Neutral ruthenium(II) complexes of phenylcyanamido ligands

Neutral Ru(II) complexes with the formula trans-[Ru(trpy*)(L 2)(pcyd)] have been prepared, where trpy* = 4,4′,4″-tri-tert-butyl-terpyridine, L 2 = 2-pyrazinecarboxylato (pca), 2-pyridinecarboxylato (pic), acetylacetonato (acac) and pcyd = 3-chlorophenylcyanamido (3-Clpcyd), 2,3-dichlorophenylcyanamido (2,3-Cl 2pcyd), 2,4,6-trichlorophenylcyanamido (2,4,6- Cl 3pcyd), 2,3,4,5-tetrachlorophenylcyanamido (2,3,4,5-Cl 4pcyd) and 3,4,5-trimethoxyphenylcyanamido (3,4,5-(OMe) 3pcyd). Spectroelectrochemistry was performed on these Ru(II) complexes to obtain the visible absorption spectrum of the Ru(III)-cyanamide ligand-to-metal charge transfer chromophore. The Ru(III)-cyanamide metal-ligand coupling elements of these complexes were compared to other Ru(III)-cyanamide complexes

Antibacterial Activity of 2-Picolyl-polypyridyl-Based Ruthenium (II/III) Complexes on Non-Drug-Resistant and Drug-Resistant Bacteria

A new hexadentate 2-picolyl-polypyridyl-based ligand (4, 4'-(butane-1, 4-diylbis(oxy))bis(N, N-bis(pyridin-2-ylmethyl)aniline)) (2BUT) (1) and its corresponding Ru(II/III) complexes were synthesized and characterized, followed by assessment of their possible bioactive properties towards drug-resistant and non-drug-resistant bacteria. Spectroscopic characterization of the ligand was done using proton NMR, FTIR, and ESI-MS, which showed that the ligand was successfully synthesized. The Ru(II/III) complexes were characterized by FTIR, UV/Vis, elemental analysis, proton NMR, ESI-MS, and magnetic susceptibility studies. The analysis of ESI-MS data of the complexes showed that they were successfully synthesized. Empirical formulae derived from elemental analysis of the complexes also indicated successful synthesis and relative purity of the complexes. The important functional groups of the ligands could be observed after complexation using FTIR. Magnetic susceptibility data and electronic spectra indicated that both complexes adopt a low spin configuration. The disc diffusion assay was used to test the compounds for antibiotic activity on two bacteria species and their drug-resistant counterparts. The compounds displayed antibiotic activity towards the two non-drug-resistant bacteria. As for the drug-resistant organisms, only [Ru2(2BUT)(DMF)2(DPA)2](BH4)43 and 2, 2-dipyridylamine inhibited the growth of MRSA. Gel electrophoresis DNA cleavage studies showed that the ligands had no DNA cleaving properties while all the complexes denatured the bacterial DNA. Therefore, the complexes may have DNA nuclease activity towards the bacterial genomic material

Human Glutaredoxin 3 Forms [2Fe-2S]-Bridged Complexes with Human BolA2

Human glutaredoxin 3 (Glrx3) is an essential [2Fe-2S]-binding protein with ill-defined roles in immune cell response, embryogenesis, cancer cell growth, and regulation of cardiac hypertrophy. Similar to other members of the CGFS monothiol glutaredoxin (Grx) family, human Glrx3 forms homodimers bridged by two [2Fe-2S] clusters that are ligated by the conserved CGFS motifs and glutathione (GSH). We recently demonstrated that the yeast homologues of human Glrx3 and the yeast BolA-like protein Fra2 form [2Fe-2S]-bridged heterodimers that play a key role in signaling intracellular iron availability. Herein, we provide biophysical and biochemical evidence that the two tandem Grx-like domains in human Glrx3 form similar [2Fe-2S]-bridged complexes with human BolA2. UV–visible absorption and circular dichroism, resonance Raman, and electron paramagnetic resonance spectroscopic analyses of recombinant [2Fe-2S] Glrx3 homodimers and [2Fe-2S] Glrx3–BolA2 complexes indicate that the Fe–S coordination environments in these complexes are virtually identical to those of the analogous complexes in yeast. Furthermore, we demonstrate that apo BolA2 binds to each Grx domain in the [2Fe-2S] Glrx3 homodimer forming a [2Fe-2S] BolA2–Glrx3 heterotrimer. Taken together, these results suggest that the unusual [2Fe-2S]-bridging Grx–BolA interaction is conserved in higher eukaryotes and may play a role in signaling cellular iron status in humans

