Orientation-selected 15N-HYSCORE detection of weakly coupled nitrogens around the archaeal rieske [2Fe-2S] center

The weakly coupled 15N atoms around a reduced Rieske [2Fe-2S] cluster of the uniformly 15N-labeled, hyperthermostable archaeal Rieske protein appear to produce readily observable cross-peaks in the HYSCORE spectra, with the well-resolved couplings of 0.3-0.4 MHz for the Nepsilon and 1.1 MHz for the peptide backbone nitrogens, in addition to the contributions from the coordinated Ndelta atoms. These features can be used for structure-mechanism studies of the biological redox protein system involving the weakly coupled nitrogens in coupled electron-proton transfer reactions.     

Characterization of the pH-dependent resonance Raman transitions of archaeal and bacterial Rieske [2Fe-2S] proteins

The pH-dependent resonance Raman (RR) spectral changes of the cytochrome bc1-associated, high-potential Rieske proteins have frequently been invoked to explain the redox-linked ionization behavior. We report herein RR spectral data of archaeal and bacterial Rieske proteins that directly demonstrate the pH-dependent changes near and above pKa,ox2, but not around pKa,ox1, of the visible circular dichroism (CD) transitions. The RR spectral changes are attributed to modification of the immediate [2Fe-2S] cluster environment due to deprotonation of some exchangeable amide groups in the polypeptide backbone, rather than previously assumed simple changes of the Fe-Nimid stretching vibrations.     

Protonation and concerted proton-electron transfer reactivity of a bis-benzimidazolate ligated [2Fe-2S] model for Rieske clusters

A model system for biological Rieske clusters that incorporates bis-benzimidazolate ligands ((Pr)bbim)(2-) has been developed ((Pr)bbimH(2) = 4,4-bis(benzimidazol-2-yl)heptane). The diferric and mixed-valence clusters have been prepared and characterized in both their protonated and deprotonated states. The thermochemistry of interconversions of these species has been measured, and the effect of protonation on the reduction potential is in good agreement to that observed in the biological systems. The mixed-valence and protonated congener [Fe(2)S(2)((Pr)bbim)((Pr)bbimH)](Et(4)N)(2) (4) reacts rapidly with TEMPO or p-benzoquinones to generate diferric and deprotonated [Fe(2)S(2)((Pr)bbim)(2)](Et(4)N)(2) (1) and 1 equiv of TEMPOH or 0.5 equiv of p-benzohydroquinones, respectively. The reaction with TEMPO is the first well-defined example of concerted proton-electron transfer (CPET) at a synthetic ferric/ferrous [Fe-S] cluster.     

Antibacterial activity of [1Fe-2S]- and [2Fe-2S]-nitrosyl complexes as nitric oxide donors

Russian Chemical Bulletin - The biological activity of a series of sulfur-nitrosyl iron complexes (NICs) depends on the structure of the ligands and the position of the functional groups in the...     

Reactions of synthetic [2Fe-2S] and [4Fe-4S] clusters with nitric oxide and nitrosothiols

The interaction of nitric oxide (NO) with iron-sulfur cluster proteins results in degradation and breakdown of the cluster to generate dinitrosyl iron complexes (DNICs). In some cases the formation of DNICs from such cluster systems can lead to activation of a regulatory pathway or the loss of enzyme activity. In order to understand the basic chemistry underlying these processes, we have investigated the reactions of NO with synthetic [2Fe-2S] and [4Fe-4S] clusters. Reaction of excess NO(g) with solutions of [Fe2S2(SR)4](2-) (R = Ph, p-tolyl (4-MeC6H4), or 1/2 (CH2)2-o-C6H4) cleanly affords the respective DNIC, [Fe(NO)2(SR)2](-), with concomitant reductive elimination of the bridging sulfide ligands as elemental sulfur. The structure of (Et4N)[Fe(NO)2(S-p-tolyl)2] was verified by X-ray crystallography. Reactions of the [4Fe-4S] clusters, [Fe4S4(SR)4](2-) (R = Ph, CH2Ph, (t)Bu, or 1/2 (CH2)-m-C6H4) proceed in the absence of added thiolate to yield Roussin's black salt, [Fe4S3(NO)7](-). In contrast, (Et4N)2[Fe4S4(SPh)4] reacts with NO(g) in the presence of 4 equiv of (Et4N)(SPh) to yield the expected DNIC. For all reactions, we could reproduce the chemistry effected by NO(g) with the use of trityl-S-nitrosothiol (Ph3CSNO) as the nitric oxide source. These results demonstrate possible pathways for the reaction of iron-sulfur clusters with nitric oxide in biological systems and highlight the importance of thiolate-to-iron ratios in stabilizing DNICs.     

