Biomedical subjects

Dennis R Dean

Publications and source records attributed to Dennis R Dean.

At least 19 recordsLinked to original sources

PubMed2006

NifX and NifEN exchange NifB cofactor and the VK-cluster, a newly isolated intermediate of the iron-molybdenum cofactor biosynthetic pathway.

The iron-molybdenum cofactor of nitrogenase (FeMo-co) is synthesized in a multistep process catalysed by several Nif proteins and is finally inserted into a pre-synthesized apo-dinitrogenase to generate mature dinitrogenase protein. The NifEN complex serves as scaffold for some steps of this synthesis, while NifX belongs to a family of small proteins that bind either FeMo-co precursors or FeMo-co during cofactor synthesis. In this work, the binding of FeMo-co precursors and their transfer between purified Azotobacter vinelandii NifX and NifEN proteins was studied to shed light on the role of NifX on FeMo-co synthesis. Purified NifX binds NifB cofactor (NifB-co), a precursor to FeMo-co, with high affinity and is able to transfer it to the NifEN complex. In addition, NifEN and NifX exchange another [Fe-S] cluster that serves as a FeMo-co precursor, and we have designated it as the VK-cluster. In contrast to NifB-co, the VK-cluster is electronic paramagnetic resonance (EPR)-active in the reduced and the oxidized states. The NifX/VK-cluster complex is unable to support in vitro FeMo-co synthesis in the absence of NifEN because further processing of the VK-cluster into FeMo-co requires the simultaneous activities of NifEN and NifH. Our in vitro studies suggest that the role of NifX in vivo is to serve as transient reservoir of FeMo-co precursors and thus help control their flux during FeMo-co synthesis.

Azotobacter vinelandii↗

PubMed2006

A methyldiazene (HN=N-CH3)-derived species bound to the nitrogenase active-site FeMo cofactor: Implications for mechanism.

Methyldiazene (HN=N-CH3) isotopomers labeled with 15N at the terminal or internal nitrogens or with 13C or 2H were used as substrates for the nitrogenase alpha-195Gln-substituted MoFe protein. Freeze quenching under turnover traps an S = (1/2) state that has been characterized by EPR and 1H-, 15N-, and 13C-electron nuclear double resonance spectroscopies. These studies disclosed the following: (i) a methyldiazene-derived species is bound to the active-site FeMo cofactor; (ii) this species binds through an [-NHx] fragment whose N derives from the methyldiazene terminal N; and (iii) the internal N from methyldiazene probably does not bind to FeMo cofactor. These results constrain possible mechanisms for reduction of methyldiazene. In the Chatt-Schrock mechanism for N2 reduction, H atoms sequentially add to the distal N before N-N bond cleavage (d-mechanism). In a d-mechanism for methyldiazene reduction, a bound [-NHx] fragment only occurs after reduction by three electrons, which leads to N-N bond cleavage and the release of the first NH3. Thus, the appearance of bound [-NHx] is compatible with the d-mechanism only if it represents a late stage in the reduction process. In contrast are mechanisms where H atoms add alternately to distal and proximal nitrogens before N-N cleavage (a-mechanism) and release of the first NH3 after reduction by five electrons. An [-NHx] fragment would be bound at every stage of methyldiazene reduction in an a-mechanism. Although current information does not rule out the d-mechanism, the a-mechanism is more attractive because proton delivery to substrate has been specifically compromised in alpha-195Gln-substituted MoFe protein.

Binding Sites↗

PubMed2006

Controlled expression and functional analysis of iron-sulfur cluster biosynthetic components within Azotobacter vinelandii.

