Geometric and Electronic Structures of the Ni^I and Methyl−Ni^III Intermediates of Methyl-Coenzyme M Reductase

Methyl-coenzyme M reductase (MCR) catalyzes the terminal step in the formation of biological methane from methyl-coenzyme M (Me-SCoM) and coenzyme B (CoBSH). The active site in MCR contains a Ni−F₄₃₀ cofactor, which can exist in different oxidation states. The catalytic mechanism of methane formation has remained elusive despite intense spectroscopic and theoretical investigations. On the basis of spectroscopic and crystallographic data, the first step of the mechanism is proposed to involve a nucleophilic attack of the Ni^I active state (MCR_red1) on Me-SCoM to form a Ni^III−methyl intermediate, while computational studies indicate that the first step involves the attack of Ni^I on the sulfur of Me-SCoM, forming a CH₃^• radical and a Ni^II−thiolate species. In this study, a combination of Ni K-edge X-ray absorption spectroscopic (XAS) studies and density functional theory (DFT) calculations have been performed on the Ni^I (MCR_red1), Ni^II (MCR_{red1−silent}), and Ni^III−methyl (MCR_Me) states of MCR to elucidate the geometric and electronic structures of the different redox states. Ni K-edge EXAFS data are used to reveal a five-coordinate active site with an open upper axial coordination site in MCR_red1. Ni K-pre-edge and EXAFS data and time-dependent DFT calculations unambiguously demonstrate the presence of a long Ni−C bond (∼2.04 Å) in the Ni^III−methyl state of MCR. The formation and stability of this species support mechanism I, and the Ni−C bond length suggests a homolytic cleavage of the Ni^III−methyl bond in the subsequent catalytic step. The XAS data provide insight into the role of the unique F₄₃₀ cofactor in tuning the stability of the different redox states of MCR

Structural Investigation of a Dimeric Variant of Pyruvate Kinase Muscle Isoform 2

Pyruvate kinase muscle isoform 2 (PKM2) catalyzes the terminal step in glycolysis, transferring a phosphoryl group from phosphoenolpyruvate to ADP, to produce pyruvate and ATP. PKM2 activity is allosterically regulated by fructose 1,6-bisphosphate (FBP), an upstream glycolytic intermediate. FBP stabilizes the tetrameric form of the enzyme. In its absence, the PKM2 tetramers dissociate, yielding a dimer–monomer mixture having lower enzymatic activity. The S437Y variant of PKM2 is incapable of binding FBP. Consistent with that defect, we find that S437Y exists in a monomer–dimer equilibrium in solution, with a <i>K</i>_d of ∼20 μM. Interestingly, however, the protein crystallizes as a tetramer, providing insight into the structural basis for impaired FBP binding of S437Y

Stoichiometry of Fe(II) binding to DddW.

<p>2 μM apo-DddW (under tight-binding conditions) was titrated with increasing concentrations of Fe(NH₄)₂(SO₄)₂ and the fluorescence intensity was monitored. The titration data were analyzed by nonlinear curve fitting using Eq (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127288#pone.0127288.e003" target="_blank">3</a>) to produce the solid line. Upon data fitting, the stoichiometric ratio of Fe(II) to DddW monomer was determined to be 1:1.</p

Spectral properties of Fe(II)-bound DddW.

<p>(A) UV-visible spectra of the reaction of as-isolated DddW in the presence of Fe(II) and Cu(II). All spectra with Fe(II) had an enzyme concentration of 370 μM. Trace in black, apo-DddW, green, apo-DddW in presence of 370 μM Fe(II) red, apo-DddW+Fe(II) after bubbling with NO gas. The absorption maximum is at 340 nm with a shoulder at 430 nm. Inset: Spectrum of 1 mM apo-DddW in the presence of Cu(II). The spectral feature at 550 nm is due to a charge transfer transition of DddW with Cu(II). (B) EPR spectra of: (top) 18 μM apo-DddW with Fe(II); (bottom) Fe(II)-DddW in the presence of 25 mM DMSP. The spectra were collected at microwave frequency, 9.43 GHz; receiver gain, 2 x 104; modulation frequency, 100 kHz; temperature, 4 K; microwave power, 200 microwatts; 83.89 s sweep time, and 16 scans.</p

<i>Bacillus anthracis</i> Prolyl 4‑Hydroxylase Interacts with and Modifies Elongation Factor Tu

Prolyl hydroxylation is a very common post-translational modification and plays many roles in eukaryotes such as collagen stabilization, hypoxia sensing, and controlling protein transcription and translation. There is a growing body of evidence that suggests that prokaryotes contain prolyl 4-hydroxylases (P4Hs) homologous to the hypoxia-inducible factor (HIF) prolyl hydroxylase domain (PHD) enzymes that act on elongation factor Tu (EFTu) and are likely involved in the regulation of bacterial translation. Recent biochemical and structural studies with a PHD from <i>Pseudomonas putida</i> (PPHD) determined that it forms a complex with EFTu and hydroxylates a prolyl residue of EFTu. Moreover, while animal, plant, and viral P4Hs act on peptidyl proline, most prokaryotic P4Hs have been known to target free l-proline; the exceptions include PPHD and a P4H from <i>Bacillus anthracis</i> (BaP4H) that modifies collagen-like proline-rich peptides. Here we use biophysical and mass spectrometric methods to demonstrate that BaP4H recognizes full-length BaEFTu and a BaEFTu 9-mer peptide for site-specific proline hydroxylation. Using size-exclusion chromatography coupled small-angle X-ray scattering (SEC–SAXS) and binding studies, we determined that BaP4H forms a 1:1 heterodimeric complex with BaEFTu. The SEC–SAXS studies reveal dissociation of BaP4H dimeric subunits upon interaction with BaEFTu. While BaP4H is unusual within bacteria in that it is structurally and functionally similar to the animal PHDs and collagen P4Hs, respectively, this work provides further evidence of its promiscuous substrate recognition. It is possible that the enzyme might have evolved to hydroxylate a universally conserved protein in prokaryotes, similar to the PHDs, and implies a functional role in <i>B. anthracis</i>

