Di- and Trinuclear Mixed-Valence Copper Amidinate Complexes from Reduction of Iodine

Molecular examples of mixed-valence copper complexes through chemical oxidation are rare but invoked in the mechanism of substrate activation, especially oxygen, in copper-containing enzymes. To examine the cooperative chemistry between two metals in close proximity to each other we began studying the reactivity of a dinuclear Cu(I) amidinate complex. The reaction of [(2,6-Me2C6H3N)2C(H)]2Cu2, 1, with I2 in tetrahydrofuran (THF), CH3CN, and toluene affords three new mixed-valence copper complexes [(2,6-Me2C6H3N)2C(H)]2Cu2(μ2-I3)(THF)2, 2, [(2,6-Me2C6H3N)2C(H)]2Cu2(μ2-I) (NCMe)2, 3, and [(2,6-Me2C6H3N)2C(H)]3Cu3(μ3-I)2, 4, respectively. The first two compounds were characterized by UV-vis and electron paramagnetic resonance spectroscopies, and their molecular structure was determined by X-ray crystallography. Both di- and trinuclear mixed-valence intermediates were characterized for the reaction of compound 1 to compound 4, and the molecular structure of 4 was determined by X-ray crystallography. The electronic structure of each of these complexes was also investigated using density functional theory

Influence of association state and DNA binding on the O2-reactivity of [4Fe-4S] fumarate and nitrate reduction (FNR) regulator

The fumarate and nitrate reduction (FNR) regulator is the master switch for the transition between anaerobic and aerobic respiration in Escherichia coli. Reaction of dimeric [4Fe-4S] FNR with O2 results in conversion of the cluster into a [2Fe-2S] form, via a [3Fe-4S] intermediate, leading to the loss of DNA binding through dissociation of the dimer into monomers. In the present paper, we report studies of two previously identified variants of FNR, D154A and I151A, in which the form of the cluster is decoupled from the association state. In vivo studies of permanently dimeric D154A FNR show that DNA binding does not affect the rate of cluster incorporation into the apoprotein or the rate of O2-mediated cluster loss. In vitro studies show that O2-mediated cluster conversion for D154A and the permanent monomer I151A FNR is the same as in wild-type FNR, but with altered kinetics. Decoupling leads to an increase in the rate of the [3Fe-4S]1+ into [2Fe-2S]2+ conversion step, consistent with the suggestion that this step drives association state changes in the wild-type protein. We have also shown that DNA-bound FNR reacts more rapidly with O2 than FNR free in solution, implying that transcriptionally active FNR is the preferred target for reaction with O2

Studies on the spin-spin interaction between flavin and iron-sulfur cluster in an iron-sulfur flavoprotein

When the di- or trimethylamine dehydrogenases (trimethylamine:(acceptor) oxidoreductase (demethylating), EC 1.5.99.7) of certain methylotrophic bacteria are reduced by two electrons with substrate unusual EPR signals arise at g = 2 and g = 4 (Steenkamp, D.J. and Beinert, H. (1982) Biochem, J. 207, 233-239; 241-252) indicative of spin-spin interaction between the EMN and iron-sulfur compounds of these enzymes. An attempt is made to understand, describe and simulate these spectra in terms of a triplet state with possible contributions from both dipolar and anisotropic exchange (J) interactions. No direct measurement of J is available, but various approaches to setting limits to J are outlined. According to these, J [approximate] 0.4 to 3 cm-1 or 15 to 50 cm-1. The spectra show, in the g = 2 region, a pair of rather sharp inner and a pair of broad outer lines; the latter broaden as well as move out from the center with increasing time (after substrate addition) and substrate concentration, while there is little change of g = 4. The best fits to such a spectra were obtained by assuming distribution of D and E values, depending on substrate effects and arriving presumably from `g-strain'. The fact that both shapes and intensities at g = 2 and g = 4 could be reproduced simultaneously at two frequencies indicates that the assumptions underlying our approaches and interpretations are permissible and reasonable, although we cannot claim their uniqueness. The distance between the centers of the spin densities of the flavin radical and the Fe-S cluster is thought to lie between the limits 3 to 5 A if the asymmetries in the spin-spin interaction are magnetic dipole-dipole in origin. Because there is an indication that the interaction is anisotropic exchange, the upper limit is less stringent.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26294/1/0000379.pd

