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L C Seefeldt

Publications and source records attributed to L C Seefeldt.

50 records · Page 3Linked to original sources
PubMed1994

Docking of nitrogenase iron- and molybdenum-iron proteins for electron transfer and MgATP hydrolysis: the role of arginine 140 and lysine 143 of the Azotobacter vinelandii iron protein.

Docking of the nitrogenase component proteins, the iron protein (FeP) and the molybdenum-iron protein (MoFeP), is required for MgATP hydrolysis, electron transfer between the component proteins, and substrate reductions catalyzed by nitrogenase. The present work examines the function of 3 charged amino acids, Arg 140, Glu 141, and Lys 143, of the Azotobacter vinelandii FeP in nitrogenase component protein docking. The function of these amino acids was probed by changing each to the neutral amino acid glutamine using site-directed mutagenesis. The altered FePs were expressed in A. vinelandii in place of the wild-type FeP. Changing Glu 141 to Gln (E141Q) had no adverse effects on the function of nitrogenase in whole cells, indicating that this charged residue is not essential to nitrogenase function. In contrast, changing Arg 140 or Lys 143 to Gln (R140Q and K143Q) resulted in a significant decrease in nitrogenase activity, suggesting that these charged amino acid residues play an important role in some function of the FeP. The function of each amino acid was deduced by analysis of the properties of the purified R140Q and K143Q FePs. Both altered proteins were found to support reduced substrate reduction rates when coupled to wild-type MoFeP. Detailed analysis revealed that changing these residues to Gln resulted in a dramatic reduction in the affinity of the altered FeP for binding to the MoFeP. This was deduced in FeP titration, NaCl inhibition, and MoFeP protection from Fe2+ chelation experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1994

A continuous, spectrophotometric activity assay for nitrogenase using the reductant titanium(III) citrate.

A continuous, spectrophotometric assay for determining electron transfer rates through nitrogenase during substrate reduction reactions was developed. The assay takes advantage of the facts that Ti(III) citrate can serve as a reductant for nitrogenase-catalyzed reduction reactions and that oxidation of Ti(III) citrate to Ti(IV) citrate results in a dramatic change in its absorption spectrum. Ti(III) citrate supported nitrogenase-catalyzed substrate (e.g., H+ or acetylene) reduction reactions at about the same rate as that supported by the reductant dithionite (S2O4(2-)). In addition, Ti(III) citrate had an absorption maximum centered at 325 nm, while oxidized Ti(IV) citrate had a much lower absorption in this wavelength region. An absorption coefficient for Ti(III) citrate of 0.73 mM-1.cm-1 at 340 nm was determined by titration with redox dyes with known absorption coefficients. Using this experimentally determined absorption coefficient, we developed an assay that provides a convenient way to determine electron transfer rates through nitrogenase in real time by spectrophotometrically following the oxidation of Ti(III) citrate to Ti(IV) citrate. Average electron transfer rates of 3749 +/- 218 nmol of electrons transferred.min-1.mg iron protein-1 for H+ reduction were determined using this assay which are directly comparable to the rates calculated from fixed time point, gas chromatographic assays of H2 formation. The utility of the Ti(III) citrate assay for nitrogenase is discussed and demonstrated using the nitrogenase inhibitors MgADP, CN-, and NO.

Azotobacter vinelandii↗
PubMed1993

Increasing nitrogenase catalytic efficiency for MgATP by changing serine 16 of its Fe protein to threonine: use of Mn2+ to show interaction of serine 16 with Mg2+.

