Biomedical subjects

L C Seefeldt

Publications and source records attributed to L C Seefeldt.

At least 37 records · Page 2Linked to original sources

PubMed1998

Electron transfer in nitrogenase analyzed by Marcus theory: evidence for gating by MgATP.

Nitrogenase-catalyzed substrate reduction reactions require electron transfer between two component proteins, the iron (Fe) protein and the molybdenum-iron (MoFe) protein, in a reaction that is coupled to the hydrolysis of MgATP. In the present work, electron transfer (Marcus) theory has been applied to nitrogenase electron transfer reactions to gain insights into possible roles for MgATP in this reaction. Evidence is presented indicating that an event associated with either MgATP binding or hydrolysis acts to gate electron transfer between the two component proteins. In addition, evidence is presented that the reaction mechanism can be fundamentally changed such that electron transfer becomes rate-limiting by the alteration of a single amino acid within the nitrogenase Fe protein (deletion of Leu 127, L127 Delta). These studies utilized the temperature dependence of intercomponent electron transfer within two different nitrogenase complexes: the wild-type nitrogenase complex that requires MgATP for electron transfer and the L127 Delta Fe protein-MoFe protein complex that does not require MgATP for electron transfer. It was found that the wild-type nitrogenase electron transfer reaction did not conform to Marcus theory, suggesting that an adiabatic event associated with MgATP interaction precedes electron transfer and is rate-limiting. Application of transition state theory provided activation parameters for this rate-limiting step. In contrast, electron transfer from the L127 Delta Fe protein variant to the MoFe protein (which does not require MgATP hydrolysis) was found to be described by Marcus theory, indicating that electron transfer was rate-limiting. Marcus parameters were determined for this reaction with a reorganization energy (lambda) of 2.4 eV, a coupling constant (HAB) of 0.9 cm-1, a free energy change (Delta G' degrees ) of -22.0 kJ/mol, and a donor-acceptor distance (r) of 14 A. These values are consistent with parameters deduced for electron transfer reactions in other protein-protein systems where electron transfer is rate-limiting. Finally, the electron transfer reaction within the L127 Delta Fe protein-MoFe protein complex was found to be reversible. These results are discussed in the context of models for how MgATP interactions might be coupled to electron transfer in nitrogenase.

Adenosine Triphosphate↗

PubMed1997

Changes in the midpoint potentials of the nitrogenase metal centers as a result of iron protein-molybdenum-iron protein complex formation.

All nitrogenase-catalyzed substrate reduction reactions require the transient association between the iron (Fe) protein component and the molybdenum-iron (MoFe) protein component with concomitant intercomponent electron transfer and MgATP hydrolysis. Understanding the effects of Fe protein-MoFe protein complex formation on the properties of the nitrogenase metal centers is thus essential to understanding the electron transfer reactions. This work presents evidence for significant shifts in midpoint potentials for two of the three nitrogenase metal centers as a result of Fe protein binding to the MoFe protein. The midpoint potentials for the three nitrogenase metal centers, namely the [4Fe-4S] cluster of the Fe protein, and the [8Fe-7S] (or P-) clusters and FeMo cofactors (or M-centers) of the MoFe protein, were determined within a nondissociating nitrogenase complex prepared with a site-specifically altered Fe protein (Leu at position 127 deleted, L127Delta). The midpoint potential for each metal center was determined by mediated redox titrations, with the redox state of each center being monitored by parallel and perpendicular mode EPR spectroscopy. The midpoint potential of the Fe protein [4Fe-4S]2+/1+ cluster couple was observed to change by -200 mV from -420 mV in the uncomplexed L127Delta Fe protein to -620 mV in the L127Delta Fe protein-MoFe protein complex. The midpoint potential of the two electron oxidized couple of the P-clusters (P2+/N) of the MoFe protein was observed to shift by -80 mV upon protein-protein complex formation. No significant change in the midpoint potential of an oxidized state of FeMoco (Mox/N) was observed upon complex formation. These results provide insights into the energetics of intercomponent electron transfer in nitrogenase, suggesting that the energy of protein-protein complex formation is coupled to an increase in the driving force for electron transfer. The results are interpreted in light of the expected changes in the protein environments of the metal centers within the nitrogenase complex.

