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1.
ACS Cent Sci ; 7(10): 1751-1755, 2021 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-34729418

RESUMO

The iron oxo unit, [Fe=O] n+ is a critical intermediate in biological oxidation reactions. While its higher oxidation states are well studied, relatively little is known about the least-oxidized form [FeIII=O]+. Here, the thermally stable complex PhB(AdIm)3Fe=O has been structurally, spectroscopically, and computationally characterized as a bona fide iron(III) oxo. An unusually short Fe-O bond length is consistent with iron-oxygen multiple bond character and is supported by electronic structure calculations. The complex is thermally stable yet is able to perform hydrocarbon oxidations, facilitating both C-O bond formation and dehydrogenation reactions.

2.
J Am Chem Soc ; 143(37): 15358-15368, 2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34498465

RESUMO

In nature, methane is oxidized to methanol by two enzymes, the iron-dependent soluble methane monooxygenase (sMMO) and the copper-dependent particulate MMO (pMMO). While sMMO's diiron metal active site is spectroscopically and structurally well-characterized, pMMO's copper sites are not. Recent EPR and ENDOR studies have established the presence of two monocopper sites, but the coordination environment of only one has been determined, that within the PmoB subunit and denoted CuB. Moreover, this recent work only focused on a type I methanotrophic pMMO, while previous observations of the type II enzyme were interpreted in terms of the presence of a dicopper site. First, this report shows that the type II Methylocystis species strain Rockwell pMMO, like the type I pMMOs, contains two monocopper sites and that its CuB site has a coordination environment identical to that of type I enzymes. As such, for the full range of pMMOs this report completes the refutation of prior and ongoing suggestions of multicopper sites. Second, and of primary importance, EPR/ENDOR measurements (a) for the first time establish the coordination environment of the spectroscopically observed site, provisionally denoted CuC, in both types of pMMO, thereby (b) establishing the assignment of this site observed by EPR to the crystallographically observed metal-binding site in the PmoC subunit. Finally, these results further indicate that CuC is the likely site of biological methane oxidation by pMMO, a conclusion that will serve as a foundation for proposals regarding the mechanism of this reaction.

3.
Chem Sci ; 12(20): 6913-6922, 2021 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-34123320

RESUMO

The electronic structure of the active-site metal cofactor (FeV-cofactor) of resting-state V-dependent nitrogenase has been an open question, with earlier studies indicating that it exhibits a broad S = 3/2 EPR signal (Kramers state) having g values of ∼4.3 and 3.8, along with suggestions that it contains metal-ions with valencies [1V3+, 3Fe3+, 4Fe2+]. In the present work, genetic, biochemical, and spectroscopic approaches were combined to reveal that the EPR signals previously assigned to FeV-cofactor do not correlate with active VFe-protein, and thus cannot arise from the resting-state of catalytically relevant FeV-cofactor. It, instead, appears resting-state FeV-cofactor is either diamagnetic, S = 0, or non-Kramers, integer-spin (S = 1, 2 etc.). When VFe-protein is freeze-trapped during high-flux turnover with its natural electron-donating partner Fe protein, conditions which populate reduced states of the FeV-cofactor, a new rhombic S = 1/2 EPR signal from such a reduced state is observed, with g = [2.18, 2.12, 2.09] and showing well-defined 51V (I = 7/2) hyperfine splitting, a iso = 110 MHz. These findings indicate a different assignment for the electronic structure of the resting state of FeV-cofactor: S = 0 (or integer-spin non-Kramers state) with metal-ion valencies, [1V3+, 4Fe3+, 3Fe2+]. Our findings suggest that the V3+ does not change valency throughout the catalytic cycle.

4.
Nat Chem ; 13(7): 683-691, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34155376

RESUMO

Mammalian oocytes undergo major changes in zinc content and localization to be fertilized, the most striking being the rapid exocytosis of over 10 billion zinc ions in what are known as zinc sparks. Here, we report that fertilization of amphibian Xenopus laevis eggs also initiates a zinc spark that progresses across the cell surface in coordination with dynamic calcium waves. This zinc exocytosis is accompanied by a newly recognized loss of intracellular manganese. Synchrotron-based X-ray fluorescence and analytical electron microscopy reveal that zinc and manganese are sequestered in a system of cortical granules that are abundant at the animal pole. Through electron-nuclear double-resonance studies, we rule out Mn2+ complexation with phosphate or nitrogenous ligands in intact eggs, but the data are consistent with a carboxylate coordination environment. Our observations suggest that zinc and manganese fluxes are a conserved feature of fertilization in vertebrates and that they function as part of a physiological block to polyspermy.


