The structural stabilization of the κ three-way junction by Mg(II) represents the first step in the folding of a group II intron.

Folding of group II introns is characterized by a first slow compaction of domain 1 (D1) followed by the rapid docking of other domains to this scaffold. D1 compaction initiates in a small subregion encompassing the κ and ζ elements. These two tertiary elements are also the major interaction sites with domain 5 to form the catalytic core. Here, we provide the first characterization of the structure adopted at an early folding step and show that the folding control element can be narrowed down to the three-way junction with the κ motif. In our nuclear magnetic resonance studies of this substructure derived from the yeast mitochondrial group II intron Sc.ai5γ, we show that a high affinity Mg(II) ion stabilizes the κ element and enables coaxial stacking between helices d' and d'', favoring a rigid duplex across the three-way junction. The κ-element folds into a stable GAAA-tetraloop motif and engages in A-minor interactions with helix d'. The addition of cobalt(III)hexammine reveals three distinct binding sites. The Mg(II)-promoted structural rearrangement and rigidification of the D1 core can be identified as the first micro-step of D1 folding.

A nickel hydride complex in the active site of methyl-coenzyme m reductase: implications for the catalytic cycle.

Methanogenic archaea utilize a specific pathway in their metabolism, converting C1 substrates (i.e., CO2) or acetate to methane and thereby providing energy for the cell. Methyl-coenzyme M reductase (MCR) catalyzes the key step in the process, namely methyl-coenzyme M (CH3-S-CoM) plus coenzyme B (HS-CoB) to methane and CoM-S-S-CoB. The active site of MCR contains the nickel porphinoid F430. We report here on the coordinated ligands of the two paramagnetic MCR red2 states, induced when HS-CoM (a reversible competitive inhibitor) and the second substrate HS-CoB or its analogue CH3-S-CoB are added to the enzyme in the active MCR red1 state (Ni(I)F430). Continuous wave and pulse EPR spectroscopy are used to show that the MCR red2a state exhibits a very large proton hyperfine interaction with principal values A((1)H) = [-43,-42,-5] MHz and thus represents formally a Ni(III)F430 hydride complex formed by oxidative addition to Ni(I). In view of the known ability of nickel hydrides to activate methane, and the growing body of evidence for the involvement of MCR in "reverse" methanogenesis (anaerobic oxidation of methane), we believe that the nickel hydride complex reported here could play a key role in helping to understand both the mechanism of "reverse" and "forward" methanogenesis.

Matrix effects on copper(II)phthalocyanine complexes. A combined continuous wave and pulse EPR and DFT study.

The effect of the electron withdrawing or donating character of groups located at the periphery of the phthalocyanine ligand, as well as the influence of polar and nonpolar solvents are of importance for the redox chemistry of metal phthalocyanines. Continuous wave and pulse electron paramagnetic resonance and pulse electron nuclear double resonance spectroscopy at X- and Q-band are applied to investigate the electronic structure of the complexes Cu(II)phthalocyanine (CuPc), copper(II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc(t)), and copper(II) 1,2,3,4,8,9,10,11,15,16,17,18,22,23,24,25-hexadecafluoro-29H,31H-phthalocyanine (CuPc(F)) in various matrices. Isotope substitutions are used to determine the g values, the copper hyperfine couplings and the hyperfine interactions with the 14N, 1H and 19F nuclei of the macrocycle and the surrounding matrix molecules. Simulations and interpretations of the spectra are shown and discussed, and a qualitative analysis of the data using previous theoretical models is given. Density functional computations facilitate the interpretation of the EPR parameters. The experimental g, copper and nitrogen hyperfine and nuclear quadrupole values are found to be sensitive to changes of the solvent and the structure of the macrocycle. To elucidate the electronic, structural and bonding properties the changes in the g principal values are related to data from UV/Vis spectroscopy and to density functional theory (DFT) computations. The analysis of the EPR data indicates that the in-plane metal-ligand sigma bonding is more covalent for CuPc(t) in toluene than in sulfuric acid. Furthermore, the out-of-plane pi bonding is found to be less covalent in the case of a polar sulfuric acid environment than with nonpolar toluene or H2Pc environment, whereby the covalency of this bonding is increased upon addition of tert-butyl groups. No contribution from in-plane pi bonding is found.