Adenosine Triphosphate↗

PubMed1997

Entropies of redox reactions between proteins and mediators: the temperature dependence of reversible electrode potentials in aqueous buffers.

The temperature dependencies of the reversible electrode potentials for a number of charge transfer reactions of redox mediators were used to evaluate the corresponding charge transfer entropies in Tris-HCl (pH 8) buffer. The redox mediator thermodynamic data, along with reaction enthalpy data for mediator redox protein electron transfer, were used to evaluate the charge transfer entropy for the cytochrome c redox couple [(cytc)ox/(cytc)red] in Tris-HCl (pH 8) buffer and were found to be equal to -16 cal/degrees K mol. Reversible electrode potentials at 298 degrees K for the redox mediator half-reactions were observed to vary from -528 to +657 mV (vs NHE). Charge transfer entropies were observed to depend upon the structure of the redox mediators and to vary from -13.8 to -29.7 cal/degrees K mol for a closely related series of organic dications (viologens) and a value of -43.6 cal/degrees K mol was observed for the [Fe(CN)6]3-/[Fe(CN)6]4- couple under the same conditions. A procedure for determining charge transfer entropies of protein redox couples which cannot be studied by direct electrochemical methods is outlined. The factors contributing to the magnitude of the charge transfer entropies are discussed.

Buffers↗

PubMed1997

Reduction of thiocyanate, cyanate, and carbon disulfide by nitrogenase: kinetic characterization and EPR spectroscopic analysis.

Nitrogenase catalyzes the reduction of N2, protons, and a number of alternative substrates that contain C-C, C-N, N-N, and N-O double and triple bonds. Recently it has been shown that nitrogenase also reduces the C==S bond of COS and the C==O bond of CO2. The current work demonstrates that the COS analogs SCN-, CS2, and OCNH are novel substrates for nitrogenase and that the reduction of these substrates produces changes in the electron paramagnetic resonance (EPR) spectrum of nitrogenase, providing insight into the mechanism of substrate reduction by nitrogenase. CH4, HCN, H2S, and NH4+ were detected as products of the nitrogenase-catalyzed reduction of SCN-. CS2 was reduced by nitrogenase to H2S, providing the first demonstration of CS2 reduction catalyzed by a purified enzyme. CO was detected as a product of KOCN reduction by nitrogenase. Interestingly, the Km for KOCN reduction to CO decreased at lower pH values, suggesting that OCNH rather than OCN- was the substrate for nitrogenase. Analysis of the EPR spectra of nitrogenase under turnover conditions in the presence of KOCN, CS2, or KSCN revealed new EPR signals. Signals with g-values corresponding to those reported for CO bound to the iron-molybdenum cofactor of nitrogenase were detected during turnover of nitrogenase in the presence of KOCN. During SCN- and CS2 reduction by nitrogenase, novel EPR inflections were observed that appear to report the interaction between nitrogenase and a bound substrate or a transient intermediate produced during the reduction of SCN- and CS2.

Argon↗

PubMed1997

Determination of rate and equilibrium constants for the reactions between electron transfer mediators and proteins by linear sweep voltammetry.

Redox proteins undergo measurable charge transfer at electrodes only under special circumstances, while they readily take part in electron transfer reactions with mediators in solution. Advantage was taken of the latter fact to develop a new method to study the kinetics and equilibria of protein-mediator electron transfer reactions. It was shown that rate and equilibrium constants for the electron exchange between electron transfer mediator and the protein can be obtained from the analysis of the perturbation of the linear sweep voltammetry (LSV) response of the mediator due to the presence of the protein. The experiments were carried out under conditions where the protein does not interact with the electrode. Theoretical data obtained by digital simulation are presented to show the conditions under which rate and equilibrium constants are accessible by the LSV technique. The electron transfer reactions between ferri- and ferrocytochrome c and N,N,N',N'-tetramethylphenylenediamine and the corresponding radical cation in phosphate-buffered saline (0.04 M phosphate, pH 7.4, 0.1 M NaCl) buffer were selected to demonstrate the technique. These studies resulted in an equilibrium constant equal to 1.0 and forward and reverse rate constants equal to 1.6 x 10(4) M-1 s-1. The data available from this method include forward and reverse rate constants for electron transfer and the formal potential for the protein redox couple.