Assuntos
Fertilização/fisiologia , Metais Pesados/metabolismo , Óvulo/metabolismo , Xenopus laevis/metabolismo , Animais , Embrião não Mamífero/metabolismo , Embrião não Mamífero/ultraestrutura , Exocitose/fisiologia , Fertilização/efeitos dos fármacos , Metais Pesados/farmacologia , Óvulo/efeitos dos fármacos , Óvulo/ultraestrutura
5.
Proc Natl Acad Sci U S A ; 118(23)2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34074779

RESUMO

Some methane-oxidizing bacteria use the ribosomally synthesized, posttranslationally modified natural product methanobactin (Mbn) to acquire copper for their primary metabolic enzyme, particulate methane monooxygenase. The operons encoding the machinery to biosynthesize and transport Mbns typically include genes for two proteins, MbnH and MbnP, which are also found as a pair in other genomic contexts related to copper homeostasis. While the MbnH protein, a member of the bacterial diheme cytochrome c peroxidase (bCcP)/MauG superfamily, has been characterized, the structure and function of MbnP, the relationship between the two proteins, and their role in copper homeostasis remain unclear. Biochemical characterization of MbnP from the methanotroph Methylosinus trichosporium OB3b now reveals that MbnP binds a single copper ion, present in the +1 oxidation state, with high affinity. Copper binding to MbnP in vivo is dependent on oxidation of the first tryptophan in a conserved WxW motif to a kynurenine, a transformation that occurs through an interaction of MbnH with MbnP. The 2.04-Å-resolution crystal structure of MbnP reveals a unique fold and an unusual copper-binding site involving a histidine, a methionine, a solvent ligand, and the kynurenine. Although the kynurenine residue may not serve as a CuI primary-sphere ligand, being positioned ∼2.9 Å away from the CuI ion, its presence is required for copper binding. Genomic neighborhood analysis indicates that MbnP proteins, and by extension kynurenine-containing copper sites, are widespread and may play diverse roles in microbial copper homeostasis.

6.
Proc Natl Acad Sci U S A ; 118(25)2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34161271

RESUMO

Desert varnish is a dark rock coating that forms in arid environments worldwide. It is highly and selectively enriched in manganese, the mechanism for which has been a long-standing geological mystery. We collected varnish samples from diverse sites across the western United States, examined them in petrographic thin section using microscale chemical imaging techniques, and investigated the associated microbial communities using 16S amplicon and shotgun metagenomic DNA sequencing. Our analyses described a material governed by sunlight, water, and manganese redox cycling that hosts an unusually aerobic microbial ecosystem characterized by a remarkable abundance of photosynthetic Cyanobacteria in the genus Chroococcidiopsis as the major autotrophic constituent. We then showed that diverse Cyanobacteria, including the relevant Chroococcidiopsis taxon, accumulate extraordinary amounts of intracellular manganese-over two orders of magnitude higher manganese content than other cells. The speciation of this manganese determined by advanced paramagnetic resonance techniques suggested that the Cyanobacteria use it as a catalytic antioxidant-a valuable adaptation for coping with the substantial oxidative stress present in this environment. Taken together, these results indicated that the manganese enrichment in varnish is related to its specific uptake and use by likely founding members of varnish microbial communities.