MgATP-binding and hydrolysis are an integral part of the nitrogenase catalytic mechanism. We are exploring the function of MgATP hydrolysis in this reaction by analyzing the properties of the Fe protein (FeP) component of Azotobacter vinelandii nitrogenase altered by site-directed mutagenesis. We have previously (Seefeldt, L.C., Morgan, T.V., Dean, D.R., & Mortenson, L.E., 1992, J. Biol. Chem. 267, 6680-6688) identified a region near the N-terminus of FeP that is involved in interaction with MgATP. This region of FeP is homologous to the well-known nucleotide-binding motif GXXXXGKS/T. In the present work, we examined the function of the four hydroxyl-containing amino acids immediately C-terminal to the conserved lysine 15 that is involved in interaction with the gamma-phosphate of MgATP. We have established, by altering independently Thr 17, Thr 18, and Thr 19 to alanine, that a hydroxyl-containing residue is not needed at these positions for FeP to function. In contrast, an hydroxyl-containing amino acid at position 16 was found to be critical for FeP function. When the strictly conserved Ser 16 was altered to Ala, Cys, Asp, or Gly, the FeP did not support N2 fixation when expressed in place of the wild-type FeP in A. vinelandii. Altering Ser 16 to Thr (S16T), however, resulted in the expression of an FeP that was partially active. This S16T FeP was purified to homogeneity, and its biochemical examination allowed us to assign a catalytic function to this hydroxyl group in the nitrogenase mechanism. Of particular importance was the finding that the S16T FeP had a significantly higher affinity for MgATP than the wild-type FeP, with a measured Km of 20 microM compared to the wild-type FeP Km of 220 microM. This increased kinetic affinity for MgATP was reflected in a significantly stronger binding of the S16T FeP for MgATP. In contrast, the affinity for MgADP, which binds at the same site as MgATP, was unchanged. The catalytic efficiency (kcat/Km) of S16T FeP was found to be 5.3-fold higher than for the wild-type FeP, with the S16T FeP supporting up to 10 times greater nitrogenase activity at low MgATP concentrations. This indicates a role for the hydroxyl group at position 16 in interaction with MgATP but not MgADP. The site of interaction of this residue was further defined by examining the properties of wild-type and S16T FePs in utilizing MnATP compared with MgATP.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗
PubMed1992

Mapping the site(s) of MgATP and MgADP interaction with the nitrogenase of Azotobacter vinelandii. Lysine 15 of the iron protein plays a major role in MgATP interaction.

Nitrogenase binds and hydrolyzes 2MgATP yielding 2MgADP and 2Pi for each electron that is transferred from the iron protein to the MoFe protein. The iron protein alone binds but does not hydrolyze 2MgATP or 2MgADP and the binding of these nucleotides is competitive. Iron protein amino acid sequences all contain a putatitive mononucleotide-binding region similar to a region found in other mononucleotide-binding proteins. To examine the role of this region in MgATP interaction, we have substituted glutamine and proline for conserved lysine 15. The amino acid substitutions, K15Q and K15P, both yielded a non-N2-fixing phenotype when the genes coding for them were substituted into the Azotobacter vinelandii chromosome in place of the wild-type gene. The iron protein from the K15Q mutant was purified to homogeneity, whereas the protein from the K15P mutant could not be purified in its native form. Unlike wild-type iron protein, the purified K15Q iron protein showed no acetylene reduction, H2 evolution, or ATP hydrolysis activities when complemented with wild-type MoFe protein. The K15Q iron protein and the normal iron protein had a similar total iron content and both proteins showed the characteristic rhombic EPR signal resulting from the reduced state of the single 4Fe-4S cluster bridging the two subunits. Unlike the wild-type iron protein, addition of MgATP to the K15Q iron protein did not result in the perturbation necessary to change the EPR signal of its 4Fe-4S center from a rhombic to an axial line shape. Also unlike the wild-type iron protein, addition of MgATP to K15Q iron protein in the presence of the iron chelator, alpha,alpha'-dipyridyl, did not result in a time-dependent transfer of iron to the chelator. Thus, even though the K15Q iron protein contains a normal 4Fe-4S center, it does not respond to MgATP like the wild-type protein. Examination of the ability of the K15Q iron protein to bind MgADP showed no change from the wild-type iron protein, but its ability to bind MgATP decreased to 35% of the wild-type protein. Thus, in A. vinelandii iron protein, lysine 15 is not needed for interaction with MgADP but is involved in the binding of ATP, presumably through charge-charge interaction with the gamma-phosphate. Based on the above data, this lysine appears to be essential for the MgATP induced conformational change of wild-type iron protein that is required for activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗
PubMed1992

Kinetic and spectroscopic analysis of the inactivating effects of nitric oxide on the individual components of Azotobacter vinelandii nitrogenase.