Spectroscopic and Functional Characterization of Iron-Bound Forms of <i>Azotobacter vinelandii</i> ^NifIscA

The ability of <i>Azotobacter vinelandii</i> ^NifIscA to bind Fe has been investigated to assess the role of Fe-bound forms in NIF-specific Fe–S cluster biogenesis. ^NifIscA is shown to bind one Fe­(III) or one Fe­(II) per homodimer and the spectroscopic and redox properties of both the Fe­(III)- and Fe­(II)-bound forms have been characterized using the UV–visible absorption, circular dichroism, and variable-temperature magnetic circular dichroism, electron paramagnetic resonance, Möss­bauer and resonance Raman spectroscopies. The results reveal a rhombic intermediate-spin (<i>S</i> = 3/2) Fe­(III) center (<i>E/D</i> = 0.33, <i>D</i> = 3.5 ± 1.5 cm^–1) that is most likely 5-coordinate with two or three cysteinate ligands and a rhombic high spin (<i>S</i> = 2) Fe­(II) center (<i>E/D</i> = 0.28, <i>D</i> = 7.6 cm^–1) with properties similar to reduced rubredoxins or rubredoxin variants with three cysteinate and one or two oxygenic ligands. Iron-bound ^NifIscA undergoes reversible redox cycling between the Fe­(III)/Fe­(II) forms with a midpoint potential of +36 ± 15 mV at pH 7.8 (versus NHE). l-Cysteine is effective in mediating release of free Fe­(II) from both the Fe­(II)- and Fe­(III)-bound forms of ^NifIscA. Fe­(III)-bound ^NifIscA was also shown to be a competent iron source for in vitro NifS-mediated [2Fe-2S] cluster assembly on the N-terminal domain of NifU, but the reaction occurs via cysteine-mediated release of free Fe­(II) rather than direct iron transfer. The proposed roles of A-type proteins in storing Fe under aerobic growth conditions and serving as iron donors for cluster assembly on U-type scaffold proteins or maturation of biological [4Fe-4S] centers are discussed in light of these results

Monothiol Glutaredoxins and A-Type Proteins: Partners in Fe–S Cluster Trafficking

Monothiol glutaredoxins (Grxs) are proposed to function in Fe-S cluster storage and delivery, based on their ability to exist as apo monomeric forms and dimeric forms containing a subunit-bridging [Fe2S2](2+) cluster, and to accept [Fe2S2](2+) clusters from primary scaffold proteins. In addition yeast cytosolic monothiol Grxs interact with Fra2 (Fe repressor of activation-2), to form a heterodimeric complex with a bound [Fe2S2](2+) cluster that plays a key role in iron sensing and regulation of iron homeostasis. In this work, we report on in vitro UV-visible CD studies of cluster transfer between homodimeric monothiol Grxs and members of the ubiquitous A-type class of Fe-S cluster carrier proteins ((Nif)IscA and SufA). The results reveal rapid, unidirectional, intact and quantitative cluster transfer from the [Fe2S2](2+) cluster-bound forms of A. thaliana GrxS14, S. cerevisiae Grx3, and A. vinelandii Grx-nif homodimers to A. vinelandii (Nif)IscA and from A. thaliana GrxS14 to A. thaliana SufA1. Coupled with in vivo evidence for interaction between monothiol Grxs and A-type Fe-S cluster carrier proteins, the results indicate that these two classes of proteins work together in cellular Fe-S cluster trafficking. However, cluster transfer is reversed in the presence of Fra2, since the [Fe2S2](2+) cluster-bound heterodimeric Grx3-Fra2 complex can be formed by intact [Fe2S2](2+) cluster transfer from (Nif)IscA. The significance of these results for Fe-S cluster biogenesis or repair and the cellular regulation of the Fe-S cluster status are discussed

The Yeast Iron Regulatory Proteins Grx3/4 and Fra2 Form Heterodimeric Complexes Containing a [2Fe-2S] Cluster with Cysteinyl and Histidyl Ligation

