26 research outputs found
Advanced paramagnetic resonance spectroscopies of ironāsulfur proteins: Electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM)
AbstractThe advanced electron paramagnetic resonance (EPR) techniques, electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) spectroscopies, provide unique insights into the structure, coordination chemistry, and biochemical mechanism of nature's widely distributed ironāsulfur cluster (FeS) proteins. This review describes the ENDOR and ESEEM techniques and then provides a series of case studies on their application to a wide variety of FeS proteins including ferredoxins, nitrogenase, and radical SAM enzymes. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases
Characterization of an Feā”NāNH_2 Intermediate Relevant to Catalytic N_2 Reduction to NH_3
The ability of certain transition metals to mediate the reduction of N_2 to NH_3 has attracted broad interest in the biological and inorganic chemistry communities. Early transition metals such as Mo and W readily bind N_2 and mediate its protonation at one or more N atoms to furnish M(N_xH_y) species that can be characterized and, in turn, extrude NH_3. By contrast, the direct protonation of FeāN_2 species to Fe(N_xH_y) products that can be characterized has been elusive. Herein, we show that addition of acid at low temperature to [(TPB)Fe(N_2)][Na(12-crown-4)] results in a new S = 1/2 Fe species. EPR, ENDOR, Mƶssbauer, and EXAFS analysis, coupled with a DFT study, unequivocally assign this new species as [(TPB)Feā”NāNH_2]^+, a doubly protonated hydrazido(2ā) complex featuring an Fe-to-N triple bond. This unstable species offers strong evidence that the first steps in Fe-mediated nitrogen reduction by [(TPB)Fe(N_2)][Na(12-crown-4)] can proceed along a distal or āChatt-typeā pathway. A brief discussion of whether subsequent catalytic steps may involve early or late stage cleavage of the NāN bond, as would be found in limiting distal or alternating mechanisms, respectively, is also provided
The Asp1 pyrophosphatase from S. pombe hosts a [2Fe-2S]2+ cluster in vivo
AbstractThe Schizosaccharomyces pombe Asp1 protein is a bifunctional kinase/pyrophosphatase that belongs to the highly conserved eukaryotic diphosphoinositol pentakisphosphate kinase PPIP5K/Vip1 family. The N-terminal Asp1 kinase domain generates specific high-energy inositol pyrophosphate (IPP) molecules, which are hydrolyzed by the C-terminal Asp1 pyrophosphatase domain (Asp1365ā920). Thus, Asp1 activities regulate the intracellular level of a specific class of IPP molecules, which control a wide number of biological processes ranging from cell morphogenesis to chromosome transmission. Recently, it was shown that chemical reconstitution of Asp1371ā920 leads to the formation of a [2Fe-2S] cluster; however, the biological relevance of the cofactor remained under debate. In this study, we provide evidence for the presence of the FeāS cluster in Asp1365ā920 inside the cell. However, we show that the FeāS cluster does not influence Asp1 pyrophosphatase activity in vitro or in vivo. Characterization of the as-isolated protein by electronic absorption spectroscopy, mass spectrometry, and X-ray absorption spectroscopy is consistent with the presence of a [2Fe-2S]2+ cluster in the enzyme. Furthermore, we have identified the cysteine ligands of the cluster. Overall, our work reveals that Asp1 contains an FeāS cluster in vivo that is not involved in its pyrophosphatase activity.</jats:p
A low spin manganese(IV) nitride single molecule magnet
Structural, spectroscopic and magnetic methods have been used to characterize the tris(carbene) borate compound PhB(MesIm)(3)Mn equivalent to N as a four-coordinate manganese(IV) complex with a low spin (S = 1/2) configuration. The slow relaxation of the magnetization in this complex, i.e. its single-molecule magnet (SMM) properties, is revealed under an applied dc field. Multireference quantum mechanical calculations indicate that this SMM behavior originates from an anisotropic ground doublet stabilized by spin-orbit coupling. Consistent theoretical and experiment data show that the resulting magnetization dynamics in this system is dominated by ground state quantum tunneling, while its temperature dependence is influenced by Raman relaxation
Free H_2 Rotation vs JahnāTeller Constraints in the Nonclassical Trigonal (TPB)CoāH_2 Complex
Proton exchange within the MāH_2 moiety of (TPB)Co(H_2) (CoāH_2; TPB = B(o-C_6H_4PiPr_2)_3) by 2-fold rotation about the MāH_2 axis is probed through EPR/ENDOR studies and a neutron diffraction crystal structure. This complex is compared with previously studied (SiP^(iPr)_3)Fe(H_2) (FeāH_2) (SiP^(iPr)_3 = [Si(o-C_6H_4PiPr_2)_3]). The g-values for CoāH_2 and FeāH_2 show that both have the JahnāTeller (JT)-active ^2E ground state (idealized C_3 symmetry) with doubly degenerate frontier orbitals, (e)^3 = [|m_L Ā± 2>]^3 = [x^2 ā y^2, xy]^3, but with stronger linear vibronic coupling for CoāH_2. The observation of ^1H ENDOR signals from the CoāHD complex, ^2H signals from the CoāD_2/HD complexes, but no ^1H signals from the CoāH_2 complex establishes that H_2 undergoes proton exchange at 2 K through rotation around the CoāH_2 axis, which introduces a quantum-statistical (Pauli-principle) requirement that the overall nuclear wave function be antisymmetric to exchange of identical protons (I = 1/2; Fermions), symmetric for identical deuterons (I = 1; Bosons). Analysis of the 1-D rotor problem indicates that CoāH_2 exhibits rotor-like behavior in solution because the underlying C_3 molecular symmetry combined with H_2 exchange creates a dominant 6-fold barrier to H_2 rotation. FeāH_2 instead shows H_2 localization at 2 K because a dominant 2-fold barrier is introduced by strong Fe(3d)ā H_2(Ļ^*) Ļ-backbonding that becomes dependent on the H_2 orientation through quadratic JT distortion. ENDOR sensitively probes bonding along the L_2āMāE axis (E = Si for FeāH_2; E = B for CoāH_2). Notably, the isotropic ^1H/^2H hyperfine coupling to the diatomic of CoāH_2 is nearly 4-fold smaller than for FeāH_2