The effects of nitric oxide (NO) on the individual components of Azotobacter vinelandii nitrogenase have been examined by kinetic and spectroscopic methods. Incubation of the Fe protein (Av2) for 1 h with stoichiometries of 4- and 8-fold molar excesses of NO to Av2 dimer resulted in a complete loss of activity of Av2 in C2H2-reduction assays. The kinetics of inactivation indicated that the minimum stoichiometry of NO to Av2 required to fully inactivate Av2 lies between 1 and 2. The rate of inactivation of Av2 activity by NO was stimulated up to 2-fold by the presence of MgATP and MgADP but was unaffected by the presence of sodium dithionite. Unexpectedly, complete inactivation of Av2 by low ratios of NO to Av2 also resulted in a complete loss of its ability to bind MgATP and MgADP. UV-visible spectroscopy indicated that the effect of NO on Av2 involves oxidation of the [4Fe-4S] center. EPR spectroscopy revealed that the loss of activity during inactivation of Av2 by NO correlated with the loss of the S = 1/2 and S = 3/2 signals. Appearance of the classical and intense iron-nitrosyl signal (g = 20.3) was only observed when Av2 was incubated with large molar excesses of NO and the appearance of this signal did not correlate with the loss of Av2 activity. The effects of NO on the MoFe protein (Av1) were more complex than for Av2. A time-dependent inactivation of Av1 activity (C2H2 reduction) was observed which required considerably higher concentrations of NO than those required to inactivate Av2 (up to 10 kPa).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1990

The identification, characterization, sequencing and mutagenesis of the genes (hupSL) encoding the small and large subunits of the H2-uptake hydrogenase of Azotobacter chroococcum.

The structural genes (hupSL) of the membrane-bound NiFe-containing H2-uptake hydrogenase (Hup) of Azotobacter chroococcum were identified by oligonucleotide screening and sequenced. The small subunit gene (hupS) encodes a signal sequence of 34 amino acids followed by a 310-amino-acid, 34156D protein containing 12 cysteine residues. The large subunit gene (hupL) overlaps hupS by one base and codes for a predicted 601-amino-acid, 66433D protein. There are two regions of strong homology with other Ni hydrogenases: a Cys-Thr-Cys-Cys-Ser motif near the N-terminus of HupS and an Asp-Pro-Cys-Leu-Ala-Cys motif near the carboxy-terminus of HupL. Strong overall homology exists between Azotobacter, Bradyrhizobium japonicum and Rhodobacter capsulatus Hup proteins but less exists between the Azotobacter proteins and hydrogenases from Desulfovibrio strains. Mutagenesis of either hupS or hupL genes of A. chroococcum yielded Hup- phenotypes but some of these mutants retained a partial H2-evolving activity. Hybridization experiments at different stages of gene segregation confirmed the multicopy nature of the Azotobacter genome.

Amino Acid Sequence↗
PubMed1989

Immunological relationship among hydrogenases.

We examined the immunological cross-reactions of 11 different hydrogenase antigens with 9 different hydrogenase antibodies. Included were antibodies and antigens of both subunits of the hydrogenases of Bradyrhizobium japonicum and Thiocapsa roseopersicina. The results showed a strong relationship among the Ni-Fe dimeric hydrogenases. The two subunits of Ni-Fe dimeric hydrogenases appeared immunologically distinct: specific interactions occurred only when antibodies to the 60- and 30-kilodalton subunits reacted with the 60- and 30-kilodalton-subunit antigens. The interspecies cross-reactions suggested that at least one conserved protein region exists among the large subunits of these enzymes, whereas the small subunits are less conserved. Antibodies to the Fe-only bidirectional hydrogenase of Clostridium pasteurianum reacted with the Desulfovibrio vulgaris bidirectional hydrogenase. Surprisingly, antibodies to the clostridial uptake hydrogenase did not react with any of the Fe-only bidirectional hydrogenases but did react with several of the Ni-Fe dimeric hydrogenases. The two hydrogenases from C. pasteurianum were found to be quite different immunologically. The possible relationship of these findings to the structure and catalytic functions of hydrogenase are discussed.

Antibodies↗
PubMed1989

Cyanide inactivation of hydrogenase from Azotobacter vinelandii.

