JBIC Journal of Biological Inorganic Chemistry (v.23, #6)

NADP+-dependent cytosolic isocitrate dehydrogenase provides NADPH in the presence of cadmium due to the moderate chelating effect of glutathione by Hyo Je Cho; Ha Yeon Cho; Jeen-Woo Park; Oh-Shin Kwon; Hyun-Shik Lee; Tae Lin Huh; Beom Sik Kang (849-860).
Cadmium (Cd2+) is toxic to living organisms because it causes the malfunction of essential proteins and induces oxidative stress. NADP+-dependent cytosolic isocitrate dehydrogenase (IDH) provides reducing energy to counteract oxidative stress via oxidative decarboxylation of isocitrate. Intriguingly, the effects of Cd2+ on the activity of IDH are both positive and negative, and to understand the molecular basis, we determined the crystal structure of NADP+-dependent cytosolic IDH in the presence of Cd2+. The structure includes two Cd2+ ions, one coordinated by active site residues and another near a cysteine residue. Cd2+ presumably inactivates IDH due to its high affinity for thiols, leading to a covalent enzyme modification. However, Cd2+ also activates IDH by providing a divalent cation required for catalytic activity. Inactivation of IDH by Cd2+ is less effective when the enzyme is activated with Cd2+ than Mg2+. Although reducing agents cannot restore activity following inactivation by Cd2+, they can maintain IDH activity by chelating Cd2+. Glutathione, a cellular sulphydryl reductant, has a moderate affinity for Cd2+, allowing IDH to be activated with residual Cd2+, unlike dithiothreitol, which has a much higher affinity. In the presence of Cd2+-consuming cellular antioxidants, cells must continually supply reductants to protect against oxidative stress. The ability of IDH to utilise Cd2+ to generate NADPH could allow cells to protect themselves against Cd2+.
Keywords: NADP+-dependent cytosolic isocitrate dehydrogenase; Cadmium; Glutathione; Crystal structure; Enzyme activity

A re-investigation of the interaction with NO of the small tetraheme protein cytochrome c 554 (C554) from Nitrosomonas europaea has shown that the 5-coordinate heme II of the two- or four-electron-reduced protein will nitrosylate reversibly. The process is first order in C554, first order in NO, and second-order overall. The rate constant for NO binding to the heme is 3000 ± 140 M−1s−1, while that for dissociation is 0.034 ± 0.009 s−1; the degree of protein reduction does not appear to significantly influence the nitrosylation rate. In contrast to a previous report (Upadhyay AK, et al. J Am Chem Soc 128:4330, 2006), this study found no evidence of C554-catalyzed NO reduction, either with $$ { ext{C}}_{554}^{2 - } $$ C5542- or with $$ { ext{C}}_{554}^{4 - } . $$ C5544-. Some sub-stoichiometric oxidation of the lowest potential heme IV was detected when $$ { ext{C}}_{554}^{4 - } $$ C5544- was exposed to an excess of NO, but this is believed to arise from partial intramolecular electron transfer that generates {Fe(NO)}8 at heme II. The vacant heme II coordination site of C554 is crowded by three non-bonding hydrophobic amino acids. After replacing one of these (Phe156) with the smaller alanine, the nitrosylation rate for F156A2− and F156A4− was about 400× faster than for the wild type, though the rate of the reverse denitrosylation process was almost unchanged. Unlike in the wild-type C554, the 6-coordinate low-spin hemes of F156A4− oxidized over the course of several minutes after exposure to NO. Concomitant formation of N2O could explain this heme oxidation, though alternative explanations are equally plausible given the available data.
Keywords: Multi-heme cytochromes; Nitric oxide reduction; Mechanistic studies

Ether cross-link formation in the R2-like ligand-binding oxidase by Julia J. Griese; Rui M. M. Branca; Vivek Srinivas; Martin Högbom (879-886).
R2-like ligand-binding oxidases contain a dinuclear metal cofactor which can consist either of two iron ions or one manganese and one iron ion, but the heterodinuclear Mn/Fe cofactor is the preferred assembly in the presence of MnII and FeII in vitro. We have previously shown that both types of cofactor are capable of catalyzing formation of a tyrosine–valine ether cross-link in the protein scaffold. Here we demonstrate that Mn/Fe centers catalyze cross-link formation more efficiently than Fe/Fe centers, indicating that the heterodinuclear cofactor is the biologically relevant one. We further explore the chemical potential of the Mn/Fe cofactor by introducing mutations at the cross-linking valine residue. We find that cross-link formation is possible also to the tertiary beta-carbon in an isoleucine, but not to the secondary beta-carbon or tertiary gamma-carbon in a leucine, nor to the primary beta-carbon of an alanine. These results illustrate that the reactivity of the cofactor is highly specific and directed.
Keywords: Di-metal carboxylate protein; Ferritin; Ribonucleotide reductase; R2-like ligand-binding oxidase; X-ray crystallography