Animals↗

PubMed1997

A mediated thin-layer voltammetry method for the study of redox protein electrochemistry.

A novel mediated thin-layer voltammetry technique that allows the rapid determination of midpoint potentials and electron transfer rate constants for small quantities of redox active proteins is described. Thin-layer voltammograms simulated for an electrolyte containing a redox active protein and an electron transfer mediator show that the rapid homogeneous electron exchange reaction between the protein and the mediator serves to mediate the charge transfer of the protein at the electrode, which does not take place in the absence of the mediator, and results in the observation of an apparently reversible redox couple. Both theoretical and experimental data are presented which suggest that the thin-layer voltammetry method will be generally applicable for the determination of protein redox potentials with the proper selection of mediators. Rate constants for the electron transfer between metalloproteins and mediators can be evaluated by comparing experimental voltammograms with theoretical data from simulations. The technique is demonstrated for the metalloproteins cytochrome c, ferredoxin, and the iron protein of nitrogenase.

Animals↗

PubMed1997

Evidence for electron transfer-dependent formation of a nitrogenase iron protein-molybdenum-iron protein tight complex. The role of aspartate 39.

Nitrogenase-catalyzed substrate reduction reactions require the association of the iron (Fe) protein and the molybdenum-iron (MoFe) protein, electron transfer from the Fe protein to the MoFe protein coupled to the hydrolysis of MgATP, followed by protein-protein complex dissociation. This work examines the role of MgATP hydrolysis and electron transfer in the dissociation of the Fe protein-MoFe protein complex. Alteration of aspartate 39 to asparagine (D39N) in the nucleotide binding site of Azotobacter vinelandii Fe protein by site-directed mutagenesis resulted in an Fe protein-MoFe protein complex that did not dissociate after electron transfer. While the D39N Fe protein-MoFe protein complex was inactive in all substrate reduction reactions, the complex catalyzed both reductant-dependent and reductant-independent MgATP hydrolysis. Once docked to the MoFe protein, the D39N Fe protein was found to transfer one electron to the MoFe protein requiring MgATP hydrolysis, with an apparent first order rate constant of 0.02 s-1 compared with 140 s-1 for the wild-type Fe protein. Only following electron transfer to the MoFe protein did the D39N Fe protein form a tight complex with the MoFe protein, with no detectable dissociation rate. This was in contrast with the dissociation rate constant of the wild-type Fe protein from the MoFe protein following electron transfer of 5 s-1. Chemically oxidized D39N Fe protein with MgADP-bound did not form a tight complex with the MoFe protein, showing a dissociation rate similar to chemically oxidized wild-type Fe protein (3 s-1 for D39N Fe protein and 6 s-1 for wild-type Fe protein). These results suggest that electron transfer from the Fe protein to the MoFe protein within the protein-protein complex normally induces conformational changes which increase the affinity of the Fe protein for the MoFe protein. A model is presented in which Asp-39 participates in a nucleotide signal transduction pathway involved in component protein-protein dissociation.

Adenosine Triphosphate↗

PubMed1996

Electron transfer from the nitrogenase iron protein to the [8Fe-(7/8)S] clusters of the molybdenum-iron protein.