Spin density and coenzyme M coordination geometry of the ox1 form of methyl-coenzyme M reductase: a pulse EPR study.

Methyl-coenzyme M reductase (MCR) catalyses the reduction of methyl-coenzyme M (CH3-S-CoM) with coenzyme B (H-S-CoB) to CH4 and CoM-S-S-CoB in methanogenic archaea. Here we present a pulse EPR study of the "ready" form MCR(ox1), providing a detailed description of the spin density and the coordination of coenzyme M (CoM) to the Ni cofactor F430. To achieve this, MCR was purified from cells grown in a 61Ni enriched medium and samples were prepared in D2O with the substrate analogue CoM either deuterated in the beta-position or with 33S in the thiol group. To obtain the magnetic parameters ENDOR and HYSCORE measurements were done at X- and Q-band, and CW EPR, at X- and W-band. The hyperfine couplings of the beta-protons of CoM indicate that the nickel to beta-proton distances in MCR(ox1) are very similar to those in Ni(II)-MCR(ox1-silent), and thus the position of CoM relative to F430 is very similar in both species. Our thiolate sulfur and nickel EPR data prove a Ni-S coordination, with an unpaired spin density on the sulfur of 7 +/- 3%. These results highlight the redox-active or noninnocent nature of the sulfur ligand on the oxidation state. Assuming that MCR(ox1) is oxidized relative to the Ni(II) species, the complex is formally best described as a Ni(III) (d7) thiolate in resonance with a thiyl radical/high-spin Ni(II) complex, Ni(III)-(-)SR <--> Ni(II)-*SR.

Characterization of the MCRred2 form of methyl-coenzyme M reductase: a pulse EPR and ENDOR study.

Methyl-coenzyme M reductase (MCR), which catalyses the reduction of methyl-coenzyme M (CH(3)-S-CoM) with coenzyme B (H-S-CoB) to CH(4) and CoM-S-S-CoB, contains the nickel porphinoid F430 as prosthetic group. The active enzyme exhibits the Ni(I)-derived axial EPR signal MCR(red1) both in the absence and presence of the substrates. When the enzyme is competitively inhibited by coenzyme M (HS-CoM) the MCR(red1) signal is partially converted into the rhombic EPR signal MCR(red2). To obtain deeper insight into the geometric and electronic structure of the red2 form, pulse EPR and ENDOR spectroscopy at X- and Q-band microwave frequencies was used. Hyperfine interactions of the four pyrrole nitrogens were determined from ENDOR and HYSCORE data, which revealed two sets of nitrogens with hyperfine couplings differing by about a factor of two. In addition, ENDOR data enabled observation of two nearly isotropic (1)H hyperfine interactions. Both the nitrogen and proton data indicate that the substrate analogue coenzyme M is axially coordinated to Ni(I) in the MCR(red2) state.

Coenzyme B induced coordination of coenzyme M via its thiol group to Ni(I) of F430 in active methyl-coenzyme M reductase.

Methyl-coenzyme M reductase (MCR) catalyzes the reaction of methyl-coenzyme M (CH3-S-CoM) with coenzyme B (HS-CoB) to methane and CoM-S-S-CoB. At the active site, it contains the nickel porphinoid F430, which has to be in the Ni(I) oxidation state for the enzyme to be active. How the substrates interact with the active site Ni(I) has remained elusive. We report here that coenzyme M (HS-CoM), which is a reversible competitive inhibitor to methyl-coenzyme M, interacts with its thiol group with the Ni(I) and that for interaction the simultaneous presence of coenzyme B is required. The evidence is based on X-band continuous wave EPR and Q-band hyperfine sublevel correlation spectroscopy of MCR in the red2 state induced with 33S-labeled coenzyme M and unlabeled coenzyme B.