7.
J Am Chem Soc ; 143(24): 9183-9190, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34110795

RESUMO

Mo-dependent nitrogenase is a major contributor to global biological N2 reduction, which sustains life on Earth. Its multi-metallic active-site FeMo-cofactor (Fe7MoS9C-homocitrate) contains a carbide (C4-) centered within a trigonal prismatic CFe6 core resembling the structural motif of the iron carbide, cementite. The role of the carbide in FeMo-cofactor binding and activation of substrates and inhibitors is unknown. To explore this role, the carbide has been in effect selectively enriched with 13C, which enables its detailed examination by ENDOR/ESEEM spectroscopies. 13C-carbide ENDOR of the S = 3/2 resting state (E0) is remarkable, with an extremely small isotropic hyperfine coupling constant, Ca = +0.86 MHz. Turnover under high CO partial pressure generates the S = 1/2 hi-CO state, with two CO molecules bound to FeMo-cofactor. This conversion surprisingly leaves the small magnitude of the 13C carbide isotropic hyperfine-coupling constant essentially unchanged, Ca = -1.30 MHz. This indicates that both the E0 and hi-CO states exhibit an exchange-coupling scheme with nearly cancelling contributions to Ca from three spin-up and three spin-down carbide-bound Fe ions. In contrast, the anisotropic hyperfine coupling constant undergoes a symmetry change upon conversion of E0 to hi-CO that may be associated with bonding and coordination changes at Fe ions. In combination with the negligible difference between CFe6 core structures of E0 and hi-CO, these results suggest that in CO-inhibited hi-CO the dominant role of the FeMo-cofactor carbide is to maintain the core structure, rather than to facilitate inhibitor binding through changes in Fe-carbide covalency or stretching/breaking of carbide-Fe bonds.

8.
Angew Chem Int Ed Engl ; 60(9): 4666-4672, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33935588

RESUMO

Radical S-adenosyl-l-methionine (SAM) enzymes initiate biological radical reactions with the 5'-deoxyadenosyl radical (5'-dAdo•). A [4Fe-4S]+ cluster reductively cleaves SAM to form the Ω organometallic intermediate in which the 5'-deoxyadenosyl moiety is directly bound to the unique iron of the [4Fe-4S] cluster, with subsequent liberation of 5'-dAdo•. Here we present synthesis of the SAM analog S-adenosyl-l-ethionine (SAE) and show SAE is a mechanistically-equivalent SAM-alternative for HydG, both supporting enzymatic turnover of substrate tyrosine and forming the organometallic intermediate Ω. Photolysis of SAE bound to HydG forms an ethyl radical trapped in the active site. The ethyl radical withstands prolonged storage at 77 K and its EPR signal is only partially lost upon annealing at 100 K, making it significantly less reactive than the methyl radical formed by SAM photolysis. Upon annealing above 77K, the ethyl radical adds to the [4Fe-4S]2+ cluster, generating an ethyl-[4Fe-4S]3+ organometallic species termed ΩE.


Assuntos
Proteínas de Escherichia coli/metabolismo , Etionina/metabolismo , Transativadores/metabolismo , Biocatálise , Espectroscopia de Ressonância de Spin Eletrônica , Proteínas de Escherichia coli/química , Etionina/análogos & derivados , Etionina/química , Radicais Livres/química , Radicais Livres/metabolismo , Modelos Moleculares , Estrutura Molecular , Transativadores/química
9.
Proc Natl Acad Sci U S A ; 118(14)2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33782122

RESUMO

Ultrafast structural dynamics with different spatial and temporal scales were investigated during photodissociation of carbon monoxide (CO) from iron(II)-heme in bovine myoglobin during the first 3 ps following laser excitation. We used simultaneous X-ray transient absorption (XTA) spectroscopy and X-ray transient solution scattering (XSS) at an X-ray free electron laser source with a time resolution of 80 fs. Kinetic traces at different characteristic X-ray energies were collected to give a global picture of the multistep pathway in the photodissociation of CO from heme. In order to extract the reaction coordinates along different directions of the CO departure, XTA data were collected with parallel and perpendicular relative polarizations of the laser pump and X-ray probe pulse to isolate the contributions of electronic spin state transition, bond breaking, and heme macrocycle nuclear relaxation. The time evolution of the iron K-edge X-ray absorption near edge structure (XANES) features along the two major photochemical reaction coordinates, i.e., the iron(II)-CO bond elongation and the heme macrocycle doming relaxation were modeled by time-dependent density functional theory calculations. Combined results from the experiments and computations reveal insight into interplays between the nuclear and electronic structural dynamics along the CO photodissociation trajectory. Time-resolved small-angle X-ray scattering data during the same process are also simultaneously collected, which show that the local CO dissociation causes a protein quake propagating on different spatial and temporal scales. These studies are important for understanding gas transport and protein deligation processes and shed light on the interplay of active site conformational changes and large-scale protein reorganization.