The reduction of substrates catalyzed by nitrogenase requires electron transfer between the iron (Fe) protein and the molybdenum-iron (MoFe) protein in a reaction that is coupled to the hydrolysis of MgATP. The [4Fe-4S] cluster of the Fe protein transfers one electron ultimately to the M-clusters (FeMoco) of the MoFe protein for substrate reduction, with the P-clusters ([8Fe-(7/8)S]) of the MoFe protein as proposed electron transfer intermediates. This work presents direct EPR evidence for primary electron transfer from the [4Fe-4S] cluster of the Fe protein to the P-clusters of the MoFe protein in a reaction that requires the MgATP-bound state of the Fe protein. An oxidized state of the MoFe protein was prepared in which the P-clusters were oxidized by 2 equiv of electrons to the P2+ state. In this oxidation state, the M-clusters (S = 3/2) and the P(2+-clusters (S > or = 3) are paramagnetic and can be observed by perpendicular and parallel mode EPR, providing the opportunity to follow electron transfer from the Fe protein to either cluster type in the MoFe protein. Electron transfer from the reduced [4Fe-4S]1+ cluster of two different Fe proteins to the P2+ clusters of the MoFe protein was observed by the disappearance of the [4Fe-4S]1+ cluster EPR signal and the conversion of the MoFe protein P-clusters from the P2+ to the P1+ oxidation state. In the first case, stoichiometric quantities of the wild-type Fe protein transferred one electron to the P-clusters only in the presence of MgATP. MgADP would not support this electron transfer reaction. In the second case, an altered Fe protein (L127 delta) that is in a conformation resembling the MgATP-bound state was found to transfer an electron to the P-clusters in the absence of MgATP. These results suggest that the first electron transferred from the Fe protein goes to the P-cluster and that the MgATP-bound protein conformation of the Fe protein, not MgATP hydrolysis, is required for this electron transfer reaction.

Adenosine Triphosphate↗

PubMed1996

The [4Fe-4S] cluster domain of the nitrogenase iron protein facilitates conformational changes required for the cooperative binding of two nucleotides.

MgATP binding and hydrolysis are central to all reduction reactions catalyzed by nitrogenase. The iron (Fe) protein component of nitrogenase is a homodimeric protein with a bridging [4Fe-4S] cluster and two nucleotide binding sites, one on each subunit. This work presents evidence that the [4Fe-4S] cluster domain of the nitrogenase Fe protein functions as a hinge region between the two nucleotide binding domains, participating in the cooperative binding of two nucleotides. Alanine residues at position 98 (located near the [4Fe-4S] cluster) of the Azotobacter vinelandii Fe protein were changed by means of site-directed mutagenesis to Val (V) and Gly (G), and the resulting altered proteins were purified and characterized. While the wild-type and A98G Fe proteins were found to bind two nucleotides (MgATP or MgADP) with strong cooperativity (Hill coefficient of 2), the A98V Fe protein was found to bind one nucleotide with no apparent cooperativity. The binding of two nucleotides to the wild-type Fe protein is known to induce protein conformational changes which are reflected as changes in the properties of the [4Fe-4S] cluster, including a change in the redox potential of the [4Fe-4S] cluster of -120 mV for MgATP binding (-300 to -420 mV) and of -160 mV for MgADP binding (-300 to -460 mV). The binding of one nucleotide to the A98V Fe protein was found to result in only half the lowering of the redox potential, with MgATP binding resulting in a -80 mV change (-280 to -360 mV) and MgADP binding resulting in a -50 mV change (-280 to -330 mV). Results from 1H NMR, EPR, and CD spectra, along with Fe chelation rates, were all consistent with the binding of a single nucleotide to the A98V Fe protein inducing a partial conformational change. Finally, the A98V Fe protein with one nucleotide bound, still bound to the molybdenum-iron protein but did not support MgATP hydrolysis, electron transfer, or substrate reduction. A model is discussed in which the [4Fe-4S] cluster domain can be viewed as a hinge region between the two nucleotide binding domains which facilitates conformational rearrangements required for the cooperative binding of a second nucleotide.

Acetylene↗

PubMed1996

Elucidating the mechanism of nucleotide-dependent changes in the redox potential of the [4Fe-4S] cluster in nitrogenase iron protein: the role of phenylalanine 135.

