Reconstitution of mitochondrial processing peptidase from the core proteins (subunits I and II) of bovine heart mitochondrial cytochrome bc(1) complex

Mature core I and core II proteins of the bovine heart mitochondrial cytochrome bc(1) complex were individually overexpressed in Escherichia coli as soluble proteins using the expression vector pET-I and pET-II, respectively. Purified recombinant core I and core II alone show no mitochondrial processing peptidase (MPP) activity. When these two proteins are mixed together, MPP activity is observed. Maximum activity is obtained when the molar ratio of these two core proteins reaches 1. This indicates that only the two core subunits of thebc(1) complex are needed for MPP activity. The properties of reconstituted MPP are similar to those of Triton X-100-activated MPP in the bovine bc(1) complex. When Rieske iron-sulfur protein precursor is used as substrate for reconstituted MPP, the processing activity stops when the amount of product formation (subunit IX) equals the amount of reconstituted MPP used in the system. Addition of Triton X-100 to the product-inhibited reaction mixture restores MPP activity, indicating that Triton X-100 dissociates bound subunit IX from the active site of reconstituted MPP. The aromatic group, rather than the hydroxyl group, at Tyr(57) of core I is essential for reconstitutive activity.     

Structural, IR, and EPR studies of the bis(methoxyacetato)diaquo-copper(II) complex

The structure, stability, and the IR, and EPR spectroscopic properties of bis(methoxyacetato)diaquo-copper(II) were studied both experimentally using FT-IR and theoretically using B3LYP/6-31G**, B3LYP/6-311G, BWP91/6-31G** methods. The same approaches were used to calculate the harmonic frequencies and to compare them to the experimental solid state values. The g-tensors are calculated using the NMR/GIAO computational method.     

Uptake of chromium(III) and chromium(VI) compounds in the yeast cell structure

The study presented in this article investigated the influence of different Cr(III) and Cr(VI) compounds in the cultivation medium on the uptake and localization of chromium in the cell structure of the yeast Candida intermedia. The morphology of the yeast cell surface was observed by the scanning electron microscopy. Results demonstrated that the growth inhibitory concentration of Cr(III) in the cultivation medium induced changes in the yeast cell shape and affected the budding pattern, while inhibitory concentration of Cr(VI) did not cause any visible effects on morphological properties of the yeast cells. The amount of total accumulated chromium in yeast cells and the distribution of chromium between the yeast cell walls and spheroplasts were determined by atomic absorption spectroscopy. No significant differences were found neither in total chromium accumulation nor in the distribution of chromium in yeast cell walls and spheroplasts between the two of Cr(VI) compounds. Conversely, substantial differences between Cr(III) compounds were demonstrated in the total uptake as well as the localization of chromium in yeast cells.     

Influence of nickel (II) and chromium (VI) on the laboratory scale rotating biological contactor

High concentration of heavy metals is toxic for most microorganisms and cause strict damage in wastewater treatment operations and often a physico-chemical pretreatment prior to biological treatment is considered necessary. However, in this study it has been shown that biological systems can adapt to Ni (II) and Cr (VI) when their concentration is below 10 and 20 mg/L, respectively. The aim of this study was to evaluate the effect of Ni (II) and Cr (VI) on the lab-scale rotating biological contactor process. It was found that, addition of Ni (II) up to 10 mg/L did not reduce the chemical oxygen demand removal efficiency and on the contrary concentrations below 10 mg/L improved the performance. The influent Ni (II) concentration of 1 mg/L was the concentration where the treatment efficiency produced a maximum COD removal of 86.5%. Moreover, Ni (II) concentration above 10 mg/L was relatively toxic to the system and produced lower treatment efficiencies than the baseline study without Ni (II). Turbidity and suspended solids removals were not stimulated to a great extent with nickel. Addition of Ni (II) did not seem to affect the pH of the system during treatment. The dissolved oxygen concentration did not drop below 4 mg/L at all concentrations of Ni (II) indicating aerobic conditions prevailed in the system. Experiments conducted with Cr (VI) revealed that addition of Cr (VI) up to 20 mg/L did not reduce the COD removal efficiency and on the contrary concentrations below 20 mg/L improved the performance. The influent Cr (VI) concentration of 1 mg/L was the concentration where the treatment efficiency produced a maximum COD removal of 88%. Turbidity and SS removals were more efficient at 5 mg/L Cr (VI) concentration, rather than 1 mg/L, which lead to the conclusion that 5 mg/L Cr (VI) concentration is the optimum concentration, in terms of COD, turbidity and SS removals. Similar with Ni (II) experiments, addition of Cr (VI) did not significantly affect the pH value of the effluent. The DO concentration remained above 5 mg/L.     