Assuntos
Monóxido de Carbono/química , Simulação de Dinâmica Molecular , Mioglobina/química , Animais , Bovinos , Heme/química , Heme/metabolismo , Ferro/química , Mioglobina/metabolismo , Ligação Proteica
10.
Science ; 371(6530)2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33574183

RESUMO

Kang et al (Reports, 19 June 2020, p. 1381) report a structure of the nitrogenase MoFe protein that is interpreted to indicate binding of N2 or an N2-derived species to the active-site FeMo cofactor. Independent refinement of the structure and consideration of biochemical evidence do not support this claim.


Assuntos
Azotobacter vinelandii , Molibdoferredoxina , Domínio Catalítico , Nitrogenase/metabolismo
11.
J Am Chem Soc ; 143(1): 335-348, 2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33372786

RESUMO

Catalysis by canonical radical S-adenosyl-l-methionine (SAM) enzymes involves electron transfer (ET) from [4Fe-4S]+ to SAM, generating an R3S0 radical that undergoes regioselective homolytic reductive cleavage of the S-C5' bond to generate the 5'-dAdo· radical. However, cryogenic photoinduced S-C bond cleavage has regioselectively yielded either 5'-dAdo· or ·CH3, and indeed, each of the three SAM S-C bonds can be regioselectively cleaved in an RS enzyme. This diversity highlights a longstanding central question: what controls regioselective homolytic S-C bond cleavage upon SAM reduction? We here provide an unexpected answer, founded on our observation that photoinduced S-C bond cleavage in multiple canonical RS enzymes reveals two enzyme classes: in one, photolysis forms 5'-dAdo·, and in another it forms ·CH3. The identity of the cleaved S-C bond correlates with SAM ribose conformation but not with positioning and orientation of the sulfonium center relative to the [4Fe-4S] cluster. We have recognized the reduced-SAM R3S0 radical is a (2E) state with its antibonding unpaired electron in an orbital doublet, which renders R3S0 Jahn-Teller (JT)-active and therefore subject to vibronically induced distortion. Active-site forces induce a JT distortion that localizes the odd electron in a single priority S-C antibond, which undergoes regioselective cleavage. In photolytic cleavage those forces act through control of the ribose conformation and are transmitted to the sulfur via the S-C5' bond, but during catalysis thermally induced conformational changes that enable ET from a cluster iron generate dominant additional forces that specifically select S-C5' for cleavage. This motion also can explain how 5'-dAdo· subsequently forms the organometallic intermediate Ω.


Assuntos
Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , S-Adenosilmetionina/química , Proteínas de Bactérias/química , Proteínas de Bactérias/efeitos da radiação , Biocatálise , Domínio Catalítico , Clostridium acetobutylicum/enzimologia , Teoria da Densidade Funcional , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/efeitos da radiação , Luz , Modelos Químicos , Estrutura Molecular , Oxirredução/efeitos da radiação , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/efeitos da radiação , Fotólise , S-Adenosilmetionina/efeitos da radiação , Thermotoga maritima/enzimologia
12.
J Am Chem Soc ; 142(52): 21679-21690, 2020 12 30.
Artigo em Inglês | MEDLINE | ID: mdl-33326225

RESUMO

Nitrogen fixation by nitrogenase begins with the accumulation of four reducing equivalents at the active-site FeMo-cofactor (FeMo-co), generating a state (denoted E4(4H)) with two [Fe-H-Fe] bridging hydrides. Recently, photolytic reductive elimination (re) of the E4(4H) hydrides showed that enzymatic re of E4(4H) hydride yields an H2-bound complex (E4(H2,2H)), in a process corresponding to a formal 2-electron reduction of the metal-ion core of FeMo-co. The resulting electron-density redistribution from Fe-H bonds to the metal ions themselves enables N2 to bind with concomitant H2 release, a process illuminated here by QM/MM molecular dynamics simulations. What is the nature of this redistribution? Although E4(H2,2H) has not been trapped, cryogenic photolysis of E4(4H) provides a means to address this question. Photolysis of E4(4H) causes hydride-re with release of H2, generating doubly reduced FeMo-co (denoted E4(2H)*), the extreme limit of the electron-density redistribution upon formation of E4(H2,2H). Here we examine the doubly reduced FeMo-co core of the E4(2H)* limiting-state by 1H, 57Fe, and 95Mo ENDOR to illuminate the partial electron-density redistribution upon E4(H2,2H) formation during catalysis, complementing these results with corresponding DFT computations. Inferences from the E4(2H)* ENDOR results as extended by DFT computations include (i) the Mo-site participates negligibly, and overall it is unlikely that Mo changes valency throughout the catalytic cycle; and (ii) two distinctive E4(4H) 57Fe signals are suggested as associated with structurally identified "anchors" of one bridging hydride, two others with identified anchors of the second, with NBO-analysis further identifying one anchor of each hydride as a major recipient of electrons released upon breaking Fe-H bonds.