The transcription of iron uptake and storage genes in S. cerevisiae is primarily regulated by the transcription factor Aft1. Nucleocytoplasmic shuttling of Aft1 is dependent upon mitochondrial Fe-S cluster biosynthesis via a signaling pathway that includes the cytosolic monothiol glutaredoxins (Grx3 and Grx4) and the BolA homologue Fra2. However the interactions between these proteins and the iron-dependent mechanism by which they control Aft1 localization are unclear. To reconstitute and characterize components of this signaling pathway in vitro, we have overexpressed yeast Fra2 and Grx3/4 in E. coli. We have shown that co-expression of recombinant Fra2 with Grx3 or Grx4 allows purification of a stable [2Fe-2S](2+) cluster-containing Fra2-Grx3 or Fra2-Grx4 heterodimeric complex. Reconstitution of a [2Fe-2S] cluster on Grx3 or Grx4 without Fra2 produces a [2Fe-2S]-bridged homodimer. UV-visible absorption and CD, resonance Raman, EPR, ENDOR, Mössbauer, and EXAFS studies of [2Fe-2S] Grx3/4 homodimers and the [2Fe-2S] Fra2-Grx3/4 heterodimers indicate that inclusion of Fra2 in the Grx3/4 Fe-S complex causes a change in the cluster stability and coordination environment. Taken together, our analytical, spectroscopic, and mutagenesis data indicate that Grx3/4 and Fra2 form a Fe-S-bridged heterodimeric complex with Fe ligands provided by the active site cysteine of Grx3/4, glutathione, and a histidine residue. Overall, these results suggest that the ability of the Fra2-Grx3/4 complex to assemble a [2Fe-2S] cluster may act as a signal to control the iron regulon in response to cellular iron status in yeast

The <i>Escherichia coli</i> BolA Protein IbaG Forms a Histidine-Ligated [2Fe-2S]-Bridged Complex with Grx4

Two ubiquitous protein families have emerged as key players in iron metabolism, the CGFS-type monothiol glutaredoxins (Grxs) and the BolA proteins. Monothiol Grxs and BolA proteins form heterocomplexes that have been implicated in Fe–S cluster assembly and trafficking. The <i>Escherichia coli</i> genome encodes members of both of these proteins families, namely, the monothiol glutaredoxin Grx4 and two BolA family proteins, BolA and IbaG. Previous work has demonstrated that <i>E. coli</i> Grx4 and BolA interact as both apo and [2Fe-2S]-bridged heterodimers that are spectroscopically distinct from [2Fe-2S]-bridged Grx4 homodimers. However, the physical and functional interactions between Grx4 and IbaG are uncharacterized. Here we show that co-expression of Grx4 with IbaG yields a [2Fe-2S]-bridged Grx4–IbaG heterodimer. <i>In vitro</i> interaction studies indicate that IbaG binds the [2Fe-2S] Grx4 homodimer to form apo Grx4–IbaG heterodimer as well as the [2Fe-2S] Grx4–IbaG heterodimer, altering the cluster stability and coordination environment. Additionally, spectroscopic and mutagenesis studies provide evidence that IbaG ligates the Fe–S cluster via the conserved histidine that is present in all BolA proteins and by a second conserved histidine that is present in the H/C loop of two of the four classes of BolA proteins. These results suggest that IbaG may function in Fe–S cluster assembly and trafficking in <i>E. coli</i> as demonstrated for other BolA homologues that interact with monothiol Grxs

Human Glutaredoxin 3 Forms [2Fe-2S]-Bridged Complexes with Human BolA2

Human glutaredoxin 3 (Glrx3) is an essential [2Fe-2S]-binding protein with ill-defined roles in immune cell response, embryogenesis, cancer cell growth, and regulation of cardiac hypertrophy. Similar to other members of the CGFS monothiol glutaredoxin (Grx) family, human Glrx3 forms homodimers bridged by two [2Fe-2S] clusters that are ligated by the conserved CGFS motifs and glutathione (GSH). We recently demonstrated that the yeast homologues of human Glrx3 and the yeast BolA-like protein Fra2 form [2Fe-2S]-bridged heterodimers that play a key role in signaling intracellular iron availability. Herein we provide biophysical and biochemical evidence that the two tandem Grx-like domains in human Glrx3 form similar [2Fe-2S]-bridged complexes with human BolA2. UV-visible absorption and CD, resonance Raman, and EPR spectroscopic analyses of recombinant [2Fe-2S] Glrx3 homodimers and [2Fe-2S] Glrx3-BolA2 complexes indicate that the Fe-S coordination environments in these complexes are virtually identical to the analogous complexes in yeast. Furthermore, we demonstrate that apo BolA2 binds to each Grx domain in the [2Fe-2S] Glrx3 homodimer forming a [2Fe-2S] BolA2-Glrx3 heterotrimer. Taken together, these results suggest that the unusual [2Fe-2S]-bridging Grx-BolA interaction is conserved in higher eukaryotes and may play a role in signaling cellular iron status in humans