A system for the controlled expression of genes in Azotobacter vinelandii by using genomic fusions to the sucrose catabolic regulon was developed. This system was used for the functional analysis of the A. vinelandii isc genes, whose products are involved in the maturation of [Fe-S] proteins. For this analysis, the scrX gene, contained within the sucrose catabolic regulon, was replaced by the contiguous A. vinelandii iscS, iscU, iscA, hscB, hscA, fdx, and iscX genes, resulting in duplicate genomic copies of these genes: one whose expression is directed by the normal isc regulatory elements (Pisc) and the other whose expression is directed by the scrX promoter (PscrX). Functional analysis of [Fe-S] protein maturation components was achieved by placing a mutation within a particular Pisc-controlled gene with subsequent repression of the corresponding PscrX-controlled component by growth on glucose as the carbon source. This experimental strategy was used to show that IscS, IscU, HscBA, and Fdx are essential in A. vinelandii and that their depletion results in a deficiency in the maturation of aconitase, an enzyme that requires a [4Fe-4S] cluster for its catalytic activity. Depletion of IscA results in a null growth phenotype only when cells are cultured under conditions of elevated oxygen, marking the first null phenotype associated with the loss of a bacterial IscA-type protein. Furthermore, the null growth phenotype of cells depleted of HscBA could be partially reversed by culturing cells under conditions of low oxygen. Conserved amino acid residues within IscS, IscU, and IscA that are essential for their respective functions and/or whose replacement results in a partial or complete dominant-negative growth phenotype were also identified using this system.

Aconitate Hydratase↗

PubMed2006

Breaking the N2 triple bond: insights into the nitrogenase mechanism.

Nitrogenase is the metalloenzyme that performs biological nitrogen fixation by catalyzing the reduction of N2 to ammonia. Understanding how the nitrogenase active site metal cofactor (FeMo-cofactor) catalyzes the cleavage of the N2 triple bond has been the focus of intense study for more than 50 years. Goals have included the determination of where and how substrates interact with the FeMo-cofactor, and the nature of reaction intermediates along the reduction pathway. Progress has included the trapping of intermediates formed during turnover of non-physiological substrates (e.g., alkynes, CS2) providing insights into how these molecules interact with the nitrogenase FeMo-cofactor active site. More recently, substrate-derived species have been trapped at high concentrations during the reduction of N2, a diazene, and hydrazine, providing the first insights into binding modes and possible mechanisms for N2 reduction. A comparison of the current state of knowledge of the trapped species arising from non-physiological substrates and nitrogenous substrates is beginning to reveal some of the intricacies of how nitrogenase breaks the N2 triple bond.

Electron Spin Resonance Spectroscopy↗

PubMed2005

Electron inventory, kinetic assignment (E(n)), structure, and bonding of nitrogenase turnover intermediates with C2H2 and CO.

Improved 1H ENDOR data from the S(EPR1) intermediate formed during turnover of the nitrogenase alpha-195Gln MoFe protein with C2(1,2)H2 in (1,2)H2O buffers, taken in context with the recent study of the intermediate formed from propargyl alcohol, indicate that S(EPR1) is a product complex, likely with C2H4 bound as a ferracycle to a single Fe of the FeMo-cofactor active site. 35 GHz CW and Mims pulsed 57Fe ENDOR of 57Fe-enriched S(EPR1) cofactor indicates that it exhibits the same valencies as those of the CO-bound cofactor of the lo-CO intermediate formed during turnover with CO, [Mo4+, Fe3+, Fe6(2+), S9(2-)(d43)](+1), reduced by m = 2 electrons relative to the resting-state cofactor. Consideration of 57Fe hyperfine coupling in S(EPR1) and lo-CO leads to a picture in which CO bridges two Fe of lo-CO, while the C2H4 of S(EPR1) binds to one of these. To correlate these and other intermediates with Lowe-Thorneley (LT) kinetic schemes for substrate reduction, we introduce the concept of an "electron inventory". It partitions the number of electrons a MoFe protein intermediate has accepted from the Fe protein (n) into the number transmitted to the substrate (s), the number that remain on the intermediate cofactor (m), and the additional number delivered to the cofactor from the P clusters (p): n = m + s - p (with p = 0 here). The cofactors of lo-CO and S(EPR1) both are reduced by m = 2 electrons, but the intermediates are not at the same LT reduction stage (E(n)): (n = 2; m = 2, s = 0) for lo-CO; (n = 4; s = 2, m = 2) for S(EPR1). This is the first proposed correlation of an LT E(n) kinetic state with a well-defined chemical state of the enzyme.