Spectroscopic characterization of the Co-substituted C-terminal domain of rubredoxin-2
Pseudomonas putida rubredoxin-2 (Rxn2) is an essential member of the alkane hydroxylation pathway and transfers electrons from a reductase to the membrane-bound hydroxylase. The regioselective hydroxylation of linear alkanes is a challenging chemical transformation of great interest for the chemical industry. Herein, we report the preparation and spectroscopic characterization of cobalt-substituted P. putida Rxn2 and a truncated version of the protein consisting of the C-terminal domain of the protein. Our spectroscopic data on the Co-substituted C-terminal domain supports a high-spin Co(II) with a distorted tetrahedral coordination environment. Investigation of the two-domain protein Rxn2 provides insights into the metal-binding properties of the N-terminal domain, the role of which is not well understood so far. Circular dichroism, electron paramagnetic resonance and X-ray absorption spectroscopies support an alternative Co-binding site within the N-terminal domain, which appears to not be relevant in nature. We have shown that chemical reconstitution in the presence of Co leads to incorporation of Co(II) into the active site of the C-terminal domain, but not the N-terminal domain of Rxn2 indicating distinct roles for the two rubredoxin domain
XAS and EPR in Situ Observation of Ru(V) Oxo Intermediate in a Ru Water Oxidation Complex.
In this study, we combine inā
situ spectroelectrochemistry coupled with electron paramagnetic resonance (EPR) and X-ray absorption spectroscopies (XAS) to investigate a molecular Ru-based water oxidation catalyst bearing a polypyridinic backbone [RuII(OH2)(Py2Metacn)]2+ . Although high valent key intermediate species arising in catalytic cycles of this family of compounds have remain elusive due to the lack of additional anionic ligands that could potentially stabilize them, mechanistic studies performed on this system proposed a water nucleophilic attack (WNA) mechanism for the O-O bond formation. Employing inā
situ experimental conditions and complementary spectroscopic techniques allowed to observe intermediates that provide support for a WNA mechanism, including for the first time a Ru(V) oxo intermediate based on the Py2Metacn ligand, in agreement with the previously proposed mechanism
Evidence for Oxygen Binding at the Active Site of Particulate Methane Monooxygenase
Particulate methane monooxygenase (pMMO) is an integral
membrane
metalloenzyme that converts methane to methanol in methanotrophic
bacteria. The enzyme consists of three subunits, pmoB, pmoA, and pmoC,
organized in an Ī±<sub>3</sub>Ī²<sub>3</sub>Ī³<sub>3</sub> trimer. Studies of intact pMMO and a recombinant soluble
fragment of the pmoB subunit (denoted as spmoB) indicate that the
active site is located within the soluble region of pmoB at the site
of a crystallographically modeled dicopper center. In this work, we
have investigated the reactivity of pMMO and spmoB with oxidants.
Upon reduction and treatment of spmoB with O<sub>2</sub> or H<sub>2</sub>O<sub>2</sub> or pMMO with H<sub>2</sub>O<sub>2</sub>, an
absorbance feature at 345 nm is generated. The energy and intensity
of this band are similar to those of the Ī¼-Ī·<sup>2</sup>:Ī·<sup>2</sup>-peroxo-Cu<sup>II</sup><sub>2</sub> species formed
in several dicopper enzymes and model compounds. The feature is not
observed in inactive spmoB variants in which the dicopper center is
disrupted, consistent with O<sub>2</sub> binding to the proposed active
site. Reaction of the 345 nm species with CH<sub>4</sub> results in
the disappearance of the spectroscopic feature, suggesting that this
O<sub>2</sub> intermediate is mechanistically relevant. Taken together,
these observations provide strong new support for the identity and
location of the pMMO active site
13C and 63,65Cu ENDOR studies of CO Dehydrogenase from Oligotropha carboxidovorans. Experimental Evidence in Support of a CopperāCarbonyl Intermediate
We report here an ENDOR study of an S = 1/2 intermediate state trapped during reduction of the binuclear Mo/Cu enzyme CO dehydrogenase by CO. ENDOR spectra of this state confirm that the (63,65)Cu nuclei exhibits strong and almost entirely isotropic coupling to the unpaired electron, show that this coupling atypically has a positive sign, aiso = +148 MHz, and indicate an apparently undetectably small quadrupolar coupling. When the intermediate is generated using (13)CO, coupling to the (13)C is observed, with aiso = +17.3 MHz. A comparison with the couplings seen in related, structurally assigned Mo(V) species from xanthine oxidase, in conjunction with complementary computational studies, leads us to conclude that the intermediate contains a partially reduced Mo(V)/Cu(I) center with CO bound at the copper. Our results provide strong experimental support for a reaction mechanism that proceeds from a comparable complex of CO with fully oxidized Mo(VI)/Cu(I) enzyme