The effects of cyanide on membrane-associated and purified hydrogenase from Azotobacter vinelandii were characterized. Inactivation of hydrogenase by cyanide was dependent on the activity (oxidation) state of the enzyme. Active (reduced) hydrogenase showed no inactivation when treated with cyanide over several hours. Treatment of reversibly inactive (oxidized) states of both membrane-associated and purified hydrogenase, however, resulted in a time-dependent, irreversible loss of hydrogenase activity. The rate of cyanide inactivation was dependent on the cyanide concentration and was an apparent first-order process for purified enzyme (bimolecular rate constant, 23.1 M-1 min-1 for CN-). The rate of inactivation decreased with decreasing pH. [14C]cyanide remained associated with cyanide-inactivated hydrogenase after gel filtration chromatography, with a stoichiometry of 1.7 mol of cyanide bound per mol of inactive enzyme. The presence of saturating concentrations of CO had no effect on the rate or extent of cyanide inactivation of hydrogenases. The results indicate that cyanide can cause a time-dependent, irreversible inactivation of hydrogenase in the oxidized, activatable state but has no effect when hydrogenase is in the reduced, active state.

Azotobacter↗
PubMed1988

Aerobic, inactive forms of Azotobacter vinelandii hydrogenase: activation kinetics and insensitivity to C2H2 inhibition.

Azotobacter vinelandii hydrogenase (EC class 1.12), either purified or membrane-associated, was obtained aerobically in an inactive state. The kinetics of activation by treatment with a reductant (H2 or dithionite) were determined. Three distinct phases of the activation were observed. Aerobically prepared, inactive hydrogenase was insensitive to acetylene inhibition, but could be rendered acetylene-sensitive by reduction with dithionite. These findings indicate that acetylene inhibition of hydrogenase requires catalytically active enzyme.

Acetylene↗
PubMed1987

Redox-dependent subunit dissociation of Azotobacter vinelandii hydrogenase in the presence of sodium dodecyl sulfate.

Hydrogenases catalyze the reversible activation of dihydrogen. We have previously demonstrated that the purified hydrogenase from the nitrogen-fixing microorganism Azotobacter vinelandii is an alpha beta dimer (98,000 Da) with subunits of 67,000 (alpha) and 31,000 (beta) daltons and that this enzyme contains iron and nickel. The enzyme can be purified anaerobically in the presence of dithionite in a fully active state that is irreversibly inactivated by exposure to O2. Analysis of this hydrogenase by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) following boiling in SDS yields two protein staining bands corresponding to the alpha and beta subunits. However, when this enzyme was treated with SDS (25-65 degrees C) for up to 30 min under anaerobic/reductive conditions and then analyzed by anaerobic SDS-PAGE, a protein staining band corresponding to an apparent molecular mass of 58,000 Da was observed that stained for hydrogenase activity. Analysis of the 58,000-Da activity staining band by a Western immunoblot or a second aerobic SDS-polyacrylamide gel revealed that this protein actually consisted of both the alpha and beta subunits. Thus, the activity staining band (apparent 58,000 Da) represents the 98,000-Da dimer migrating abnormally on SDS-PAGE. Treatment of the anaerobically purified hydrogenase with SDS under aerobic conditions or under anaerobic conditions with electron acceptors prior to electrophoresis resulted in no activity staining band and the separated alpha and beta subunits. A. vinelandii hydrogenase was also purified under aerobic conditions in an inactive O2 stable form that can be activated by removal of oxygen followed by addition of reductant. This enzyme (as isolated), the activated form, and the reoxidized form were analyzed for their stability toward denaturation by SDS. We conclude that the dissociation of the A. vinelandii hydrogenase subunits in SDS is controlled by the redox state of the enzyme suggesting an important role of one or more redox sites in controlling the structure of this enzyme.

2,6-Dichloroindophenol↗
PubMed1986

Reversible inactivation of the O2-labile hydrogenases from Azotobacter vinelandii and Rhizobium japonicum.

Hydrogenases catalyze the reversible activation of dihydrogen. The hydrogenases from the aerobic, N2-fixing microorganisms Azotobacter vinelandii and Rhizobium japonicum are nickel- and iron-containing dimers that belong to the group of O2-labile enzymes. Exposure of these hydrogenases to O2 results in an irreversible inactivation; therefore, these enzymes are purified anaerobically in a fully active state. We describe in this paper an electron acceptor-requiring and pH-dependent, reversible inactivation of these hydrogenases. These results are the first example of an anaerobic, reversible inactivation of the O2-labile hydrogenases. The reversible inactivation required the presence of an electron acceptor. The rate of inactivation was first-order, with similar rates observed for methylene blue, benzyl viologen, and phenazine-methosulfate. The rate of inactivation was also dependent on the pH. However, increasing the pH of the enzyme in the absence of an electron acceptor did not result in inactivation. Thus, the reversible inactivation was not a result of high pH alone. The inactive enzyme could not be reactivated by H2 or other reductants at high pH. Titration of enzyme inactivated at high pH back to low pH was also ineffective at reactivating the enzyme. However, if reductants were present during this titration, the enzyme could be fully reactivated. The temperature dependence of inactivation yielded an activation energy of 44 kJ X mol-1. Gel filtration chromatography of active and inactive hydrogenase indicated that neither dissociation nor aggregation of the dimer hydrogenase was responsible for this reversible inactivation. We propose a four-state model to describe this reversible inactivation.

