Thiol reactivity of the nitrogenase Fe-protein from Azotobacter vinelandii

A procedure has been developed to examine some of the functional roles of the 14 cysteinyl residues in the nitrogenase Fe-protein (Av2) from Azotobacter vinelandii. The reduced form of Av2 was alkylated with iodo[2-14C]acetic acid under a variety of experimental conditions, e.g. reaction in the presence of nucleotides, alpha,alpha'-dipyridyl and nucleotides, or denaturants. The labeled cysteinyl residues were identified and quantified using an analytical DEAE-Sepharose ion exchange chromatography peptide mapping technique based upon the known amino acid sequence (Hausinger, R. P., and Howard, J. B. (1982) J. Biol. Chem. 257, 2483-2490). From the results of the labeling experiments, the following features of the Av2 structure have been proposed. 1) Av2 contains no disulfides, hyperreactive thiols, or surface thiols as defined by reaction with iodoacetic acid. 2) Cysteines 97 and 132 are the probable ligands for the Av2 Fe:S center which is bound symmetrically between subunits. 3) MgATP partially protects cysteine 85 from carboxymethylation by iodoacetic acid and may be part of the nucleotide-binding site. 4) Of the five nonligand thiols only cysteines 5 and 184 are completely alkylated when Av2 is denatured in hexamethylphosphoramide, whereas all five nonligand thiols appear to rapidly exchange at the Fe:S center if the protein is denatured in the absence of alkylating reagents. 5) Both Av2 and apo-Av2 appear to undergo a reversible conformational change upon binding MgATP.     

Cross-linking site in Azotobacter vinelandii complex

The Fe-protein and the MoFe-protein of the Azotobacter vinelandii nitrogenase complex can be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Willing, A., Georgiadis, M.M., Rees, D. C., and Howard, J. B. (1989) J. Biol. Chem. 264, 8499-8503). In this reaction, one of the identical subunits of the Fe-protein dimer is linked by an isopeptide bond to each beta-subunit of the MoFe-protein tetramer. The reaction has been found to be highly specific with greater than 85% of amino acid residues Glu-112 (Fe-protein) and Lys-399 (MoFe-protein) cross-linked to each other. Although Glu-112 is located in a highly conserved amino acid sequence, it is found in only half of the known Fe-protein sequences. Likewise, Lys-399 is not a conserved residue in the MoFe-protein. Glu-112 appears to be part of an anionic cluster of nine carboxylic acids which is located between the proposed thiol ligands for the Fe:S center. In contrast, the basic residue cluster which includes Lys-399 has been found in only in the Azotobacter MoFe-protein. Thus, this crosslinking reaction either is unique to Azotobacter nitrogenase or must involve other residues in the MoFe-protein of other species. Because Lys-399 and Glu-112 form a specific cross-link, it is probable that they are part of the interaction site leading to productive complex formation. This information should be useful for the model building of the complex from the crystallographic structures of the individual components.     

The [4Fe–4S]-cluster coordination of [FeFe]-hydrogenase maturation protein HydF as revealed by EPR and HYSCORE spectroscopies

[FeFe] hydrogenases are key enzymes for bio(photo)production of molecular hydrogen, and several efforts are underway to understand how their complex active site is assembled. This site contains a [4Fe–4S]-2Fe cluster and three conserved maturation proteins are required for its biosynthesis. Among them, HydF has a double task of scaffold, in which the dinuclear iron precursor is chemically modified by the two other maturases, and carrier to transfer this unit to a hydrogenase containing a preformed [4Fe–4S]-cluster. This dual role is associated with the capability of HydF to bind and dissociate an iron–sulfur center, due to the presence of the conserved FeS-cluster binding sequence CxHx_46–53HCxxC. The recently solved three-dimensional structure of HydF from Thermotoga neapolitana described the domain containing the three cysteines which are supposed to bind the FeS cluster, and identified the position of two conserved histidines which could provide the fourth iron ligand. The functional role of two of these cysteines in the activation of [FeFe]-hydrogenases has been confirmed by site-specific mutagenesis. On the other hand, the contribution of the three cysteines to the FeS cluster coordination sphere is still to be demonstrated. Furthermore, the potential role of the two histidines in [FeFe]-hydrogenase maturation has never been addressed, and their involvement as fourth ligand for the cluster coordination is controversial. In this work we combined site-specific mutagenesis with EPR (electron paramagnetic resonance) and HYSCORE (hyperfine sublevel correlation spectroscopy) to assign a role to these conserved residues, in both cluster coordination and hydrogenase maturation/activation, in HydF proteins from different microorganisms.     

