trans-Diacetonitrile­tetra­kis(1H-pyrazole-κN 2)nickel(II) dinitrate

In the title complex, [Ni(CH3CN)2(C3H4N2)4](NO3)2, the cation lies on an inversion center and adopts an octa­hedral coordination geometry about the Ni atom. The two acetonitrile ligands are in a trans conformation. N—H⋯O hydrogen bonds between cations and anions link the complex mol­ecules into one-dimensional chains running parallel to [100]

The P174L mutation in human Sco1 severely compromises Cox17-dependent metallation but does not impair copper binding.

International audienceSco1 is a metallochaperone that is required for copper delivery to the Cu(A) site in the CoxII subunit of cytochrome c oxidase. The only known missense mutation in human Sco1, a P174L substitution in the copper-binding domain, is associated with a fatal neonatal hepatopathy; however, the molecular basis for dysfunction of the protein is unknown. Immortalized fibroblasts from a SCO1 patient show a severe deficiency in cytochrome c oxidase activity that was partially rescued by overexpression of P174L Sco1. The mutant protein retained the ability to bind Cu(I) and Cu(II) normally when expressed in bacteria, but Cox17-mediated copper transfer was severely compromised both in vitro and in a yeast cytoplasmic assay. The corresponding P153L substitution in yeast Sco1 was impaired in suppressing the phenotype of cells harboring the weakly functional C57Y allele of Cox17; however, it was functional in sco1delta yeast when the wild-type COX17 gene was present. Pulse-chase labeling of mitochondrial translation products in SCO1 patient fibroblasts showed no change in the rate of CoxII translation, but there was a specific and rapid turnover of CoxII protein in the chase. These data indicate that the P174L mutation attenuates a transient interaction with Cox17 that is necessary for copper transfer. They further suggest that defective Cox17-mediated copper metallation of Sco1, as well as the subsequent failure of Cu(A) site maturation, is the basis for the inefficient assembly of the cytochrome c oxidase complex in SCO1 patients

Investigation of the kinetic mechanism and the activation of methyl -coenzyme M reductase: The catalyst for the final step in methanogenesis

Methyl Coenzyme M Reductase (MCR) from Methanothermobacter marburgensis (Mtm) catalyzes the final step of methane formation in methanogenesis. This enzyme uses Coenzyme B (HS-CoB) as the electron donor to catalyze the two-electron reduction of methyl-Coenzyme M (CH 3-SCoM) to form methane and the heterodisulfide, CoM-SS-CoB. At the active site of MCR is cofactor F430, a nickel tetrapyrrole. Several states of MCR have been characterized. The only active form of the enzyme, called “MCRred1,” is formed by in vitro activation of the ready “MCRox1” state with a low-potential reductant, titanium(III) citrate. Based on EPR, ENDOR, and X-ray absorption spectroscopic studies and cryoreduction experiments, both the MCRox1 and MCRred1 forms of the enzyme are in the low valent Ni(I) state. Thus, the requirement of the low potential reductant titanium(III) citrate to convert Ni(I)-MCRox1 to Ni(I)-MCRred1 was puzzling. The 40 nm blue shift accompanying conversion of the ox1 to the red1 state hinted that changes in the tetrapyrrole ring might be involved in activation. Resonance Raman spectra of MCRred1, lack a vibrational band attributed to a C=N bond that is present in Ni(II)-F430, Ni(II) states of MCR, and MCRox1. These results indicate that, in the red1 state of MCR and in Ni(I)-F430, one of the conjugated C=N bonds of the tetrapyrrole ring has undergone reduction. EPR and kinetic studies of the reaction of 3-bromopropanesulfonate (BPS, I50 = 50 nM) with MCR ox1 and MCRred1 are consistent with this conclusion. Reaction of the red1 state of MCR with BPS forms a Ni(I) EPR signal called MCR BPS and propane sulfonate as a product. Reaction of the ox1 state of MCR forms an identical EPR signal without an intermediate red1 state and without forming product. Thus, the red1 state is indeed more reduced than the MCR ox1 state. These results also imply that similar redox changes may be involved in methane formation from the natural substrate, methyl-SCoM. Furthermore, we have studied the kinetic mechanism of MCR by single-turn-over kinetics. Our results indicate that Coenzyme B must react with MCR-bound methyl-Coenzyme M before even a single turnover of methane is formed

Mechanistic Studies of Methane Biogenesis by Methyl-Coenzyme M Reductase: Evidence that Coenzyme B Participates in Cleaving the C-S Bond of Methyl-Coenzyme M