Azotobacter↗
PubMed1986

Purification to homogeneity of Azotobacter vinelandii hydrogenase: a nickel and iron containing alpha beta dimer.

Azotobacter vinelandii hydrogenase has been purified to homogeneity from membranes. The enzyme was solubilized with Triton X-100 followed by ammonium sulfate-hexane extractions to remove lipids and detergent. The enzyme was then purified by carboxymethyl-Sepharose and octyl-Sepharose column chromatography. All purification steps were performed under anaerobic conditions in the presence of dithionite and dithiothreitol. The enzyme was purified 143-fold from membranes to a specific activity of 124 mumol of H2 uptake . min-1 . mg protein-1. Nondenaturing polyacrylamide gel electrophoresis of the hydrogenase revealed a single band which stained for both activity and protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands corresponding to peptides of 67,000 and 31,000 daltons. Densitometric scans of the SDS-gel indicated a molar ratio of the two bands of 1.07 +/- 0.05. The molecular weight of the native enzyme was determined by three different methods. While gel permeation gave a molecular weight of 53,000, sucrose density gradient centrifugation and native polyacrylamide gel electrophoresis gave molecular weights of 98,600 +/- 10,000 and 98,600 +/- 2,000, respectively. We conclude that the A. vinelandii hydrogenase is an alpha beta dimer (98,000 daltons) with subunits of 67,000 and 31,000 daltons. Analyses for nickel and iron indicated 0.68 +/- 0.01 mol Ni/mol hydrogenase and 6.6 +/- 0.5 mol Fe/mol hydrogenase. The isoelectric point of the enzyme was 6.1 +/- 0.01. In addition, several catalytic properties of the enzyme have been examined. The Km for H2 was 0.86 microM, and H2 evolution was observed in the presence of reduced methyl viologen. The pH profile of enzyme activity with methylene blue as the electron acceptor has been determined, along with the Km and Vmax for various electron acceptors.

Azotobacter↗
PubMed1985

Molecular and immunological comparison of membrane-bound, H2-oxidizing hydrogenases of Bradyrhizobium japonicum, Alcaligenes eutrophus, Alcaligenes latus, and Azotobacter vinelandii.

The membrane-bound hydrogenases of Bradyrhizobium japonicum, Alcaligenes eutrophus, Alcaligenes latus, and Azotobacter vinelandii were purified extensively and compared. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of each hydrogenase revealed two prominent protein bands, one near 60 kilodaltons and the other near 30 kilodaltons. The migration distances during nondenaturing polyacrylamide gel electrophoresis were similar for all except A. vinelandii hydrogenase, which migrated further than the other three. The amino acid composition of each hydrogenase was determined, revealing substantial similarity among these enzymes. This was confirmed by calculation of S delta Q values, which ranged from 8.0 to 26.7 S delta Q units. S delta Q is defined as sigma j(Xi,j-Xk,j)2, where i and k identify the proteins compared and Xj is the content (residues per 100) of a given amino acid of type j. The hydrogenases of this study were also compared with an enzyme-linked immunosorbent assay. Antibody raised against B. japonicum hydrogenase cross-reacted with all four hydrogenases, but to various degrees and in the order B. japonicum greater than A. latus greater than A. eutrophus greater than A. vinelandii. Antibody raised against A. eutrophus hydrogenase also cross-reacted with all four hydrogenases, following the pattern of cross-reaction A. eutrophus greater than A. latus = B. japonicum greater than A. vinelandii. Antibody raised against B. japonicum hydrogenase inhibited B. japonicum hydrogenase activity to a greater extent than the A. eutrophus and A. latus activities; no inhibition of A. vinelandii hydrogenase activity was detected. The results of these experiments indicated remarkable homology of the hydrogenases from these four microorganisms.

Alcaligenes↗