Antisymmetric exchange in [2Fe-2S]1+ clusters: EPR of the Rieske protein from Thermus thermophilus at pH 14

[2Fe-2S] clusters found in the xanthine oxidase family of proteins exhibit an S = 1/2 EPR feature, called signal II, for which one g-value is significantly above g = 2.0. The g-values of signal II cannot be explained with the standard spin coupling model that has been so successful in describing the g = 1.94 signals of [2Fe-2S] ferredoxins. We have studied the EPR spectra of the Rieske protein from Thermus thermophilus at pH 14 and observed a signal II-type EPR spectrum, with g-values at 1.81, 1.94, and 2.14. It is shown that the g-values of signal II can be explained by including an antisymmetric exchange term, d.S1xS2, in the spin Hamiltonian. The presence of this term is sensed by EPR if the isotropic exchange coupling constant J is sufficiently small. For the Rieske protein we determined J = 43 cm-1 which is at least 4 times smaller than the J values reported for [2Fe-2S] clusters that yield standard g = 1.94 signals.     

Mechanism of glutaredoxin-ISU [2Fe-2S] cluster exchange

Exchange of [2Fe-2S] centers between Grx2 and the cluster scaffold protein ISU, and characterization of two mutually exclusive Grx2 binding sites on ISU by isothermal titration calorimetry supports a direct link for Grx and glutathione involvement in ISU promoted Fe-S cluster biosynthesis.     

Secondary bonding interactions in biomimetic [2Fe-2S] clusters

A series of synthetic [2Fe-2S] complexes with terminal thiophenolate ligands and tethered ether or thioether moieties has been prepared and investigated in order to provide models for the potential interaction of additional donor atoms with the Fe atoms in biological [2Fe-2S] clusters. X-ray crystal structures have been determined for six new complexes that feature appended Et (1(C)), OMe (1(O)), or SMe (1(S)) groups, or with a methylene group (2(C) ), an ether-O (2(O)), or an thioether-S (2(S)) linking two aryl groups. The latter two systems provide a constrained chelate arrangement that induces secondary bonding interactions with the ether-O and thioether-S, which is confirmed by density functional theory (DFT) calculations that also reveal significant spin density on those fifth donor atoms. Structural consequences of the secondary bonding interactions are analyzed in detail, and effects on the spectroscopic and electronic properties are probed by UV-vis, M?ssbauer, and (1)H NMR spectroscopy, as well by SQUID measurements and cyclic voltammetry. The potential relevance of the findings for biological [2Fe-2S] sites is considered.     

An Efficient Synthesis of [15N]-Carbazole from [15N]-Aniline

A reaction sequence involving ortho-lithiation of [¹⁵N]-(tert-Butoxy-carbonyl)aniline, quenching with Bu₃SnCl, palladium catalyzed coupling with bromobenzene, and deprotection provides efficient access to [¹⁵N]-2-aminobiphenyl (4). Compound 4 is converted to [¹⁵N]-carbazole via diazotization, treatment with NaN₃, and heating to promote intramolecular nitrene insertion.     

