport proteins ModA for molybdate and TupA for tungstate. Its structural gene is present in the genome of numerous archaea and some bacteria. The identification of this new tungstate and molybdate binding protein clarifies the mechanism of tungstate and molybdate transport in organisms that lack the known uptake systems associated with the ModA and TupA proteins, like many archaea. The periplasmic protein of this ABC transporter, WtpA (PF0080), was cloned and expressed in Escherichia coli. Using isothermal titration calorimetry, WtpA was observed to bind tungstate (dissociation constant [K(D)] of 17 +/- 7 pM) and molybdate (K(D) of 11 +/- 5 nM) with a stoichiometry of 1.0 mol oxoanion per mole of protein. These low K(D) values indicate that WtpA has a higher affinity for tungstate than do ModA and TupA and an affinity for molybdate similar to that of ModA. A displacement titration of molybdate-saturated WtpA with tungstate showed that the tungstate effectively replaced the molybdate in the binding site of the protein.     

Iron-molybdenum cofactor biosynthesis in Azotobacter vinelandii requires the iron protein of nitrogenase

Nitrogenase is composed of two separately purified proteins called the Fe protein and the MoFe protein. In Azotobacter vinelandii the genes encoding these structural components are clustered and ordered: nifH (Fe protein)-nifD (MoFe protein alpha subunit)-nifK (MoFe protein beta subunit). The MoFe protein contains an ironmolybdenum cofactor (FeMo cofactor) whose biosynthesis involves the participation of at least five gene products, nifQ, nifB, nifN, nifE, and nifV. In this study an A. vinelandii mutant strain, which contains a defined deletion within the nifH (Fe protein) gene, was isolated and studied. This mutant is still able to accumulate significant amounts of MoFe protein subunits. However, extracts of this nifH deletion strain have only very low levels of MoFe protein acetylene reduction activity. Fully active MoFe protein can be reconstituted by simply adding isolated FeMo cofactor to the extracts. Fe protein is not necessary to stabilize or insert this preformed FeMo cofactor into the FeMo cofactor-deficient MoFe protein synthesized by the nifH deletion strain. Extracts of the nifH deletion strain can carry out molybdate and ATP-dependent in vitro FeMo cofactor biosynthesis provided Fe protein is added, demonstrating that they contain the products encoded by the FeMo cofactor biosynthetic genes. These data demonstrate that the Fe protein is physically required for the biosynthesis of FeMo cofactor in A. vinelandii.     

The molybdate binding protein Mop from Haemophilus influenzae--biochemical and thermodynamic characterisation

The protein Mop from Haemophilus influenzae is a member of the molbindin family of proteins. Using isothermal titration calorimetry (ITC), Mop was observed to bind molybdate at two distinct sites with a stoichiometry of 8 mol molybdate per Mop hexamer. Six moles of molybdate bound endothermically at high affinity sites (K(a)=8.5 x 10(7)M(-1)), while 2 mol of molybdate bound exothermically at lower affinity sites (K(a)=3.7 x 10(7)M(-1)). Sulphate was also found to bind weakly at the higher affinity sites. ITC revealed that the affinity of molybdate binding to the endothermic site decreased with increasing pH and was accompanied by the transfer from the buffer to the protein of one proton per Mop monomer. These kinetic and thermodynamic results are interpreted with reference to molbindin crystal structures and data concerning molbindin binding affinities. Mop binds molybdate with high specificity, capacity, and affinity which indicates that Mop has a role as an intracellular molybdate binding protein involved in oxyanion homeostasis.     

Distorted octahedral coordination of tungstate in a subfamily of specific binding proteins

Bacteria and archaea import molybdenum and tungsten from the environment in the form of the oxyanions molybdate (MoO₄ ²⁻) and tungstate (WO₄ ²⁻). These substrates are captured by an external, high-affinity binding protein, and delivered to ATP binding cassette transporters, which move them across the cell membrane. We have recently reported a crystal structure of the molybdate/tungstate binding protein ModA/WtpA from Archaeoglobus fulgidus, which revealed an octahedrally coordinated central metal atom. By contrast, the previously determined structures of three bacterial homologs showed tetracoordinate molybdenum and tungsten atoms in their binding pockets. Until then, coordination numbers above four had only been found for molybdenum/tungsten in metalloenzymes where these metal atoms are part of the catalytic cofactors and coordinated by mostly non-oxygen ligands. We now report a high-resolution structure of A. fulgidus ModA/WtpA, as well as crystal structures of four additional homologs, all bound to tungstate. These crystal structures match X-ray absorption spectroscopy measurements from soluble, tungstate-bound protein, and reveal the details of the distorted octahedral coordination. Our results demonstrate that the distorted octahedral geometry is not an exclusive feature of the A. fulgidus protein, and suggest distinct binding modes of the binding proteins from archaea and bacteria. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. K. Hollenstein and M. Comellas-Bigler contributed equally to this work.     