Adjacent cysteines are capable of ligating the same tetranuclear iron-sulfur cluster

The mechanism of the energy-converting NADH (beta-nicotinamide adenine dinucleotide, reduced form):ubiquinone oxidoreductase, which is also called respiratory complex I, is largely unknown due to lack of a high-resolution structure and the most complicated construction of the enzyme. Electron transport is carried out by one flavin mononucleotide (FMN) and up to 9 Fe/S clusters. The Fe/S cluster N2, which is believed to be directly involved in redox-coupled proton-translocation, is located on subunit NuoB (the homologue of the mitochondrial PSST subunit). This subunit contains a conserved binding motif for a [4Fe/4S] cluster with two adjacent cysteines. It was questioned whether these adjacent cysteines could be ligands of the same cluster due to a possible steric hinderance. However, mutagenesis of either of these cysteines led to a loss of cluster N2. We used the known structure of the homologous small subunit of hydrogenases containing a regular cysteine motif to generate an in silico mutant with two consecutive cysteines. Molecular dynamics simulation showed that the conformation of these cysteines does not meet the topological requirements for coordination of a [4Fe/4S] cluster when the protein backbone conformation is kept constant. In comparison, the simulation of a dipeptide amide using a "template forcing" approach resulted in a conformation compatible to an optimal coordination of the two cluster positions in question. Thus, a slight main-chain conformational change would allow two adjacent cysteines to coordinate a [4Fe/4S] cluster.     

The iron-sulfur center of biotin synthase: site-directed mutants

Biotin synthase contains an essential [4Fe-4S]+ cluster that is thought to provide an electron for the cleavage of S-adenosylmethionine, a cofactor required for biotin formation. The conserved cysteine residues Cys53, Cys57 and Cys60 have been proposed as ligands to the [4Fe-4S] cluster. These residues belong to a C-X3-C-X2-C motif which is also found in pyruvate formate lyase-activating enzyme, lysine 2,3-aminomutase and the anaerobic ribonucleotide reductase-activating component. To investigate the role of the cysteine residues, Cys-->Ala mutants of the eight cysteine residues of Escherichia coli biotin synthase were prepared and assayed for activity. Our results show that six cysteines are important for biotin formation. Only two mutant proteins, C276A and C288A, closely resembled the wild-type protein, indicating that the corresponding cysteines are not involved in iron chelation and biotin formation. The six other mutant proteins, C53A, C57A, C60A, C97A, C128A and C188A, were inactive but capable of assembling a [4Fe-4S] cluster, as shown by M?ssbauer spectroscopy. The C53A, C57A and C60A mutant proteins are unique in that their cluster could not undergo reduction to the [4Fe-4S]+ state, as shown by EPR and M?ssbauer spectroscopy. On this basis and by analogy with pyruvate formate lyase-activating enzyme and the anaerobic ribonucleotide reductase-activating component, it is suggested that the corresponding cysteines coordinate the cluster even though one cannot fully exclude the possibility that other cysteines play that role as well. Therefore it appears that for activity biotin synthase absolutely requires cysteines that are not involved in iron chelation.     

Sequence motif-specific assignment of two [2Fe-2S] clusters in rat xanthine oxidoreductase studied by site-directed mutagenesis

The sequence motif-specific assignment of the two distinct [2Fe-2S] clusters in rat xanthine oxidoreductase (XOR) was unequivocally established by site-directed mutagenesis of recombinant enzymes expressed in a baculovirus-insect cell system and electron paramagnetic resonance (EPR) spectroscopy. The conserved cysteine residues, including Cys-115, in the unusual C-terminal -Cys-Xaa(2)-Cys-//-Cys-Xaa(1)-Cys- motif serve as ligands to the Fe/S I center, which is probably located in close proximity to the Mo-pterin center. Other conserved cysteine residues, including Cys-43 and Cys-51, in the N-terminal plant ferredoxin-like motif serve as ligands to the Fe/S II center, which is distantly located from the Mo-pterin center. The present sequence motif-specific assignment of the Fe/S I and II centers is discussed in the light of the structural features of XOR.     