Synthesis, characterization and reactivity of palladium(II) compounds containing terdentate [Csp, N, S] or [Csp, N, S] ligands

The study of the reactivity of R---CH=N---(C₆H₄-2-SMe) with R=C₆H₅ or 2,4,6-Me₃-C₆H₂ with palladium(II) salts is reported. These studies have allowed us to prepare and characterize the coordination complexes: cis-[Pd{R---CH=N---(C₆H₄-2-SMe)}Cl₂] {R=C₆H₅ or 2,4,6-Me₃-C₆H₂} and the cyclopalladated compounds [Pd{C₆H₄---CH=N---(C₆H₄-2-SMe)}Cl] and [Pd{(2-CH₂-4,6-Me₂-C₆H₂)---CH=N---(C₆H₄-2-SMe)}Cl]. The X-ray crystal structures of the latter complexes reveal that the thioimines act as a [Csp², phenyl,N,S]⁻ and as a [Csp³, N,S]⁻ terdentate group, respectively. The study of the reactions of the cyclopalladated compounds with PPh₃ is also reported.     

The vibrational spectra of [15N2]-succinonitrile

For the first time, the infrared and Raman spectra of [15N2]-succinonitrile are presented and discussed in detail. Assignments of the vibrational bands of its two rotational conformers gauche and trans, respectively, have been made for both infrared and Raman spectra. The assignments were based on a recent ab-initio force field calculation for succinonitrile, taking into account the vibrational frequencies of other succinonitrile isotopomers. There are differences in the frequencies of the vibrational bands due to the mass increase in the cyanide groups, which have been analysed in depth.     

Permselective-membrane pyrolytic graphite electrode for the study of microvolumes of [2Fe-2S] ferredoxin

A permselective-membrane pyrolytic graphite electrode was used to study the electrochemistry of [2Fe-2S] ferredoxin entrapped between the membrane and the graphite surface, in the presence of poly(l-Lysine). Factors influencing the electrode response [such as the concentration of poly(l-lysine), ionic strength and pH] were explored. The analytical performance of this permselective-membrane electrode, which allows 20 pmol of ferredoxin to be detected, was examined.     

Direct detection of a hydrogen ligand in the [NiFe] center of the regulatory H2-sensing hydrogenase from Ralstonia eutropha in its reduced state by HYSCORE and ENDOR spectroscopy

The regulatory H2-sensing [NiFe] hydrogenase of the beta-proteobacterium Ralstonia eutropha displays an Ni-C "active" state after reduction with H2 that is very similar to the reduced Ni-C state of standard [NiFe] hydrogenases. Pulse electron nuclear double resonance (ENDOR) and four-pulse ESEEM (hyperfine sublevel correlation, HYSCORE) spectroscopy are applied to obtain structural information on this state via detection of the electron-nuclear hyperfine coupling constants. Two proton hyperfine couplings are determined by analysis of ENDOR spectra recorded over the full magnetic field range of the EPR spectrum. These are associated with nonexchangeable protons and belong to the beta-CH(2) protons of a bridging cysteine of the NiFe center. The signals of a third proton exhibit a large anisotropic coupling (Ax = 18.4 MHz, Ay = -10.8 MHz, Az = -18 MHz). They disappear from the 1H region of the ENDOR spectra after exchange of H2O with 2H2O and activation with 2H2 instead of H2 gas. They reappear in the 2H region of the ENDOR and HYSCORE spectra. Based on a comparison with the spectroscopically similar [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F, for which the g-tensor orientation of the Ni-C state with respect to the crystal structure is known (Foerster et al. J. Am. Chem. Soc. 2003, 125, 83-93), an assignment of the 1H hyperfine couplings is proposed. The exchangeable proton resides in a bridging position between the Ni and Fe and is assigned to a formal hydride ion. After illumination at low temperature (T = 10 K), the Ni-L state is formed. For the Ni-L state, the strong hyperfine coupling observed for the exchangeable hydrogen in Ni-C is lost, indicating a cleavage of the metal-hydride bond(s). These experiments give first direct information on the position of hydrogen binding in the active NiFe center of the regulatory hydrogenase. It is proposed that such a binding situation is also present in the active Ni-C state of standard hydrogenases.     

1H, 13C and 15N NMR spectra of [1,2-15N2]pyrazole derivatives

The chemical shifts and coupling constants of [1,2-¹⁵N₂]pyrazole, 2-(1-[1,2- ¹⁵N₂]pyrazolyl)-2-[l,3-²H₆]propanol, 1-nitro[1,2¹⁵N₂] and 3-nitro[1,2-¹⁵N₂]pyrazole are reported.