Preparation and characterization of metal-substituted carotenoid cleavage oxygenases by Xuewu Sui; Erik R. Farquhar; Hannah E. Hill; Johannes von Lintig; Wuxian Shi; Philip D. Kiser (887-901).
Carotenoid cleavage oxygenases (CCO) are non-heme iron enzymes that catalyze oxidative cleavage of alkene bonds in carotenoid and stilbenoid substrates. Previously, we showed that the iron cofactor of CAO1, a resveratrol-cleaving member of this family, can be substituted with cobalt to yield a catalytically inert enzyme useful for trapping active site-bound stilbenoid substrates for structural characterization. Metal substitution may provide a general method for identifying the natural substrates for CCOs in addition to facilitating structural and biophysical characterization of CCO-carotenoid complexes under normal aerobic conditions. Here, we demonstrate the general applicability of cobalt substitution in a prototypical carotenoid cleaving CCO, apocarotenoid oxygenase (ACO) from Synechocystis. Among the non-native divalent metals investigated, cobalt was uniquely able to stably occupy the ACO metal binding site and inhibit catalysis. Analysis by X-ray crystallography and X-ray absorption spectroscopy demonstrate that the Co(II) forms of both ACO and CAO1 exhibit a close structural correspondence to the native Fe(II) enzyme forms. Hence, cobalt substitution is an effective strategy for generating catalytically inert but structurally intact forms of CCOs.
Keywords: Non-heme iron enzyme; Cobalt substitution; X-ray absorption spectroscopy; Crystal structure

Targeting the mitochondrial VDAC in hepatocellular carcinoma using a polyclonal antibody-conjugated to a nitrosyl ruthenium complex by Loyanne C. B. Ramos; Fernando P. Rodrigues; Juliana C. Biazzotto; Sergio de Paula Machado; Leonardo D. Slep; Michael R. Hamblin; Roberto S. da Silva (903-916).
The rational design of anti-cancer agents includes a new approach based on ruthenium complexes that can act as nitric oxide (NO) donor agents against specific cellular targets. One of the most studied classes of those compounds is based on bis(bipyridine) ruthenium fragment and its derivative species. In this work, we present the chemical and cytotoxicity properties against the liver hepatocellular carcinoma cell line HepG2 of cis-[RuII(NO+)Cl(dcbpy)2]2− conjugated to a polyclonal antibody IgG (anti-VDAC) recognizing a cell surface marker. UV–visible bands of the ruthenium complex were assigned with the aid of density functional theory, which also allowed estimation of the structures that explain the biological effects of the ruthenium complex–IgG conjugate. The interaction of cis-[RuII(NO+)Cl(dcbpy)2]3− with mitochondria was evaluated due to the potential of these organelles as anti-cancer targets, and considering they interact with the anti-VDAC antibody. The cytotoxicity of cis-[RuII(NO+)Cl(dcbpy)2]3−-anti-VDAC antibody was up to 80% greater in comparison to the free cis-[RuII(NO+)Cl(dcbpy)2]3− complex. We suggest that this effect is due to site-specific interaction of the complex followed by NO release.
Keywords: Nitrosyl ruthenium complexes; Conjugated ruthenium-antibody complex; Nitric oxide delivery agent

Antenna effect and phosphorescence spectra to find the location of drug tetracycline in bovine β-lactoglobulin A by Moumita Mukherjee; Pinki Saha Sardar; Pritam Roy; Swagata Dasgupta; Maitrayee Basu Roy; Sanjib Ghosh (917-927).
A ternary system comprising of a Eu(III) complex of the drug Tetracycline hydrochloride (Eu3TC) bound to bovine β-lactoglobulin variant A (BLGA) in aqueous buffer at physiological pH (pH = 7.4) has been investigated to exploit the enhanced “antenna effect” to locate the bound drug and find the microenvironment of the binding site. Steady-state and time-resolved emission studies at room temperature as well as at 77 K have been carried out to evaluate the binding parameters in the binary system consisting of BLGA and tetracycline hydrochloride (TC). Low-temperature phosphorescence studies at 77 K of pure BLGA confirm Trp 19 to be the emitting residue, while Trp 61 is silent. Enhancement of BLGA phosphorescence emission in the ternary system at 77 K indicates that Trp 19 is very close to Eu(III) in the Eu3TC complex. The molecular docking results further confirm that TC binds close to Trp 19 in a hydrophobic domain. The results thus obtained can provide guidelines to design and synthesize target-oriented drugs as well as suitable bio-probes.
Keywords: Antenna effect; Bovine β-lactoglobulin; Tetracycline; Fluorescence; Phosphorescence; Accessible surface area