Affinity-chromatographic purification of sixteen cysteine-substituted maltoporin variants: thiol reactivity and cross-linking in an outer membrane protein of Escherichia coli

Wild-type and 16 variant maltoporins with site-directed cysteine substitutions at 14 sites were purified by a novel one-step affinity-chromatographic procedure. The trimer stability of purified proteins with C22S, C38S and G103C substitutions was reduced compared to wild-type maltoporin. Quantitative labelling with N-ethyl[14C]maleimide, cross-linking with bifunctional bismaleimides and disulphide formation was used to test the reactivity of cysteines in the folded protein. The maleimide reactivity of the residues was in the order: 152 approximately equal to 153 greater than 265 greater than 30 approximately equal to 103 approximately equal to 120 approximately equal to 154 approximately equal to 382 greater than 57 approximately equal to 146, with the other sites (22, 38, 97, 184) poorly labelled. Only cysteines at 152 or 153 permitted the formation of inter-subunit disulphide bonds suggesting these residues are located within 0.5-0.9 nm of each other in homotrimers of maltoporin. S152C and S153C as well as S154C permitted the formation of inter-subunit cross-links using bifunctional bismaleimides. The cross-linkability and the high reactivity to N-ethylmaleimide of the 150 region was consistent with the current model of the structure of maltoporin in the outer membrane; the reactivity of the other sites is also discussed within the context of this model.     

Modular organization and identification of a mononuclear iron-binding site within the NifU protein

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. Received: 27 October 1999 / Accepted: 30 November 1999     

Identification of a second site compensatory mutation in the Fe-protein that allows diazotrophic growth of Azotobacter vinelandii UW97

Azotobacter vinelandii UW97 is defective in nitrogen fixation due to a replacement of serine at position 44 by phenylalanine in the Fe-protein [Pulakat, L., Hausman, B.S., Lei, S. and Gavini, N. (1996) J. Biol. Chem. 271, 1884-1889]. Serine residue 44 is located in a conserved domain that links the nucleotide binding site and the MoFe-protein docking surface of the Fe-protein. Therefore, it is possible that the loss of function by A. vinelandii UW97-Fe-protein may be caused by global conformational disruption or disruption of the conformational change upon MgATP binding. To determine whether it is possible to generate a functional nitrogenase complex via a compensating second site mutation(s) in the Fe-protein, we have attempted to isolate genetic revertants of A. vinelandii UW97 that can grow on nitrogen-free medium. One such revertant, designated A vinelandii BG9, encoded a Fe-protein that retained the Ser44Phe mutation and also had a second mutation that caused the replacement of a lysine at position 170 by glutamic acid. Lysine 170 is highly conserved and is located in a conserved region of the Fe-protein. This region is implicated in stabilizing the MgATP-induced conformation of the Fe-protein and in docking to the MoFe-protein. Further complementation analysis showed that the Fe-protein mutant that retained serine 44 but contained the substitution of lysine at position 170 by glutamic acid was also non-functional. Thus, neither Ser44Phe nor Lys170Glu mutants of Fe-protein were functional; however, the Fe-protein in A. vinelandii BG9 that contained both substitutions could support diazotrophic growth on the strain.     

Mutational analysis of the mitochondrial Rieske iron-sulfur protein of Saccharomyces cerevisiae. III. Import, protease processing, and assembly into the cytochrome bc1 complex of iron-sulfur protein lacking the iron-sulfur cluster.

We have used site-directed mutagenesis of the Saccharomyces cerevisiae Rieske iron-sulfur protein gene (RIP 1) to convert cysteines 159, 164, 178, and 180 to serines, and to convert histidines 161 and 181 to arginines. These 4 cysteines and 2 histidines are conserved in all Rieske proteins sequenced to date, and 4 of these 6 residues are thought to ligate the iron-sulfur cluster to the apoprotein. We have also converted histidine 184 to arginine. This histidine is conserved only in respiring organisms. The site-directed mutations of the six fully conserved putative iron-sulfur cluster ligands result in an inactive iron-sulfur protein, lacking iron-sulfur cluster, and failure of the yeast to grow on nonfermentable carbon sources. In contrast, when histidine 184 is replaced by arginine, the iron-sulfur cluster is assembled properly and the yeast grow on nonfermentable carbon sources. The site-directed mutations of the 6 fully conserved residues do not prevent post-translational import of iron-sulfur protein precursor into mitochondria, nor do the mutations prevent processing of iron-sulfur protein precursor to mature size protein by mitochondrial proteases. Optical spectra of mitochondria from the six mutants indicate that cytochrome b is normal, in contrast to the deranged spectrum of cytochrome b which results when the iron-sulfur protein gene is deleted. In addition, mature size iron-sulfur apoprotein is associated with cytochrome bc1 complex purified from a site-directed mutant in which iron-sulfur cluster is not inserted. These results indicate that mature size iron-sulfur apoprotein, lacking iron-sulfur cluster, is inserted into the cytochrome bc1 complex, where it interacts with and preserves the optical properties of cytochrome b. Insertion of the iron-sulfur cluster is not an obligatory prerequisite to processing of the protein to its final size. Either the processing protease cannot distinguish between iron-sulfur protein with or without the iron-sulfur cluster, or insertion of the iron-sulfur cluster occurs after the protein is processed to its mature size, possibly after it is assembled in the cytochrome bc1 complex.     