Nucleotide binding to the nitrogenase iron (Fe) protein results in a lowering of the redox potential of its [4Fe-4S] cluster by over 100 mV, and this is thought to be essential for electron transfer to the molybdenum-iron (MoFe) protein for substrate reduction. This work presents evidence for an important role of the strictly conserved phenylalanine at position 135, located near the [4Fe-4S] cluster of nitrogenase Fe protein, in defining both the redox potential and the nucleotide-induced changes in the redox potential of the [4Fe-4S] cluster. Phe 135 was changed by means of site-directed mutagenesis to the amino acids Tyr (F135Y), Ile (F135I), Trp (F135W), and His (F135H), and the altered proteins were purified to homogeneity. Minor changes in the UV/visible and EPR spectra arising from the [4Fe-4S] cluster were detected in the altered proteins, while dramatic changes were observed in the visible region circular dichroism (CD) spectrum, suggesting that Phe 135 contributes significantly to the chiroptical properties of the [4Fe-4S] cluster. Likewise, significant changes in the redox potentials of the Phe altered Fe proteins were observed, with shifts of +50 to +120 mV compared to the redox potential of the wild-type Fe protein (-300 mV). The shifts in redox potential for the altered Fe proteins appeared to correlate with changes in isotropically shifted proton NMR resonances assigned to cluster ligands. All of the Phe 135 altered Fe proteins were found to bind either MgADP or MgATP, while the reduced and oxidized states of the F135W and F135H altered Fe proteins had significantly higher affinities for binding MgATP when compared to the wild-type Fe protein. While MgATP binding to the wild-type and Phe 135 altered Fe proteins resulted in approximately -100 mV shifts in the redox potentials for all proteins, MgADP binding resulted in only -30 to -50 mV shifts for the altered proteins compared to a -160 mV shift for the wild-type Fe protein. The current results suggest that Phe 135 is important in defining the redox potential of the [4Fe-4S] cluster in the Fe protein and influences the MgADP (but not MgATP) induced modulation of the redox potential.

Adenosine Diphosphate↗

PubMed1996

Evidence for electron transfer from the nitrogenase iron protein to the molybdenum-iron protein without MgATP hydrolysis: characterization of a tight protein-protein complex.

MgA TP hydrolysis has been proposed to be absolutely required for electron transfer from the nitrogenase iron (Fe) protein to the molybdenum-iron (MoFe) protein. This work presents evidence for primary electron transfer from the Azotobacter vinelandii nitrogenase Fe protein to the MoFe protein in the absence of MgATP hydrolysis. Deletion of an amino acid (Leu 127) in a signal transduction pathway in the Fe protein resulted in an Fe protein conformation resembling the MgATP-bound state. This altered Fe protein (L127delta) was found to bind to the MoFe protein in the absence of MgATP, forming a tight protein complex. Both steady state and stopped-flow transient kinetic measurements suggest that two L127delta Fe proteins bind to one MoFe protein with an extremely high affinity. From pre-steady state kinetic determinations of the rate of complex dissociation, the affinity was found to be at least 350 times tighter than that of the wild-type A. vinelandii nitrogenase complex and at least 20 times tighter than that of the heterologous Clostridium pasteurianum Fe protein-A. vinelandii MoFe protein complex. The L127delta Fe protein-MoFe protein complex was isolated by gel filtration liquid chromatography. Scanning densitometry of an SDS gel of the complex isolated from the gel filtration column revealed a stoichiometry of 1.7 L 127 delta Fe proteins bound per MoFe protein. The L 127 delta Fe protein was found to transfer a single electron from its [4Fe-4S] cluster to the MoFe protein at a rate of 0.2s-1. This compares with the MgATP dependent electron transfer rate of 140 s-1 observed for transfer of an electron from the wild-type Fe protein to the MoFe protein. No substrate reduction (H+ or C2H2) was detected when wild-type MoFe protein was complemented with L 127 delta Fe protein. The MgATP-independent electron transfer from the L 127 delta Fe protein to the MoFe protein required active MoFe protein and was not inhibited by MgADP. EPR spectroscopy of the complex was employed to confirm the electron transfer reaction. These results show that Fe protein in a conformation resembling the MgATP-bound state can transfer at least one electron to the MoFe protein without the need for MgATP hydrolysis.

Acetylene↗

PubMed1996

Elucidation of a MgATP signal transduction pathway in the nitrogenase iron protein: formation of a conformation resembling the MgATP-bound state by protein engineering.