The role of superoxide radical in chromium (VI)-generated hydroxyl radical: the Cr(VI) Haber-Weiss cycle.

To understand the role of the superoxide (O-2) radical in chromate-related genotoxicity, we investigated whether Cr(VI) can catalyze the Haber-Weiss cycle in vitro: O-2 + Cr(VI)----Cr(V) + O2 Cr(V) + H2O2----Cr(VI) + .OH + OH-. ESR and spin trapping techniques were utilized to monitor the O-2 (produced using xanthine/xanthine oxidase), .OH, and Cr(V) species. Superoxide dismutase as well as catalase inhibited the .OH radical radical formation, attesting to the direct involvement of O-2 and H2O2 in the process. ESR measurements also provided direct evidence for the formation of Cr(V). Kinetic measurements were consistent with the role of Cr(V) and H2O2 as intermediates in .OH formation. These results indicate that in cellular media, especially during chromate phagocytosis, the O-2 radical can become a significant source of .OH radicals and hence a significant factor in the biochemical mechanism of cellular damage due to Cr(VI) exposure.     

Identification of inner- and outer-sphere reaction pathways in the reduction of iron-sulphur proteins with a chromium(II)-macrocycle complex

When parsley [2Fe-2S] and $\text{C. pasteurianum}$ 2[4Fe-4S] proteins in the normal oxidised state are reduced 1:1 with Cr(II) (15-aneN₄) (H₂O)₂²⁺ the Cr(III) product remains attached to the protein and reduction is by an inner-sphere mechanism. With $\text{Chromatium}$ high potential [4Fe-4S] protein and $\text{C. pasteurianum}$ rubredoxin the Cr(III) product is not attached to the protein and the mechanism is outer-sphere. Results are discussed in the context of protein crystallographic information. The Cr(III) product is not attached to the Fe₂S₂ core (extrusion experiments) or to the cysteinyl S-atoms (ESR). Negative patches close to the active site remain possible alternatives.     

Carbonic anhydrase subunits of the mitochondrial NADH dehydrogenase complex (complex I) in plants

The mitochondrial nicotinamide adenine dinucleotide, reduced (NADH) dehydrogenase complex (complex I) of plants has a molecular mass of about 1000 kDa and is composed of more than 40 distinct protein subunits. About three quarter of these subunits are homologous to complex I subunits of heterotrophic eukaryotes, whereas the remaining subunits are unique to plants. Among them are three to five structurally related proteins that resemble an archaebacterial γ-type carbonic anhydrase (γCA). The γCA subunits are attached to the membrane arm of complex I on the matrix-exposed side and form an extra spherical domain. At the same time, they span the inner mitochondrial membrane and are essential for assembly of the protein complex. Expression of the genes encoding γCA subunits is reduced if plants are cultivated in the presence of elevated CO₂ concentration. The functional role of these subunits within plant mitochondria is currently unknown but might be related to photorespiration. We propose that the complex I–integrated γCAs are involved in mitochondrial HCO₃⁻ formation to allow efficient recycling of inorganic carbon for CO₂ fixation in chloroplasts under high light conditions.     

Mucopolysaccharidosis I, II, and VI: Brief review and guidelines for treatment

Mucopolysaccharidoses (MPS) are rare genetic diseases caused by the deficiency of one of the lysosomal enzymes involved in the glycosaminoglycan (GAG) breakdown pathway. This metabolic block leads to the accumulation of GAG in various organs and tissues of the affected patients, resulting in a multisystemic clinical picture, sometimes including cognitive impairment. Until the beginning of the XXI century, treatment was mainly supportive. Bone marrow transplantation improved the natural course of the disease in some types of MPS, but the morbidity and mortality restricted its use to selected cases. The identification of the genes involved, the new molecular biology tools and the availability of animal models made it possible to develop specific enzyme replacement therapies (ERT) for these diseases. At present, a great number of Brazilian medical centers from all regions of the country have experience with ERT for MPS I, II, and VI, acquired not only through patient treatment but also in clinical trials. Taking the three types of MPS together, over 200 patients have been treated with ERT in our country. This document summarizes the experience of the professionals involved, along with the data available in the international literature, bringing together and harmonizing the information available on the management of these severe and progressive diseases, thus disclosing new prospects for Brazilian patients affected by these conditions.     