7-Iron ferredoxin revisited

The crystal structure of the 7Fe ferredoxin from Azotobacter vinelandii has been redetermined using area detector data to 2.7-A resolution and a new derivative. Tetragonal crystals of the protein were maintained at pH 8.0. The results show that the structure previously reported was in error and confirms a recent independent report of the structure (Stout, G.H., Turley, S., Sieker, L. C., and Jensen, L. H. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, in press). The protein fold is similar to the homologous 8Fe ferredoxin structure for the N-terminal half of the protein; the C-terminal residues wrap around this structure. The structure contains a 3Fe cluster coordinated by cysteines 8, 16, and 49 and a 4Fe cluster coordinated by cysteines 20, 39, 42, and 45. However, there are two free sulfhydryls, cysteines 11 and 24, in the new model. Cysteine 24 is in contact with the [4Fe-4S] cluster. Cysteine 11 is shielded from solvent by residues 86-90.     

Desulfovibrio desulfuricans iron hydrogenase: the structure shows unusual coordination to an active site Fe binuclear center.

BACKGROUND: Many microorganisms have the ability to either oxidize molecular hydrogen to generate reducing power or to produce hydrogen in order to remove low-potential electrons. These reactions are catalyzed by two unrelated enzymes: the Ni-Fe hydrogenases and the Fe-only hydrogenases. RESULTS: We report here the structure of the heterodimeric Fe-only hydrogenase from Desulfovibrio desulfuricans - the first for this class of enzymes. With the exception of a ferredoxin-like domain, the structure represents a novel protein fold. The so-called H cluster of the enzyme is composed of a typical [4Fe-4S] cubane bridged to a binuclear active site Fe center containing putative CO and CN ligands and one bridging 1, 3-propanedithiol molecule. The conformation of the subunits can be explained by the evolutionary changes that have transformed monomeric cytoplasmic enzymes into dimeric periplasmic enzymes. Plausible electron- and proton-transfer pathways and a putative channel for the access of hydrogen to the active site have been identified. CONCLUSIONS: The unrelated active sites of Ni-Fe and Fe-only hydrogenases have several common features: coordination of diatomic ligands to an Fe ion; a vacant coordination site on one of the metal ions representing a possible substrate-binding site; a thiolate-bridged binuclear center; and plausible proton- and electron-transfer pathways and substrate channels. The diatomic coordination to Fe ions makes them low spin and favors low redox states, which may be required for catalysis. Complex electron paramagnetic resonance signals typical of Fe-only hydrogenases arise from magnetic interactions between the [4Fe-4S] cluster and the active site binuclear center. The paucity of protein ligands to this center suggests that it was imported from the inorganic world as an already functional unit.     

An iron-sulfur cluster in the family 4 uracil-DNA glycosylases

The 25-kDa Family 4 uracil-DNA glycosylase (UDG) from Pyrobaculum aerophilum has been expressed and purified in large quantities for structural analysis. In the process we observed it to be colored and subsequently found that it contained iron. Here we demonstrate that P. aerophilum UDG has an iron-sulfur center with the EPR characteristics typical of a 4Fe4S high potential iron protein. Interestingly, it does not share any sequence similarity with the classic iron-sulfur proteins, although four cysteines (which are strongly conserved in the thermophilic members of Family 4 UDGs) may represent the metal coordinating residues. The conservation of these residues in other members of the family suggest that 4Fe4S clusters are a common feature. Although 4Fe4S clusters have been observed previously in Nth/MutY DNA repair enzymes, this is the first observation of such a feature in the UDG structural superfamily. Similar to the Nth/MutY enzymes, the Family 4 UDG centers probably play a structural rather than a catalytic role.     