Resonance Raman spectroscopy of cytochrome oxidase and electron transport particles with excitation near the Soret band

We report the resonance Raman spectra of cytochrome oxidase, both solubilized and in electron transport particles using laser excitation near the Soret band. As in the spectra of other hemoproteins, such as cytochrome , the shape and intensity of a number of bands change when the oxidation state is varied. However, one of the hemes of solubilized cytochrome oxidase shows redox behavior which is anomalous. Spectra of electron transport particles are dominated by cytochrome oxidase. There are, however, definite differences between spectra of solubilized cytochrome oxidase and electron transport particles in the oxidized states.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33860/1/0000121.pd

A Conserved Mechanism for Sulfonucleotide Reduction

Sulfonucleotide reductases are a diverse family of enzymes that catalyze the first committed step of reductive sulfur assimilation. In this reaction, activated sulfate in the context of adenosine-5′-phosphosulfate (APS) or 3′-phosphoadenosine 5′-phosphosulfate (PAPS) is converted to sulfite with reducing equivalents from thioredoxin. The sulfite generated in this reaction is utilized in bacteria and plants for the eventual production of essential biomolecules such as cysteine and coenzyme A. Humans do not possess a homologous metabolic pathway, and thus, these enzymes represent attractive targets for therapeutic intervention. Here we studied the mechanism of sulfonucleotide reduction by APS reductase from the human pathogen Mycobacterium tuberculosis, using a combination of mass spectrometry and biochemical approaches. The results support the hypothesis of a two-step mechanism in which the sulfonucleotide first undergoes rapid nucleophilic attack to form an enzyme-thiosulfonate (E-Cys-S-SO(3) (−)) intermediate. Sulfite is then released in a thioredoxin-dependent manner. Other sulfonucleotide reductases from structurally divergent subclasses appear to use the same mechanism, suggesting that this family of enzymes has evolved from a common ancestor

Biochemical properties of Paracoccus denitrificans FnrP:Reactions with molecular oxygen and nitric oxide

In Paracoccus denitrificans, three CRP/FNR family regulatory proteins, NarR, NnrR and FnrP, control the switch between aerobic and anaerobic (denitrification) respiration. FnrP is a [4Fe-4S] cluster containing homologue of the archetypal O2 sensor FNR from E. coli and accordingly regulates genes encoding aerobic and anaerobic respiratory enzymes in response to O2, and also NO, availability. Here we show that FnrP undergoes O2-driven [4Fe-4S] to [2Fe-2S] cluster conversion that involves up to 2 O2 per cluster, with significant oxidation of released cluster sulfide to sulfane observed at higher O2 concentrations. The rate of the cluster reaction was found to be ~6-fold lower than that of E. coli FNR, suggesting that FnrP can remain transcriptionally active under microaerobic conditions. This is consistent with a role for FnrP in activating expression of the high O2 affinity cytochrome c oxidase under microaerobic conditions. Cluster conversion resulted in dissociation of the transcriptionally active FnrP dimer into monomers. Therefore, along with E. coli FNR, FnrP belongs to the subset of FNR proteins in which cluster type is correlated with association state. Interestingly, two key charged residues, Arg140 and Asp154, that have been shown to play key roles in the monomer-dimer equilibrium in E. coli FNR are not conserved in FnrP, indicating that different protomer interactions are important for this equilibrium. Finally, the FnrP [4Fe-4S] cluster is shown to undergo reaction with multiple NO molecules, resulting in iron nitrosyl species and dissociation into monomers