The present work defines one MgATP signal transduction pathway in the nitrogenase iron (Fe) protein. Deletion of an amino acid (Leu 127) by site-directed mutagenesis in the protein chain between Asp 125, located in the ATP binding site, and Cys 132, a ligand to the [4Fe-4S] cluster, resulted in protein conformational changes resembling the MgATP-bound state in the absence of any bound nucleotides. Specifically, 1H nuclear magnetic resonance, electron paramagnetic resonance, and circular dichroism spectroscopic properties, along with Fe chelation assays, suggested that deletion of Leu 127 in the Fe protein resulted in changes in the electronic properties of the [4Fe-4S] cluster similar to those normally observed upon MgATP binding to the wild-type Fe protein. Deletion of Leu 127 of the Fe protein lowered the redox potential of of the [4FE-4S] cluster by 112 mV compared to the wild-typeFe protein (-412mV compared to -294 mV). A nearly identical lowering of the redox potential by 120 mV occurs in the wild-type Fe protein upon binding MgATP (-294 mV compared to 420 mV). The L127delta Fe protein did not contain bound nucleotides which could account for the observed conformational changes. The present results support a model in which the protein chain from ASP 125 to Cys 132 acts as one pathway for MgATP signal transduction and suggests a mechanism for this transduction to the [4Fe-4S] cluster. The L127delta Fe protein was found to still bind 2 MgATP or 2 MgADP molecules/Fe protein. Unlike the wild-type Fe protein, the L127delta Fe protein bound 2 ADP molecules/Fe protein in the absence of Mg2+. Finally, the L127delta protein was found to bind to the MoFe protein, although the complex did not catalyze MgATP hydrolysis or substrate reduction. In concurrence with previous models, homologies between the Asp 125 to Cys 132 transduction pathway in Fe protein and the switch II region of the broad class of GTPase signal transduction proteins (G-proteins) are discussed.

Adenosine Triphosphate↗

PubMed1996

Circular dichroism and x-ray spectroscopies of Azotobacter vinelandii nitrogenase iron protein. MgATP and MgADP induced protein conformational changes affecting the [4Fe-4S] cluster and characterization of a [2Fe-2S] form.

Nucleotide interactions with nitrogenase are a central part of the mechanism of nitrogen reduction. Previous studies have suggested that MgATP or MgADP binding to the nitrogenase iron protein (Fe protein) induce protein conformational changes that control component protein docking, interprotein electron transfer, and substrate reduction. In the present study, we have investigated the effects of MgATP or MgADP binding to the Azotobacter vinelandii nitrogenase Fe protein on the properties of the [4Fe-4S] cluster using circular dichroism (CD) and x-ray absorption spectroscopies. Previous CD and magnetic CD studies on nitrogenase Fe protein suggested that binding of either MgATP or MgADP to the Fe protein resulted in identical changes in the CD spectrum arising from transitions of the [4Fe-4S]2+ cluster. We present evidence that MgADP or MgATP binding to the oxidized nitrogenase Fe protein results in distinctly different CD spectra, suggesting distinct changes in the environment of the [4Fc-4S] cluster. The present results are consistent with previous studies such as chelation assays, electron paramagnetic resonance, and NMR, which suggested that MgADP or MgATP binding to the nitrogenase Fe protein induced different conformational changes. The CD spectrum of a [2Fe-2S]2+ form of the nitrogenase Fe protein was also investigated to address the possibility that the MgATP- or MgADP-induced changes in the CD spectrum of the native enzyme were the result of a partial conversion from a [4Fe-4S] cluster to a [2Fe-2S] cluster. No evidence was found for a contribution of a [2Fe-2S]2+ cluster to the CD spectrum of oxidized Fe protein in the absence or presence of nucleotides. A novel two-electron reduction of the [2Fe-2S]2+ cluster in Fe protein was apparent from absorption, CD, and electron paramagnetic resonance data. Fe K-edge x-ray absorption spectra of the oxidized Fe protein revealed no changes in the structure of the [4Fe-4S] cluster upon MgATP binding to the Fe protein. The present results reveal that MgATP or MgADP binding to the oxidized state of the Fe protein result in different conformational changes in the environment around the [4Fe-4S] cluster.

Absorptiometry, Photon↗

PubMed1995

Proton NMR investigation of the [4Fe--4S]1+ cluster environment of nitrogenase iron protein from Azotobacter vinelandii: defining nucleotide-induced conformational changes.