Characterization of genes for an alternative nitrogenase in the cyanobacterium Anabaena variabilis.

Anabaena variabilis ATCC 29413 is a heterotrophic, nitrogen-fixing cyanobacterium that has been reported to fix nitrogen and reduce acetylene to ethane in the absence of molybdenum. DNA from this strain hybridized well at low stringency to the nitrogenase 2 (vnfDGK) genes of Azotobacter vinelandii. The hybridizing region was cloned from a lambda EMBL3 genomic library of A. variabilis, mapped, and sequenced. The deduced amino acid sequences of the vnfD and vnfK genes of A. variabilis showed only about 56% similarity to the nifDK genes of Anabaena sp. strain PCC 7120 but were 76 to 86% similar to the anfDK or vnfDK genes of A. vinelandii. The organization of the vnf gene cluster in A. variabilis was similar to that of A. vinelandii. However, in A. variabilis, the vnfG gene was fused to vnfD; hence, this gene is designated vnfDG. A vnfH gene was not contiguous with the vnfDG gene and has not yet been identified. A mutant strain, in which a neomycin resistance cassette was inserted into the vnf cluster, grew well in a medium lacking a source of fixed nitrogen in the presence of molybdenum but grew poorly when vanadium replaced molybdenum. In contrast, the parent strain grew equally well in media containing either molybdenum or vanadium. The vnf genes were transcribed in the absence of molybdenum, with or without vanadium. The vnf gene cluster did not hybridize to chromosomal DNA from Anabaena sp. strain PCC 7120 or from the heterotrophic strains, Nostoc sp. strain Mac and Nostoc sp. strain ATCC 29150. A hybridizing ClaI fragment very similar in size to the A. variabilis ClaI fragment was present in DNA isolated from several independent, cultured isolates of Anabaena sp. from the Azolla symbiosis.     

Structure of the molybdenum site of Rhodobacter sphaeroides biotin sulfoxide reductase

Conditions for heterologous expression of Rhodobacter sphaeroides biotin sulfoxide reductase in Escherichia coli were modified, resulting in a significant improvement in the yield of recombinant enzyme and enabling structural studies of the molybdenum center. Quantitation of the guanine and the molybdenum as compared to that found in R. sphaeroides DMSO reductase demonstrated the presence of the bis(MGD)molybdenum cofactor. UV-visible absorption spectra were obtained for the oxidized, NADPH-reduced, and dithionite-reduced enzyme. EPR spectra were obtained for the Mo(V) state of the enzyme. X-ray absorption spectroscopy at the molybdenum K-edge has been used to probe the molybdenum coordination of the enzyme. The molybdenum site of the oxidized protein possesses a Mo(VI) mono-oxo site (Mo=O at 1.70 A) with additional coordination by approximately four thiolate ligands at 2.41 A and probably one oxygen or nitrogen at 1.95 A. The NADPH- and dithionite-reduced Mo(IV) forms of the enzyme are des-oxo molybdenum sites with approximately four thiolates at 2.33 A and two different Mo-O/N ligands at 2.19 and 1.94 A.     

Cyanide and methylisocyanide binding to the isolated iron-molybdenum cofactor of nitrogenase

19F NMR and x-ray absorption experiments have been performed with both the isolated FeMo cofactor and the MoFe protein of nitrogenase in search of direct evidence for substrate or inhibitor binding. Using 19F NMR as a probe and p-CF3C6H4S- as the receptor ligand, the data show that the nitrogenase inhibitors CN- and CH3NC bind to the isolated FeMo cofactor-RFS- complex in N-methylformamide with a finite formation constant. Their binding increases the electronic relaxation time of the complex and increases the life-time of the FeMo cofactor-p-CF3C6H4S- bond, Parallel molybdenum K edge and extended x-ray absorption fine structure experiments show that CH3NC does not bind to molybdenum. Although CO and N3- both relieve CN- and CH3NC inhibition of electron flow through nitrogenase, unlike the latter, they do not appear to bind to isolated FeMo cofactor. In experiments with the dithionite-reduced MoFe protein, we did not detect any changes in the molybdenum K edge or extended x-ray absorption fine structure spectra upon addition of CO, N2, C2H2, NaCN, CH3NC, or azide demonstrating that either these substrates and inhibitors do not bind to molybdenum or that the FeMo cofactor site of nitrogenase is inaccessible to substrate binding except under turnover conditions.