Acetylene↗

PubMed2005

Intermediates trapped during nitrogenase reduction of N triple bond N, CH3-N=NH, and H2N-NH2.

A high population intermediate has been trapped on the nitrogenase active site FeMo cofactor during reduction of N2. In addition, intermediates have been trapped during reduction of CH3-N=NH by the alpha-195Gln variant and during reduction of H2N-NH2 by the alpha-70Ala/alpha-195Gln variant. Each of these trapped states shows an EPR signal arising from an S = 1/2 state of the FeMo cofactor. 15N ENDOR shows that each intermediate has a nitrogenous species bound to the FeMo cofactor, with a single type of N seen for each bound intermediate. The g tensors are unique to each intermediate, g(e) = [2.084, 1.993, 1.969], g(m) = [2.083, 2.021, 1.993], g(l) = [2.082, 2.015, 1.987], as are the 15N hyperfine couplings at g1, which suggests that three distinct stages of NN reduction may have been trapped. The 1H ENDOR spectra show that the N2 intermediate is at a distinct and earlier stage of reduction from the other two, so at least two stages of NN reduction have been trapped. Some possible structures of the hydrazine intermediate are presented.

Amides↗

PubMed2005

NifS-mediated assembly of [4Fe-4S] clusters in the N- and C-terminal domains of the NifU scaffold protein.

NifU is a homodimeric modular protein comprising N- and C-terminal domains and a central domain with a redox-active [2Fe-2S](2+,+) cluster. It plays a crucial role as a scaffold protein for the assembly of the Fe-S clusters required for the maturation of nif-specific Fe-S proteins. In this work, the time course and products of in vitro NifS-mediated iron-sulfur cluster assembly on full-length NifU and truncated forms involving only the N-terminal domain or the central and C-terminal domains have been investigated using UV-vis absorption and Mössbauer spectroscopies, coupled with analytical studies. The results demonstrate sequential assembly of labile [2Fe-2S](2+) and [4Fe-4S](2+) clusters in the U-type N-terminal scaffolding domain and the assembly of [4Fe-4S](2+) clusters in the Nfu-type C-terminal scaffolding domain. Both scaffolding domains of NifU are shown to be competent for in vitro maturation of nitrogenase component proteins, as evidenced by rapid transfer of [4Fe-4S](2+) clusters preassembled on either the N- or C-terminal domains to the apo nitrogenase Fe protein. Mutagenesis studies indicate that a conserved aspartate (Asp37) plays a critical role in mediating cluster transfer. The assembly and transfer of clusters on NifU are compared with results reported for U- and Nfu-type scaffold proteins, and the need for two functional Fe-S cluster scaffolding domains on NifU is discussed.

Bacterial Proteins↗

PubMed2005

Role of conserved cysteines in mediating sulfur transfer from IscS to IscU.

The role of the three conserved cysteine residues on Azotobacter vinelandii IscU in accepting sulfane sulfur and forming a covalent complex with IscS has been evaluated using electrospray-ionization mass spectrometry studies of variants involving individual cysteine-to-alanine substitutions. The results reveal that IscS can transfer sulfur to each of the three alanine-substituted forms of IscU to yield persulfide or polysulfide species, and formation of a heterodisulfide covalent complex between IscS and Cys(37) on IscU. It is concluded that S transfer from IscS to IscU does not involve a specific cysteine on IscU or the formation of an IscS-IscU heterodisulfide complex.

Azotobacter vinelandii↗

PubMed2005

The interstitial atom of the nitrogenase FeMo-cofactor: ENDOR and ESEEM evidence that it is not a nitrogen.

X-ray crystallographic study of the nitrogenase MoFe protein revealed electron density from an atom (denoted X) inside the active-site metal cluster, the [MoFe7S9:homocitrate] FeMo-cofactor. The electron density associated with X is consistent with a single N, O, or C atom. We now have tested whether X is an N or not by comparing the Q-band ENDOR and ESEEM signals from resting-state (S = 3/2) MoFe protein and NMF-extracted FeMo-co from bacteria grown with either 14N or 15N as the exclusive N source. All of the 14N or 15N signals associated with the protein are lost upon extraction of the FeMo-co. We interpret this as strong evidence that X is not an N.