X-ray structure of the [FeFe]-hydrogenase maturase HydE from Thermotoga maritima

Maturation of the [FeFe]-hydrogenase active site depends on at least the expression of three gene products called HydE, HydF, and HydG. We have solved the high resolution structure of recombinant, reconstituted S-adenosine-L-methionine-dependent HydE from Thermotoga maritima. Besides the conserved [Fe(4)S(4)] cluster involved in the radical-based reaction, this HydE was reported to have a second [Fe(4)S(4)] cluster coordinated by three Cys residues. However, in our crystals, depending on the reconstitution and soaking conditions, this second cluster is either a [Fe(2)S(2)] center, with water occupying the fourth ligand site or is absent. We have carried out site-directed mutagenesis studies on the related HydE from Clostridium acetobutylicum, along with in silico docking and crystal soaking experiments, to define the active site region and three anion-binding sites inside a large, positive cavity, one of which binds SCN(-) with high affinity. Although the overall triose-phosphate isomerase-barrel structure of HydE is very similar to that of biotin synthase, the residues that line the internal cavity are significantly different in the two enzymes.     

Primary structure of hydrogenase I from Clostridium pasteurianum

Peptides obtained by cleavage of Clostridium pasteurianum hydrogenase I have been sequenced. The data allowed design of oligonucleotide probes which were used to clone a 2310-bp Sau3A fragment containing the hydrogenase encoding gene. The latter has been sequenced and was found to translate into a protein composed of 574 amino acids (Mr = 63,836), including 22 cysteines. C. pasteurianum hydrogenase is homologous to, but longer than, the large subunit of Desulfovibrio vulgaris (Hildenborough) [Fe] hydrogenase. It includes an additional N-terminal domain of ca. 110 amino acids which contains eight cysteine residues and which therefore could accommodate two of its postulated four [4Fe-4S] clusters. C. pasteurianum hydrogenase is most similar in length, cysteine positions, and sequence altogether to the translation product of a putative hydrogenase encoding gene from D. vulgaris (Hildenborough). Comparisons of the available [Fe] hydrogenase sequences show that these enzymes constitute a structurally rather homogeneous family. While they differ in the length of their N-termini and in the number of their [4Fe-4S] clusters, they are highly similar in their C-terminal halves, which are postulated to harbor the hydrogen-activating H cluster. Five conserved cysteine residues occurring in this domain are likely ligands of the H cluster. Possible ligation by other residues, and in particular by methionine, is discussed. The comparisons carried out here show that the H clusters most probably possess a common structural framework in all [Fe] hydrogenases. On the basis of the available data on these proteins and on the current developments in iron-sulfur chemistry, the H clusters possibly contain six to eight iron atoms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ligands to the 2Fe iron-sulfur center in succinate dehydrogenase

Membrane-bound succinate oxidoreductases are flavoenzymes containing one each of a 2Fe, a 3Fe and a 4Fe iron-sulfur center. Amino acid sequence homologies indicate that all three centers are located in the Ip (B) subunit. From polypeptide and gene analysis of Bacillus subtilis succinate dehydrogenase-defective mutants combined with earlier EPR spectroscopic data, we show that four conserved cysteine residues in the first half of Ip are the ligands to the [2Fe-2S] center. These four residues have previously been predicted to be the ligands. Our results also suggest that the N-terminal part of B. subtilis Ip constitutes a domain which can incorporate separately the 2Fe center and interact with Fp, the flavin-containing subunit of the dehydrogenase.     

An EPR/HYSCORE, Mössbauer, and resonance Raman study of the hydrogenase maturation enzyme HydF: a model for N-coordination to [4Fe–4S] clusters