This work presents the complete assignment of the isotropically shifted 1H NMR resonances of Azotobacter vinelandii nitrogenase iron protein (Fe protein) to beta-CH2 and alpha-CH protons of the [4Fe--4S]1+ cluster cysteinyl ligands. Four resonances were observed for the reduced Fe protein with chemical shifts of 49, 23, 17, and 13 ppm. T1 measurements and analysis of relative peak areas coupled with one-dimensional nuclear Overhauser effect (NOE) difference spectra were used to assign the two most downfield-shifted resonances (49 and 23 ppm) to cysteinyl ligand beta-CH2 protons and the 17 and 14 ppm resonances to cysteinyl ligand alpha-CH protons. Temperature dependent studies of the isotropically shifted protons revealed both Curie and anti-Curie behavior. These results, along with previous Mossbauer studies of the Fe protein, allowed the assignment of signal A (49 ppm) to four beta-CH2 protons and signal C (17 ppm) to 2 alpha-CH protons of two cysteinyl ligands bound to a mixed-valence iron pair (Fe3(+)--Fe2+) of the [4Fe--4S]1+ cluster. Signal B (23 ppm) was assigned to four beta-CH2 protons, and signal C (17 ppm) and D (13 ppm) were assigned to two alpha-CH protons of two cysteinyl ligands bound to a ferrous pair of irons (2Fe2+). The effects of MgATP, MgADP, and Mg-adenosine-beta, gamma-methylene-5'-triphosphate binding to the Fe protein on the assigned resonances were established and are discussed in the context of nucleotide-induced changes in the protein environment of the [4Fe--4S] cluster.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗

PubMed1995

Nucleotide hydrolysis and protein conformational changes in Azotobacter vinelandii nitrogenase iron protein: defining the function of aspartate 129.

The biological reduction of dinitrogen catalyzed by nitrogenase requires the hydrolysis of a minimum of 16 MgATP for each N2 reduced. The present work examines the role of a strictly conserved aspartic acid residue of nitrogenase iron protein (Fe protein) in coupling MgATP hydrolysis to electron transfer and substrate reduction. The aspartic acid residue at position 129 in the Azotobacter vinelandii Fe protein has been suggested to participate in nucleotide interactions from its location in the X-ray structure near several amino acids previously identified to participate in nucleotide binding and protein conformational changes. The function of this amino acid was probed by changing aspartic acid to glutamic acid (D129E) and asparagine (D129N) by site-directed mutagenesis. The D129N Fe protein proved to be unstable and could not be purified. Characterization of the purified D129E Fe protein revealed a central role for Asp 129 in the nucleotide-induced protein conformational changes in the Fe protein and possibly in the mechanism of MgATP hydrolysis. Data from EPR, circular dichroism spectroscopy, and Fe2+ chelation rates and the chemical shifts of isotropically shifted protons in the 1H NMR spectra implicate Asp 129 in the nucleotide-induced conformational changes in the Fe protein, which are reflected in changes in the environment of the [4Fe-4S] cluster. The D129E Fe protein was found to bind both MgATP and MgADP with high affinity. The Kd determined for MgADP binding (Kd = 131 microM) was comparable to that found for wild-type Fe protein (128 microM). The affinity for MgATP binding was 1.6 times tighter than that for wild-type Fe protein (370 compared to 580 microM). The midpoint reduction potential of the [4Fe-4S] cluster was similar to that determined for the wild-type Fe protein (-290 mV for wild-type Fe protein and -300 mV for D129E Fe protein). Upon the addition of MgATP or MgADP, the midpoint potentials for wild-type and D129E Fe proteins shifted to -430 and -440 mV, respectively. The D129E Fe protein was also found to bind to the molybdenum-iron protein (MoFe protein) with normal affinity, although it could not support electron transfer to the MoFe protein or MoFe protein-stimulated MgATP hydrolysis.

2,2'-Dipyridyl↗

PubMed1995

Evidence for a central role of lysine 15 of Azotobacter vinelandii nitrogenase iron protein in nucleotide binding and protein conformational changes.