Crystallography, X-Ray↗

PubMed2005

Trapping a hydrazine reduction intermediate on the nitrogenase active site.

A major challenge in understanding the mechanism of nitrogenase, the enzyme responsible for the biological fixation of N(2) to two ammonias, is to trap a nitrogenous substrate at the enzyme active site in a state that is amenable to further characterization. In the present work, a strategy is described that results in the trapping of the substrate hydrazine (H(2)N-NH(2)) as an adduct bound to the active site metal cluster of nitrogenase, and this bound adduct is characterized by EPR and ENDOR spectroscopies. Earlier work has been interpreted to indicate that nitrogenous (e.g., N(2) and hydrazine) as well as alkyne (e.g., acetylene) substrates can bind at a common FeS face of the FeMo-cofactor composed of Fe atoms 2, 3, 6, and 7. Substitution of alpha-70(Val) that resides over this FeS face by the smaller amino acid alanine was also previously shown to improve the affinity and reduction rate for hydrazine. We now show that when alpha-195(His), a putative proton donor near the active site, is substituted by glutamine in combination with substitution of alpha-70(Val) by alanine, and the resulting doubly substituted MoFe protein (alpha-70(Ala)/alpha-195(Gln)) is turned over with hydrazine as substrate, the FeMo-cofactor can be freeze-trapped in a S = (1)/(2) state in high yield ( approximately 70%). The presumed hydrazine-FeMo-cofactor adduct displays a rhombic EPR signal with g = [2.09, 2.01, 1.93]. The optimal pH for the population of this state was found to be 7.4. The EPR signal showed a Curie law temperature dependence similar to the resting state EPR signal. Mims pulsed ENDOR spectroscopy at 35 GHz using (15)N-labeled hydrazine reveals that the trapped intermediate incorporates a hydrazine-derived species bound to the FeMo-cofactor; in spectra taken at g(1) this species gives a single observed (15)N signal, A(g(1)) = 1.5 MHz.

Azotobacter vinelandii↗

PubMed2005

Trapping H- bound to the nitrogenase FeMo-cofactor active site during H2 evolution: characterization by ENDOR spectroscopy.

We here show that the iron-molybdenum (FeMo)-cofactor of the nitrogenase alpha-70(Ile) molybdenum-iron (MoFe) protein variant accumulates a novel S = (1)/(2) state that can be trapped during the reduction of protons to H(2). (1,2)H-ENDOR measurements disclose the presence of two protons/hydrides (H(+/)(-)) whose hyperfine tensors have been determined from two-dimensional field-frequency (1)H ENDOR plots. The two H(+/)(-) have large isotropic hyperfine couplings, A(iso)( )() approximately 23 MHz, which shows they are bound to the cofactor. The favored analysis for these plots indicates that the two H(+/)(-) have the same principal values, which indicates that they are chemically equivalent. The tensors are further related to each other by a permutation of the tensor components, which indicates an underlying symmetry of binding relative to the cofactor. At present, no model for the structure of the iron-molybdenum (FeMo)-cofactor in the S = (1)/(2) state trapped during the reduction of H(+) can be shown unequivocally to satisfy all of the constraints generated by the ENDOR analysis. The data disfavors any model that involves protonation of sulfides, and thus suggests that the intermediate instead contains two chemically equivalent bound hydrides; it appears unlikely that these are terminal monohydrides.

Binding Sites↗

PubMed2005

Substrate interactions with the nitrogenase active site.

The chemical mechanism for biological cleavage of the N(2) triple bond at ambient pressure and temperature has been the subject of intense study for many years. The site of substrate activation and reduction has been localized to a complex cofactor, called FeMo cofactor, yet until now the complexity of the system has denied information concerning exactly where and how substrates interact with the metal-sulfur framework of the active site. In this Account, we describe a combined genetic, biophysical, and biochemical approach that was used to provide direct and detailed information concerning where alternative alkyne substrates interact with FeMo cofactor during catalysis. The relevance and limitations of this work with respect to N(2) binding and reduction also are discussed.