[[abstract]]Methyl-coenzyme M reductase (MCR), the key enzyme in methanogenesis, catalyzes methane formation from methyl-coenzyme M (methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoBSH). Steady-state and presteady-state kinetics have been used to test two mechanistic models that contrast in the role of CoBSH in the MCR-catalyzed reaction. In class 1 mechanisms, CoBSH is integrally involved in methane formation and in C-S (methyl-SCoM) bond cleavage. On the other hand, in class 2 mechanisms, methane is formed in the absence of CoBSH, which functions to regenerate active MCR after methane is released. Steady-state kinetic studies are most consistent with a ternary complex mechanism in which CoBSH binds before methane is formed, as found earlier [Bonacker et al. (1993) Eur. J. Biochem. 217, 587-595]. Presteady-state kinetic experiments at high MCR concentrations are complicated by the presence of tightly bound CoBSH in the purified enzyme. Chemical quench studies in which (14)CH(3)-SCoM is rapidly reacted with active MCRred1 in the presence versus the absence of added CoBSH indicate that CoBSH is required for a single-turnover of methyl-SCoM to methane. Similar single turnover studies using a CoBSH analogue leads to the same conclusion. The results are consistent with class 1 mechanisms in which CoBSH is integrally involved in methane formation and in C-S (methyl-SCoM) bond cleavage and are inconsistent with class 2 mechanisms in which CoBSH binds after methane is formed. These are the first reported pre-steady-state kinetic studies of MCR

Human Sco1 and Sco2 Function as Copper-binding Proteins

[[abstract]]The function of human Sco1 and Sco2 is shown to be dependent on copper ion binding. Expression of soluble domains of human Sco1 and Sco2 either in bacteria or the yeast cytoplasm resulted in the recovery of copper-containing proteins. The metallation of human Sco1, but not Sco2, when expressed in the yeast cytoplasm is dependent on the co-expression of human Cox17. Two conserved cysteines and a histidyl residue, known to be important for both copper binding and in vivo function in yeast Sco1, are also critical for in vivo function of human Sco1 and Sco2. Human and yeast Sco proteins can bind either a single Cu(I) or Cu(II) ion. The Cu(II) site yields S-Cu(II) charge transfer transitions that are not bleached by weak reductants or chelators. The Cu(I) site exhibits trigonal geometry, whereas the Cu(II) site resembles a type II Cu(II) site with a higher coordination number. To identify additional potential ligands for the Cu(II) site, a series of mutant proteins with substitutions in conserved residues in the vicinity of the Cu(I) site were examined. Mutation of several conserved carboxylates did not alter either in vivo function or the presence of the Cu(II) chromophore. In contrast, replacement of Asp238 in human or yeast Sco1 abrogated the Cu(II) visible transitions and in yeast Sco1 attenuated Cu(II), but not Cu(I), binding. Both the mutant yeast and human proteins were nonfunctional, suggesting the importance of this aspartate for normal function. Taken together, these data suggest that both Cu(I) and Cu(II) binding are critical for normal Sco function

Specific Copper Transfer from the Cox17 Metallochaperone to Both Sco1 and Cox11 in the Assembly of Yeast Cytochrome c Oxidase

[[abstract]]The assembly of the copper sites in cytochrome c oxidase involves a series of accessory proteins, including Cox11, Cox17, and Sco1. The two mitochondrial inner membrane proteins Cox11 and Sco1 are thought to be copper donors to the CuB and CuA sites of cytochrome oxidase, respectively, whereas Cox17 is believed to be the copper donor to Sco1 within the intermembrane space. In this report we show Cox17 is a specific copper donor to both Sco1 and Cox11. Using in vitro studies with purified proteins, we demonstrate direct copper transfer from CuCox17 to Sco1 or Cox11. The transfer is specific because no transfer occurs to heterologous proteins, including bovine serum albumin and carbonic anhydrase. In addition, a C57Y mutant of Cox17 fails to transfer copper to Sco1 but is competent for copper transfer to Cox11. The in vitro transfer studies were corroborated by a yeast cytoplasm expression system. Soluble domains of Sco1 and Cox11, lacking the mitochondrial targeting sequence and transmembrane domains, were expressed in the yeast cytoplasm. Metallation of these domains was strictly dependent on the co-expression of Cox17. Thus, Cox17 represents a novel copper chaperone that delivers copper to two proteins

Preparation of a Reversible Redox-Controlled Cage-Type Molecule Linked by Disulfide Bonds

[[abstract]]A unique trithiol macromolecule, with an intrinsic conformational propensity for dimerization (cage) instead of oligomerization upon oxidation, was prepared straightforwardly through rational design. The quantitative conversion and the reversibility between the cage and trithiols through redox reactions were assayed by 1H NMR spectroscopic analysis. The X-ray structure of the synthesized cage-type molecule represents the first successful example of a redox-controlled reversible dimeric capsule linked through covalent disulfide bonds

Preparation of a Reversible Redox-Controlled Cage-Type Molecule Linked by Disulfide Bonds

[[abstract]]A unique trithiol macromolecule, with an intrinsic conformational propensity for dimerization (cage) instead of oligomerization upon oxidation, was prepared straightforwardly through rational design. The quantitative conversion and the reversibility between the cage and trithiols through redox reactions were assayed by 1H NMR spectroscopic analysis. The X-ray structure of the synthesized cage-type molecule represents the first successful example of a redox-controlled reversible dimeric capsule linked through covalent disulfide bonds

N-[2-(Methylsulfanyl)phenyl]-2-sulfanylbenzamide

In the title compound, C14H13NOS2, the S atom with the methyl group is involved in an intramolecular hydrogen bond with the amido H atom. In the crystal, the sulfanyl H atoms form intermolecular hydrogen bonds with the O atoms, connecting the molecules into zigzag chains along the c axis. The two aromatic rings exhibit a small interplanar angle of 16.03 (9)°