Assuntos
Hidrogênio/química , Molibdoferredoxina/química , Nitrogenase/química , Animais , Azotobacter vinelandii/enzimologia , Domínio Catalítico , Transporte de Elétrons
13.
J Inorg Biochem ; 213: 111278, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33068967

RESUMO

Three known nitrogenase isozymes, Mo-, V-, and Fe-, catalyze biological reduction of dinitrogen (N2) to ammonia (NH3). All three utilize the same reductive elimination mechanism: an intermediate with two metal-bound hydrides reductively-eliminates hydrogen gas (H2) in a reaction coupled to binding and activation of N2. Nonetheless, the three isozymes show dramatically different relative rates of H2 formation and N2 reduction, revealing important differences in reactivity with substrates. Carbon monoxide (CO) has been characterized as both an inhibitor and substrate for Mo- and V­nitrogenases, but not for the Fe­nitrogenase. Here, we present a comparative study of the reactivity of the three isozymes with CO, examining CO both as a substrate and as an inhibitor of proton (H+) reduction under steady-state conditions. For Mo­nitrogenase, there is neither detectable reduction of CO nor inhibition of H+ reduction. Fe- and V­nitrogenase show CO reduction and inhibition of H+ reduction that depends on the CO partial pressure. For V­nitrogenase, ethylene (C2H4) is the major reduction product with a maximum specific activity of ~7.5 nmol C2H4/nmol VFe protein/min at 1 atm CO. The major product of CO reduction for Fe­nitrogenase is methane (CH4) with a maximum specific activity of ~4.8 nmol CH4/nmol FeFe protein/min at 0.05 atm CO. The rate of CH4 production by Fe­nitrogenase progressively increases to a maximum at 0.05 atm CO and then declines by ~90% with increasing CO partial pressure up to 1 atm. CO does not inhibit proton reduction in Mo­nitrogenase but shows 16% inhibition for V­nitrogenase and 35% for Fe­nitrogenase.


Assuntos
Monóxido de Carbono/química , Hidrogênio/química , Ferro/química , Molibdênio/química , Nitrogenase/química , Vanádio/química , Catálise , Oxirredução
14.
Science ; 370(6514): 356-359, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33060362

RESUMO

High-valent iron species are key intermediates in oxidative biological processes, but hexavalent complexes apart from the ferrate ion are exceedingly rare. Here, we report the synthesis and structural and spectroscopic characterization of a stable Fe(VI) complex (3) prepared by facile one-electron oxidation of an Fe(V) bis(imido) (2). Single-crystal x-ray diffraction of 2 and 3 revealed four-coordinate Fe centers with an unusual "seesaw" geometry. 57Fe Mössbauer, x-ray photoelectron, x-ray absorption, and electron-nuclear double resonance (ENDOR) spectroscopies, supported by electronic structure calculations, support a low-spin (S = 1/2) d3 Fe(V) configuration in 2 and a diamagnetic (S = 0) d2 Fe(VI) configuration in 3 Their shared seesaw geometry is electronically dictated by a balance of Fe-imido σ- and π-bonding interactions.