Alkynes↗

PubMed2005

Structure, function, and formation of biological iron-sulfur clusters.

Iron-sulfur [Fe-S] clusters are ubiquitous and evolutionary ancient prosthetic groups that are required to sustain fundamental life processes. Owing to their remarkable structural plasticity and versatile chemical/electronic features [Fe-S] clusters participate in electron transfer, substrate binding/activation, iron/sulfur storage, regulation of gene expression, and enzyme activity. Formation of intracellular [Fe-S] clusters does not occur spontaneously but requires a complex biosynthetic machinery. Three different types of [Fe-S] cluster biosynthetic systems have been discovered, and all of them are mechanistically unified by the requirement for a cysteine desulfurase and the participation of an [Fe-S] cluster scaffolding protein. Important mechanistic questions related to [Fe-S] cluster biosynthesis involve the molecular details of how [Fe-S] clusters are assembled on scaffold proteins, how [Fe-S] clusters are transferred from scaffolds to target proteins, how various accessory proteins participate in [Fe-S] protein maturation, and how the biosynthetic process is regulated.

Amino Acid Sequence↗

PubMed2004

Substrate interaction at an iron-sulfur face of the FeMo-cofactor during nitrogenase catalysis.

Nitrogenase catalyzes biological dinitrogen fixation, the reduction of N(2) to 2NH(3). Recently, the binding site for a non-physiological alkyne substrate (propargyl alcohol, HC triple bond C-CH(2)OH) was localized to a specific Fe-S face of the FeMo-cofactor approached by the MoFe protein amino acid alpha-70(Val). Here we provide evidence to indicate that the smaller alkyne substrate acetylene (HC triple bond CH), the physiological substrate dinitrogen, and its semi-reduced form hydrazine (H(2)N-NH(2)) interact with the same Fe-S face of the FeMo-cofactor. Hydrazine is a relatively poor substrate for the wild-type (alpha-70(Val)) MoFe protein. Substitution of the alpha-70(Val) residue by an amino acid having a smaller side chain (alanine) dramatically enhanced hydrazine reduction activity. Conversely, substitution of alpha-70(Val) by an amino acid having a larger side chain (isoleucine) significantly lowered the capacity of the MoFe protein to reduce dinitrogen, hydrazine, or acetylene.

Acetylene↗

PubMed2004

An organometallic intermediate during alkyne reduction by nitrogenase.

Nitrogenase is the metalloenzyme that catalyzes the nucleotide-dependent reduction of N(2), as well as reduction of a variety of other triply bonded substrates, including the alkyne, acetylene. Substitution of the alpha-70(Val) residue in the nitrogenase MoFe protein by alanine expands the range of substrates to include short-chain alkynes not reduced by the unaltered protein. Rapid freezing of the alpha-70(Ala) nitrogenase MoFe protein during reduction of the alkyne propargyl alcohol (HC triple bond CH(2)OH; PA) traps an S = (1)/(2) intermediate state of the active-site metal cluster, the FeMo-cofactor. We have combined CW and pulsed (13)C ENDOR (electron-nuclear double resonance) with two quantitative 35 GHz (1,2)H ENDOR techniques, Mims pulsed ENDOR and the newly devised "stochastic field-modulated" ENDOR, to study this intermediate prepared with isotopically substituted ((13)C, (1,2)H) propargyl alcohol in H(2)O and D(2)O buffers. These measurements allow the first description of a trapped nitrogenase reduction intermediate. The S = (1)/(2) turnover intermediate generated during the reduction of PA contains the 3-carbon chain of PA and exhibits resolved (1,2)H ENDOR signals from three protons, two strongly coupled (H(a)) and one weakly coupled (H(b)); H(a)(c) originates as the C3 proton of PA, while H(a)(s) and H(b) are solvent-derived. The two H(a) protons have identical hyperfine tensors, despite having different origins. The equality of the (H(a)(s), H(a)(c)) hyperfine tensors strongly constrains proposals for the structure of the cluster-bound reduced PA. Through consideration of model structures found in the Cambridge Structural Database, we propose that the intermediate contains a novel bio-organometallic complex in which a reduction product of propargyl alcohol binds as a metalla-cyclopropane ring to a single Fe atom of the Fe-S face of the FeMo-cofactor that is composed of Fe atoms 2, 3, 6, and 7. Of the two most attractive structures, one singly reduced at C3 (4), the other being the doubly reduced allyl alcohol product (6), we tentatively favor 6 because of the "natural" assignment it affords for H(b).