Proposed mechanisms for the mononuclear iron dependent DMSP lyase, DddW.

<p>DddW binds to Fe(II) cofactor to which the substrate can coordinate in either monodentate or bidentate modes. (A) His81 can act as a nucleophile to remove a hydrogen atom from the α-carbon of DMSP to form acrylate. (B) A hypothetical water molecule can be activated by His81, which then acts as a nucleophile in initiating catalysis. (C) Tyr89 located near the active site can initiate the elimination reaction cleaving DMSP.</p

The pH dependence of DddW lyase activity.

<p>The optimal pH was determined by comparing the initial velocities (V_i) of reactions containing 2 μM apo-DddW, 2 μM Fe(II), and 10 mM DMSP in varying buffer solutions. The buffers used are as follows: 50 mM MES 20 mM NaCl (pH 5.5, 6.0, 6.5), 50 mM HEPES 20 mM NaCl (pH 7.0, 7.5, 8.0), 50 mM Tris-HCl 20 mM NaCl (pH 8.5, 9.0).</p

Cupin motifs and metal binding residues of DddW.

<p>(A) Sequence alignment of cupin regions of selected DddW, DddQ and DddL proteins using sequences deposited at NCBI and CLUSTAL 2.1 for the alignment. The two conserved cupin motifs 1 (GX₅HXHX_3,4EX₆G) and 2 (GX₅PXGX₂HX₃N), containing residues that bind metal ions and are catalytically important are highlighted in green. Tyr residues playing catalytic role in <i>Ruegeria lacuscaerulensis</i> DddQ are marked cyan and other conserved residues in the cupin motifs are colored yellow. The sequences are from: W1 = DddW, <i>Ruegeria pomeroyi</i> DSS-3 (SPO0453); W2 = DddW, <i>Roseobacter sp</i>. MED193, (MED193_09710); Q1 = DddQ, <i>Ruegeria pomeroyi</i> DSS-3 (SPO1596); Q2 = DddQ, <i>Ruegeria lacuscaerulensis</i> (ITI-1157); L1 = DddL, <i>Sulfitobacter sp</i>. EE-36 (EE36_11918); L2 = DddL, <i>Rhodobacter sphaeroides</i> 2.4.1 (RSP_1433); L3 = DddL, <i>Roseibacterium elongatum</i> DSM 19469 (roselon_02436); L4 = DddL, <i>Caenispirillum salinarum</i> (C882_2645). (B) Homology model of <i>Ruegeria pomeroyi</i> DddW (grey) (generated using Phyre 2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127288#pone.0127288.ref052" target="_blank">52</a>]) superimposed on the Zn(II)-bound structure of <i>Ruegeria lacuscaerulensis</i> DddQ (cyan) (PDB 4LA2). The homology model of DddW shows the catalytic residues H81, H83, E87, and H121. Most of these residues of DddW (H83, E87, and H121) superimpose well on the zinc-coordinating DddQ residues (H125, E129, and H163). While Tyr usually is not involved in metal ion binding in cupin proteins, the DddQ structure shows a Zn-coordinated Tyr residue (Tyr131) and this Tyr superimposes on Tyr89 of DddW. The side chain residues are shown in ball and stick with oxygens in red, nitrogens in blue, zinc in slate, and carbons are similar to protein backbone.</p

Metal binding affinities (K_d) of DddW.

<p>Titration of apo-DddW with increasing concentrations of the metal ions Fe(NH₄)₂(SO₄)₂ (added anaerobically), FeCl₃, CoCl₂, MnCl₂, CuCl₂ or NiCl₂ was done. The binding was monitored by saturation of the fluorescence intensities. The concentration of enzyme used is as follows: (A) 0.5 μM apo-DddW, (B)-(F) 2 μM apo-DddW. The K_d values were: (A) Fe(II), 4.7 ± 0.0 nM; (B) Fe(III), 89.3 ± 4.3 μM; (C) Mn(II), 32.7 ± 5.0 μM; (D) Co(II), 2.5 ± 0.4 μM; (E) Ni(II), 1.0 ± 0.1 μM; (F) Cu(II), 1.9 ± 0.2 μM.</p

DMSP lyase activity of DddW variants compared to wild-type enzyme.

<p>Activity assays were performed anaerobically using 2 μM DddW, 2 μM Fe(NH₄)₂(SO₄)₂ and 10 mM DMSP. The reactions were incubated for 15 mins before quenching.</p