15.
J Am Chem Soc ; 142(43): 18652-18660, 2020 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-32966073

RESUMO

Spore photoproduct lyase is a radical S-adenosyl-l-methionine (SAM) enzyme with the unusual property that addition of SAM to the [4Fe-4S]1+ enzyme absent substrate results in rapid electron transfer to SAM with accompanying homolytic S-C5' bond cleavage. Herein, we demonstrate that this unusual reaction forms the organometallic intermediate Ω in which the unique Fe atom of the [4Fe-4S] cluster is bound to C5' of the 5'-deoxyadenosyl radical (5'-dAdo•). During catalysis, homolytic cleavage of the Fe-C5' bond liberates 5'-dAdo• for reaction with substrate, but here, we use Ω formation without substrate to determine the thermal stability of Ω. The reaction of Geobacillus thermodenitrificans SPL (GtSPL) with SAM forms Ω within ∼15 ms after mixing. By monitoring the decay of Ω through rapid freeze-quench trapping at progressively longer times we find an ambient temperature decay time of the Ω Fe-C5' bond of τ ≈ 5-6 s, likely shortened by enzymatic activation as is the case with the Co-C5' bond of B12. We have further used hand quenching at times up to 10 min, and thus with multiple SAM turnovers, to probe the fate of the 5'-dAdo• radical liberated by Ω. In the absence of substrate, Ω undergoes low-probability conversion to a stable protein radical. The WT enzyme with valine at residue 172 accumulates a Val•; mutation of Val172 to isoleucine or cysteine results in accumulation of an Ile• or Cys• radical, respectively. The structures of the radical in WT, V172I, and V172C variants have been established by detailed EPR/DFT analyses.


Assuntos
Radicais Livres/química , Proteínas/química , S-Adenosilmetionina/química , Domínio Catalítico , Teoria da Densidade Funcional , Desoxiadenosinas/química , Espectroscopia de Ressonância de Spin Eletrônica , Geobacillus/enzimologia , Proteínas Ferro-Enxofre/química , Modelos Moleculares , Proteínas/genética , Proteínas/metabolismo , S-Adenosilmetionina/metabolismo
16.
J Am Chem Soc ; 142(36): 15362-15370, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32786751

RESUMO

EPR and Electron Nuclear Double Resonance spectroscopies here characterize CO binding to the active-site A cluster of wild-type (WT) Acetyl-CoA Synthase (ACS) and two variants, F229W and F229A. The A-cluster binds CO to a proximal Ni (Nip) that bridges a [4Fe-4S] cluster and a distal Nid. An alcove seen in the ACS crystal structure near the A-cluster, defined by hydrophobic residues including F229, forms a cage surrounding a Xe mimic of CO. Previously, we only knew WT ACS bound a single CO to form the Ared-CO intermediate, containing Nip(I)-CO with CO located on the axis of the dz2 odd-electron orbital (g⊥ > g|| ∼ 2). Here, the two-dimensional field-frequency pattern of 2K-35 GHz 13C-ENDOR spectra collected across the Ared-CO EPR envelope reveals a second CO bound in the dz2 orbital's equatorial plane. This WT A-cluster conformer dominates the nearly conservative F229W variant, but 13C-ENDOR reveals a minority "A" conformation with (g|| > g⊥ ∼ 2) characteristic of a "cloverleaf" (e.g., dx2-y2) odd-electron orbital, with Nip binding two, apparently "in-plane" CO. Disruption of the alcove through introduction of the smaller alanine residue in the F229A variant diminishes conversion to Ni(I) ∼ 10-fold and introduces extensive cluster flexibility. 13C-ENDOR shows the F229A cluster is mostly (60%) in the "A" conformation but with ∼20% each of the WT conformer and an "O" state in which dz2 Nip(I) (g⊥ > g|| ∼ 2) surprisingly lacks CO. This paper thus demonstrates the importance of an intact alcove in forming and stabilizing the Ni(I)-CO intermediate in the Wood-Ljungdahl pathway of anaerobic CO and CO2 fixation.


Assuntos
Acetilcoenzima A/química , Monóxido de Carbono/química , Ressonância Magnética Nuclear Biomolecular , Acetilcoenzima A/metabolismo , Sítios de Ligação , Isótopos de Carbono , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Modelos Moleculares , Conformação Molecular
17.
Chem Rev ; 120(12): 5082-5106, 2020 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-32176472

RESUMO

Nitrogenase is the enzyme that catalyzes biological N2 reduction to NH3. This enzyme achieves an impressive rate enhancement over the uncatalyzed reaction. Given the high demand for N2 fixation to support food and chemical production and the heavy reliance of the industrial Haber-Bosch nitrogen fixation reaction on fossil fuels, there is a strong need to elucidate how nitrogenase achieves this difficult reaction under benign conditions as a means of informing the design of next generation synthetic catalysts. This Review summarizes recent progress in addressing how nitrogenase catalyzes the reduction of an array of substrates. New insights into the mechanism of N2 and proton reduction are first considered. This is followed by a summary of recent gains in understanding the reduction of a number of other nitrogenous compounds not considered to be physiological substrates. Progress in understanding the reduction of a wide range of C-based substrates, including CO and CO2, is also discussed, and remaining challenges in understanding nitrogenase substrate reduction are considered.