Motivated by the role of copper ions in biological processes the aim of this study was to elucidate the impact of copper ions bound to hydroxyapatite on l-serine (l-Ser) and O-phospho-l-serine (O-Ph-l-Ser) adsorption at different acidity of aqueous solutions. The adsorption phenomenon was studied by FTIR, UV, and AA spectroscopy, XRD and thermal analysis methods together with the evolved gases analysis taking into consideration the ionic state of the amino acids as well as the apatite surface state, which are tightly correlated with the solution pH. In acidic solution, the main process involves apatite dissolution releasing calcium and copper ions. At pH > 5 the complexation of amino acids with Ca2+ or Cu2+ ions is more important leading also to the release of cations. The ability of copper ions to form water soluble complexes with l-Ser and O-Ph-l-Ser leads to an important loss of these ions, while calcium release is very low at this pH. Therefore, the use of copper ions substituting calcium in the apatite structure to enhance the ability of amino acids adsorption on the apatite surface seems problematic even at pH > 5.
Keywords: Calcium hydroxyapatite; Copper modified hydroxyapatite; l-Serine; O-Phospho-l-serine; Adsorption

Low dosage of arsenic trioxide (As2O3) inhibits angiogenesis in epithelial ovarian cancer without cell apoptosis by Dehong Luo; Xiaoyuan Zhang; Renle Du; Wenjuan Gao; Na Luo; Shuangtao Zhao; Yi Li; Rui Chen; Hui Wang; Yonghua Bao; Wancai Yang; Daishun Liu; Wenzhi Shen (939-947).
Arsenic trioxide (As2O3) induces cell apoptosis and reduces the invasive and metastatic activities in various cancer types. However, the role of As2O3 in ovarian cancer angiogenesis remains unclear. In this study, we investigated the role of As2O3 in ovarian cancer angiogenesis and found that a low concentration of As2O3 causes no effects on epithelial ovarian cancer cell viability or apoptosis. Moreover, we found that As2O3-treated epithelial ovarian cancer cells demonstrate a reduced tube formation of endothelial cells in Matrigel. In addition, As2O3-treated epithelial ovarian cancer cells show a decreased VEGFA, VEGFR2 and CD31 mRNA expression. As per the underlying mechanisms involved in As2O3 treatment, we found that As2O3 inhibits VEGFA and VEGFR2 expression that thereby inhibits the VEGFA–VEGFR2–PI3K/ERK signaling pathway. This leads to a suppression in both VEGFA synthesis and angiogenesis-related gene expression. A decreased VEGFA synthesis and secretion also inhibits the VEGFA–VEGFR2–PI3K/ERK signaling pathway in human umbilical vein endothelial cells (HUVECs). In summary, our results may provide strategies for the use of As2O3 in the prevention of tumor angiogenesis.
Keywords: As2O3 ; Angiogenesis; VEGFA; VEGFR2; Apoptosis; Epithelial ovarian cancer

Copper 8-hydroxyquinoline-2-carboxaldehyde-thiosemicarbazide complex (CuHQTS) is a copper complex with strong anticancer activity against cisplatin-resistant neuroblastoma and prostate cancer cells in vitro by cell proliferation assay or fluorescent microscopic imaging. This study aimed to evaluate anti-prostate cancer activity of CuHQTS in vivo by bioluminescence imaging (BLI) and tumor size measurement, using athymic nu/nu mice implanted with prostate cancer cells carrying luciferase reporter gene (Luc-PC3). Growth of Luc-PC3 cells (1 × 105 cells) implanted in athymic nu/nu mice treated with CuHQTS for 2 weeks was suppressed by measurement of luciferase signals (6.18 × 107 to 5.36 × 107 p/s/cm2/sr) with BLI, compared with luciferase signals of Luc-PC3 cells (4.66 × 107 to 1.51 × 108 p/s/cm2/sr, p < 0.05) in the mice treated with normal saline of placebo control. Moreover, the size of PC-3 xenograft tumor (126.5 ± 34.2 mm3) in athymic nu/nu mice treated with CuHQTS was significantly smaller than the size of PC-3 xenograft tumor (218.6 ± 48.0 mm3, p < 0.05) in athymic nu/nu mice treated with normal saline of placebo control, suggesting in vivo tumor growth inhibition activity of CuHQTS on prostate cancer. The findings of this study support further investigation of CuHQTS as a promising new anticancer agent for the treatment of metastatic prostate cancer refractory to anticancer drugs currently available.
Keywords: Prostate cancer; Anticancer copper complex; CuHQTS; Bioluminescence Imaging