Alkynes↗

PubMed2004

Localization of a catalytic intermediate bound to the FeMo-cofactor of nitrogenase.

Nitrogenase catalyzes the biological reduction of N(2) to ammonia (nitrogen fixation) as well as the reduction of a number of alternative substrates, including acetylene (HC identical with CH) to ethylene (H2C=CH2). It is known that the metallocluster FeMo-cofactor located within the nitrogenase MoFe protein component provides the site of substrate reduction, but the exact site where substrates bind and are reduced on the FeMo-cofactor remains unknown. We have recently shown that the alpha-70 residue of the MoFe protein plays a significant role in defining substrate access to the active site; alpha-70 approaches one face of the FeMo-cofactor, and when valine is substituted by alanine at this position, the substituted nitrogenase is able to accommodate a reduction of the larger alkyne propargyl alcohol (HC identical with CCH(2)OH, propargyl-OH). During this reduction, a substrate-derived intermediate can be trapped on the FeMo-cofactor resulting in an S = 1/2 spin system with a novel electron paramagnetic resonance spectrum. In the present work, trapping of the propargyl-OH-derived or propargyl amine (HC identical with CCH(2)NH(2), propargyl-NH(2))-derived intermediates is shown to be dependent on pH and the presence of histidine at position alpha-195. It is concluded that these catalytic intermediates are stabilized and thereby trapped by H-bonding interactions between either the-OH group or the-NH(3)(+)group and the imidazole epsilon-NH of alpha-195(His). Thus, for the first time it is possible to establish the location of a bound substrate-derived intermediate on the FeMo-cofactor. Refinement of the binding mode and site was accomplished by the use of density functional and force field calculations pointing to an eta(2) coordination at Fe-6 of the FeMo-cofactor.

Acetylene↗

PubMed2004

Iron-sulfur cluster assembly: NifU-directed activation of the nitrogenase Fe protein.

The NifU protein is a homodimer that is proposed to provide a molecular scaffold for the assembly of [Fe-S] clusters uniquely destined for the maturation of the nitrogenase catalytic components. There are three domains contained within NifU, with the N-terminal domain exhibiting a high degree of primary sequence similarity to a related family of [Fe-S] cluster biosynthetic scaffolds designated IscU. The C-terminal domain of NifU exhibits sequence similarity to a second family of proposed [Fe-S] cluster biosynthetic scaffolds designated Nfu. Genetic experiments described here involving amino acid substitutions within the N-terminal and C-terminal domains of NifU indicate that both domains can separately participate in nitrogenase-specific [Fe-S] cluster formation, although the N-terminal domain appears to have the dominant function. These in vivo experiments were supported by in vitro [Fe-S] cluster assembly and transfer experiments involving the activation of an apo-form of the nitrogenase Fe protein.

Bacterial Proteins↗

PubMed2004

Substrate interactions with nitrogenase: Fe versus Mo.

Biological nitrogen reduction is catalyzed by a complex two-component metalloenzyme called nitrogenase. For the Mo-dependent enzyme, the site of substrate reduction is provided by a [7Fe-9S-Mo-X-homocitrate] metallocluster, where X is proposed to be an N atom. Recent progress with organometallic model compounds, theoretical calculations, and biochemical, kinetic, and biophysical studies on nitrogenase has led to the formulation of two opposing models of where N(2) or alternative substrates might bind during catalysis. One model involves substrate binding to the Mo atom, whereas the other model involves the participation of one or more Fe atoms located in the central region of the metallocluster. Recently gathered evidence that has provided the basis for both models is summarized, and a perspective on future research in resolving this fundamental mechanistic question is presented.

Bacterial Proteins↗