Structure and Molecular Evolution of CDGSH Iron-Sulfur Domains

The recently discovered CDGSH iron-sulfur domains (CISDs) are classified into seven major types with a wide distribution throughout the three domains of life. The type 1 protein mitoNEET has been shown to fold into a dimer with the signature CDGSH motif binding to a [2Fe-2S] cluster. However, the structures of all other types of CISDs were unknown. Here we report the crystal structures of type 3, 4, and 6 CISDs determined at 1.5 Å, 1.8 Å and 1.15 Å resolution, respectively. The type 3 and 4 CISD each contain one CDGSH motif and adopt a dimeric structure. Although similar to each other, the two structures have permutated topologies, and both are distinct from the type 1 structure. The type 6 CISD contains tandem CDGSH motifs and adopts a monomeric structure with an internal pseudo dyad symmetry. All currently known CISD structures share dual iron-sulfur binding modules and a β-sandwich for either intermolecular or intramolecular dimerization. The iron-sulfur binding module, the β-strand N-terminal to the module and a proline motif are conserved among different type structures, but the dimerization module and the interface and orientation between the two iron-sulfur binding modules are divergent. Sequence analysis further shows resemblance between CISD types 4 and 7 and between 1 and 2. Our findings suggest that all CISDs share common ancestry and diverged into three primary folds with a characteristic phylogenetic distribution: a eukaryote-specific fold adopted by types 1 and 2 proteins, a prokaryote-specific fold adopted by types 3, 4 and 7 proteins, and a tandem-motif fold adopted by types 5 and 6 proteins. Our comprehensive structural, sequential and phylogenetic analysis provides significant insight into the assembly principles and evolutionary relationship of CISDs

The iron electron-nuclear double resonance (ENDOR) of two-iron ferredoxins from spinach, parsley, pig adrenal cortex and Pseudomonas putida

The iron electron-nuclear double resonance (ENDOR) spectra of reduced iron-sulfur proteins (two-iron ferredoxins) from spinach, parsley, pig adrenal cortex and Pseudomonas putida unequivocally show two inequivalent iron atoms at the active sites of each of these proteins. The frequencies of the ENDOR lines establish the total electronic spin in the ground state to be . The principal values of the hyperfine tensor have been determined for each of the iron atoms and these values are consistent with and lend considerable support to the model of a high-spin Fe(III) atom and a high-spin Fe(II) atom antiferromagnetically coupled to form an system. The measured principal axis components of the effective hyperfine tensors for are as follows (1 and 2 refer to the inequivalent iron sites): These data are consistent with Site 1 being ferric and Site 2, ferrous iron. The primes indicate that the A-tensor principal axes for Site 1 (Fe(III)) are apparently rotated about the x-axis with respect to the g-tensor axes by an angle [theta] (20[deg] [les] [theta] [les] 40[deg]). The orientations of the A-tensors for Site 2 (Fe(II)) have not been determined and hence the values presented are the observed values of the A-tensors along the x, y, and z-axes of the g-tensor for this complex.A brief introduction to the theory of ENDOR is given.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33533/1/0000032.pd

Recent developments in German corporate governance.

This paper provides an overview of the German corporate governance system. We review the governance role of large shareholders, creditors, the product market and the supervisory board. We also discuss the importance of mergers and acquisitions, the market in block trades, and the lack of a hostile takeover market. Given that Germany is often referred to as a bank-based economy, we pay particular attention to the role of the universal banks (Hausbanken). We show that the German system is characterised by a market for partial corporate control, large shareholders and bank/creditor monitoring, a two-tier (management and supervisory) board with co-determination between shareholders and employees on the supervisory board, a disciplinary product-market, and corporate governance regulation largely based on EU directives but with deep roots in the German codes and legal doctrine. Another important feature of the German system is its corporate governance efficiency criterion which is focused on the maximisation of stakeholder value rather than shareholder value. However, the German corporate governance system has experienced many important changes over the last decade. First, the relationship between ownership or control concentration and profitability has changed over time. Second, the pay-for-performance relation is influenced by large shareholder control: in firms with controlling blockholders and when a universal bank is simultaneously an equity- and debtholder, the pay-for-performance relation is lower than in widely-held firms or blockholder-controlled firms. Third, since 1995 several major regulatory initiatives (including voluntary codes) have increased transparency and accountability