Assuntos
Nitrogenase/metabolismo , Biocatálise , Dióxido de Carbono/química , Dióxido de Carbono/metabolismo , Monóxido de Carbono/química , Monóxido de Carbono/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Modelos Moleculares , Nitrogênio/química , Nitrogênio/metabolismo , Nitrogenase/química , Oxirredução , Especificidade por Substrato
18.
Biochemistry ; 59(7): 901-910, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32022556

RESUMO

Hydrogen tunneling in enzymatic C-H activation requires a dynamical sampling among ground-state enzyme-substrate (E-S) conformations, which transiently generates a tunneling-ready state (TRS). The TRS is characterized by a hydrogen donor-acceptor distance (DAD) of 2.7 Å, ∼0.5 Å shorter than the dominant DAD of optimized ground states. Recently, a high-resolution, 13C electron-nuclear double-resonance (ENDOR) approach was developed to characterize the ground-state structure of the complex of the linoleic acid (LA) substrate with soybean lipoxygenase (SLO). The resulting enzyme-substrate model revealed two ground-state conformers with different distances between the target C11 of LA and the catalytically active cofactor [Fe(III)-OH]: the active conformer "a", with a van der Waals DAD of 3.1 Å between C11 and metal-bound hydroxide, and an inactive conformer "b", with a distance that is almost 1 Å longer. Herein, the structure of the E-S complex is examined for a series of six variants in which subtle structural modifications of SLO have been introduced either at a hydrophobic side chain near the bound substrate or at a remote residue within a protein network whose flexibility influences hydrogen transfer. A remarkable correlation is found between the ENDOR-derived population of the active ground-state conformer a and the kinetically derived differential enthalpic barrier for D versus H transfer, ΔEa, with the latter increasing as the fraction of conformer a decreases. As proposed, ΔEa provides a "ruler" for the DAD within the TRS. ENDOR measurements further corroborate the previous identification of a dynamical network coupling the buried active site of SLO to the surface. This study shows that subtle imperfections within the initial ground-state structures of E-S complexes are accompanied by compromised geometries at the TRS.


Assuntos
Ácido Linoleico/química , Lipoxigenase/química , Soja/enzimologia , Isótopos de Carbono/química , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Hidrogênio/química , Lipoxigenase/genética , Mutação , Conformação Proteica
20.
J Am Chem Soc ; 141(40): 16117-16124, 2019 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-31509404

RESUMO

Radical SAM (RS) enzymes use S-adenosyl-l-methionine (SAM) and a [4Fe-4S] cluster to initiate a broad spectrum of radical transformations throughout all kingdoms of life. We report here that low-temperature photoinduced electron transfer from the [4Fe-4S]1+ cluster to bound SAM in the active site of the hydrogenase maturase RS enzyme, HydG, results in specific homolytic cleavage of the S-CH3 bond of SAM, rather than the S-C5' bond as in the enzyme-catalyzed (thermal) HydG reaction. This result is in stark contrast to a recent report in which photoinduced ET in the RS enzyme pyruvate formate-lyase activating enzyme cleaved the S-C5' bond to generate a 5'-deoxyadenosyl radical, and provides the first direct evidence for homolytic S-CH3 bond cleavage in a RS enzyme. Photoinduced ET in HydG generates a trapped •CH3 radical, as well as a small population of an organometallic species with an Fe-CH3 bond, denoted ΩM. The •CH3 radical is surprisingly found to exhibit rotational diffusion in the HydG active site at temperatures as low as 40 K, and is rapidly quenched: whereas 5'-dAdo• is stable indefinitely at 77 K, •CH3 quenches with a half-time of ∼2 min at this temperature. The rapid quenching and rotational/translational freedom of •CH3 shows that enzymes would be unable to harness this radical as a regio- and stereospecific H atom abstractor during catalysis, in contrast to the exquisite control achieved with the enzymatically generated 5'-dAdo•.


Assuntos
Hidrolases/química , Proteínas Ferro-Enxofre/química , Metano/análogos & derivados , S-Adenosilmetionina/química , Acetiltransferases/química , Acetiltransferases/metabolismo , Domínio Catalítico , Transporte de Elétrons , Ativação Enzimática , Hidrolases/metabolismo , Metano/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fotólise
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