The biosynthesis of the organometallic H cluster of [Fe–Fe] hydrogenase requires three accessory proteins, two of which (HydE and HydG) belong to the radical S-adenosylmethionine enzyme superfamily. The third, HydF, is an Fe–S protein with GTPase activity. The [4Fe–4S] cluster of HydF is bound to the polypeptide chain through only the three, conserved, cysteine residues present in the binding sequence motif CysXHisX_(46-53)HisCysXXCys. However, the involvement of the two highly conserved histidines as a fourth ligand for the cluster coordination is controversial. In this study, we set out to characterize further the [4Fe–4S] cluster of HydF using Mössbauer, EPR, hyperfine sublevel correlation (HYSCORE), and resonance Raman spectroscopy in order to investigate the influence of nitrogen ligands on the spectroscopic properties of [4Fe–4S]^2+/+ clusters. Our results show that Mössbauer, resonance Raman, and EPR spectroscopy are not able to readily discriminate between the imidazole-coordinated [4Fe–4S] cluster and the non-imidazole-bound [4Fe–4S] cluster with an exchangeable fourth ligand that is present in wild-type HydF. HYSCORE spectroscopy, on the other hand, detects the presence of an imidazole/histidine ligand on the cluster on the basis of the appearance of a specific spectral pattern in the strongly coupled region, with a coupling constant of approximately 6 MHz. We also discovered that a His-tagged version of HydF, with a hexahistidine tag at the N-terminus, has a [4Fe–4S] cluster coordinated by one histidine from the tag. This observation strongly indicates that care has to be taken in the analysis of data obtained on tagged forms of metalloproteins.     

Human lipoprotein lipase. Analysis of the catalytic triad by site-directed mutagenesis of Ser-132, Asp-156, and His-241.

Lipoprotein lipase (LPL) plays a central role in normal lipid metabolism as the key enzyme involved in the hydrolysis of triglycerides present in chylomicrons and very low density lipoproteins. LPL is a member of a family of hydrolytic enzymes that include hepatic lipase and pancreatic lipase. Based on primary sequence homology of LPL to pancreatic lipase, Ser-132, Asp-156, and His-241 have been proposed to be part of a domain required for normal enzymic activity. We have analyzed the role of these potential catalytic residues by site-directed mutagenesis and expression of the mutant LPL in human embryonic kidney-293 cells. Substitution of Ser-132, Asp-156, and His-241 by several different residues resulted in the expression of an enzyme that lacked both triolein and tributyrin esterase activities. Mutation of other conserved residues, including Ser-97, Ser-307, Asp-78, Asp-371, Asp-440, His-93, and His-439 resulted in the expression of active enzymes. Despite their effect on LPL activity, substitutions of Ser-132, Asp-156, and His-241 did not change either the heparin affinity or lipid binding properties of the mutant LPL. In summary, mutation of Ser-132, Asp-156, and His-241 specifically abolishes total hydrolytic activity without disrupting other important functional domains of LPL. These combined results strongly support the conclusion that Ser-132, Asp-156, and His-241 form the catalytic triad of LPL and are essential for LPL hydrolytic activity.     

Mutagenesis study of the 2Fe-2S center and the FAD binding site of the Na(+)-translocating NADH:ubiquinone oxidoreductase from Vibrio cholerae

Many marine and pathogenic bacteria have a unique sodium-translocating NADH:ubiquinone oxidoreductase (Na(+)-NQR), which generates an electrochemical Na(+) gradient during aerobic respiration. Na(+)-NQR consists of six subunits (NqrA-F) and contains five known redox cofactors: two covalently bound FMNs, one noncovalently bound FAD, one riboflavin, and one 2Fe-2S center. A stable neutral flavin-semiquinone radical is observed in the air-oxidized enzyme, while the NADH- or dithionite-reduced enzyme exhibits a stable anionic flavin-semiquinone radical. The NqrF subunit has been implicated in binding of both the 2Fe-2S cluster and the FAD. Four conserved cysteines (C70, C76, C79, and C111) in NqrF match the canonical 2Fe-2S motif, and three conserved residues (R210, Y212, S246) have been predicted to be part of a flavin binding domain. In this work, these two motifs have been altered by site-directed mutagenesis of individual residues and are confirmed to be essential for binding, respectively, the 2Fe-2S cluster and FAD. EPR spectra of the FAD-deficient mutants in the oxidized and reduced forms exhibit neutral and anionic flavo-semiquinone radical signals, respectively, demonstrating that the FAD in NqrF is not the source of either radical signal. In both the FAD and 2Fe-2S center mutants the line widths of the neutral and anionic flavo-semiquinone EPR signals are unchanged from the wild-type enzyme, indicating that neither of these centers is nearby or coupled to the radicals. Measurements of steady-state turnover using NADH, Q-1, and the artificial electron acceptor ferricyanide strongly support an electron transport pathway model in which the noncovalently bound FAD in the NqrF subunit is the initial electron acceptor and electrons then flow to the 2Fe-2S center.