Biological nitrogen fixation catalyzed by purified nitrogenase requires the hydrolysis of a minimum of 16 MgATP for each N2 reduced. In the present study, we demonstrate a central function for Lys-15 of Azotobacter vinelandii nitrogenase iron protein (FeP) in the interaction of nucleotides with nitrogenase. Changing Lys-15 of the FeP to Arg resulted in an FeP with a dramatically reduced affinity for both MgATP and MgADP. From equilibrium column binding experiments at different nucleotide concentrations, apparent dissociation constants (Kd) for wild type FeP binding of MgADP (143 microM) and MgATP (571 microM) were determined. Over the same nucleotide concentration ranges, the K15R FeP showed no significant affinity for either nucleotide. This contrasts sharply with previous results with an FeP in which Lys-15 was changed to Gln (K15Q) where it was found that the K15Q FeP bound MgADP with the same affinity as wild type FeP and MgATP with a slightly reduced affinity. Analysis of K15R FeP by EPR, circular dichroism (CD), and microcoulometry revealed that the [4Fe-4S] cluster was unaffected by the amino acid change and that addition of either MgADP or MgATP did not result in the protein conformational changes normally detected by these techniques. These results are integrated into a model for how MgATP and MgADP bind and induce conformational changes within the FeP.

Adenosine Diphosphate↗

PubMed1995

Carbonyl sulfide and carbon dioxide as new substrates, and carbon disulfide as a new inhibitor, of nitrogenase.

Nitrogenase is the metalloenzyme responsible for the biological reduction of N2 to NH3. Nitrogenase has been shown to reduce a variety of substrates in addition to N2 and protons. General properties of alternative substrates for nitrogenase are the presence of N-N, N-O, N-C, and C-C triple or double bonds. In the present work, we demonstrate that Azotobacter vinelandii nitrogenase can reduce both C-S and C-O bonds. Nitrogenase was found to reduce carbonyl sulfide (COS), to CO and H2S at a maximum rate of 37.2 +/- 2.0 nmol min-1 (mg of protein)-1 with a Km of 3.1 +/- 0.6 mM. The formation of CO from nitrogenase reduction of COS was monitored spectrophotometrically in real time by following the formation of carboxyhemoglobin. In this assay, the change in the visible absorption spectrum of reduced hemoglobin upon binding CO provided a sensitive way to quantify CO formation and to remove CO, which is a potent inhibitor of nitrogenase, from solution. COS reduction by nitrogenase required the molybdenum-iron protein (MoFeP), the iron protein (FeP), and MgATP. The reduction reaction was inhibited by MgADP, acetylene, and N2, while H2 was not an inhibitor of COS reduction. The pH optimum for COS reduction was 6.5. Nitrogenase was also found to reduce carbon dioxide (CO2) to CO and H2O. CO2 was reduced at a maximum rate of 0.8 +/- 0.07 nmole min-1 (mg of protein)-1 with a calculated Km for CO2 of 23.3 +/- 3.7 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Azotobacter vinelandii↗

PubMed1995

Analysis of a gene region required for dihydrogen oxidation in Azotobacter vinelandii.

The putative products of six Azotobacter vinelandii chromosomal open reading frames (ORFs) were suggested to be involved in dihydrogen (H2) metabolism [Chen and Mortenson (1992) Biochim Biophys Acta 1131, 199-202]. A promoterless lacZ-containing cassette was used to disrupt the ORFs. Qualitative analysis revealed that the lacZ genes were expressed only in those mutants where the directions of the inserted lacZ were identical to those of the ORFs, showing that the six ORFs were transcribed as predicted. Unlike wildtype (w.t.), none of the mutants could perform dioxygen (O2)-dependent H2-oxidation, even though Western immunoanalyses showed that the hydrogenase large subunit was present although in amounts less than in w.t. Only one of the mutants (a hypB mutant), grown in nickel-enriched media, showed meaningful restoration of the H2-oxidizing ability. From the above observations it is concluded that (a) the six-ORF region is transcriptionally active and involved in H2-oxidation, (b) the product of hypB is needed for nickel activation of hydrogenase, and (c) the six ORFs (genes) belong to two or more operons. Possible roles of the gene products for the assembly, modification, and processing of hydrogenase from its apoproteins and metal centers are discussed.

Azotobacter vinelandii↗