A radical-bridged bis(ferrocenylcopper(I)) complex: Structural identity, multifrequency EPR, and spectroelectrochemistry

The paramagnetic blue complex {(μ-adcO^tBu)[Cu(dppf)]₂}(PF₆) contains two heterodinuclear 1,1′-bis(diphenylphosphinoferrocene)copper(I) moieties, bridged by the radical anion of di-tert-butylazodicarboxylic ester (adcO^tBu). The assignment of oxidation states is supported by crystal structure determination and by variable frequency EPR spectroscopy which provides hyperfine (9.5 GHz) and g anisotropy information (285 GHz). Cyclic voltammetry and UV/Vis/NIR spectroelectrochemistry indicate reversible one-electron oxidation of the adcO^tBu⁻ bridge prior to the quasi-reversible two-electron oxidation of the well separated (12.55 Å) ferrocene termini. One-electron reduction to the adcO^tBu²⁻ bridged system occurs with concomitant loss of the long-wavelength metal-to-ligand charge transfer absorption.     

Allosteric nucleotide-binding site in the mitochondrial NADH:ubiquinone oxidoreductase (respiratory complex I)

The rotenone-insensitive NADH:hexaammineruthenium III (HAR) oxidoreductase reactions catalyzed by bovine heart and Yarrowia lipolytica submitochondrial particles or purified bovine complex I are stimulated by ATP and other purine nucleotides. The soluble fraction of mammalian complex I (FP) and prokaryotic complex I homolog NDH-1 in Paracoccus denitrificans plasma membrane lack stimulation of their activities by ATP. The stimulation appears as a decrease in apparent K(m) values for NADH and HAR. Thus, the "accessory" subunits of eukaryotic complex I bear an allosteric ATP-binding site.     

Formation of the paramagnetic complex [Cr(OH)5O2]5- in the reaction between chromium(VI) oxide, and hydrogen peroxide or superoxide anion radicals studied by EPR spectroscopy.

Hydrogen peroxide or superoxide anion radicals form a paramagnetic complex in the reaction with chromium(VI) oxide in an alkaline water solution at room temperature. The complex [Cr(OH)5O2]5- with the g-value equal to 1.9734 is believed to contain hydroxyl groups derived from the alkaline solution and dioxygen derived from hydrogen peroxide or superoxide anion radicals.     

The inhibition of mitochondrial complex I (NADH:ubiquinone oxidoreductase) by Zn2+

NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria is a highly complicated, membrane-bound enzyme. It is central to energy transduction, an important source of cellular reactive oxygen species, and its dysfunction is implicated in neurodegenerative and muscular diseases and in aging. Here, we describe the effects of Zn2+ on complex I to define whether complex I may contribute to mediating the pathological effects of zinc in states such as ischemia and to determine how Zn2+ can be used to probe the mechanism of complex I. Zn2+ inhibits complex I more strongly than Mg2+, Ca2+, Ba2+, and Mn2+ to Cu2+ or Cd2+. It does not inhibit NADH oxidation or intramolecular electron transfer, so it probably inhibits either proton transfer to bound quinone or proton translocation. Thus, zinc represents a new class of complex I inhibitor clearly distinct from the many ubiquinone site inhibitors. No evidence for increased superoxide production by zinc-inhibited complex I was detected. Zinc binding to complex I is mechanistically complicated. During catalysis, zinc binds slowly and progressively, but it binds rapidly and tightly to the resting state(s) of the enzyme. Reactivation of the inhibited enzyme upon the addition of EDTA is slow, and inhibition is only partially reversible. The IC50 value for the Zn2+ inhibition of complex I is high (10-50 microm, depending on the enzyme state); therefore, complex I is unlikely to be a major site for zinc inhibition of the electron transport chain. However, the slow response of complex I to a change in Zn2+ concentration may enhance any physiological consequences.     

A trigonal-prismatic Ag(I) complex and an octahedral Cu(II) complex incorporating with oxamide Ni(II) complex ligand: Syntheses, crystal structures and oxidative nuclease activities

A mononuclear macrocyclic complex Ni^IIL^3a (L^3a = dianion of 2,3-dioxo-5,6:13,14-dibenzo-9,10-cyclohexyl-7,12-bis(methoxycarbonyl)-1,4,8,11-tetraazacyclotetradeca-7,11-diene), which shows high DNA cleavage activity in the presence of H₂O₂, was reported in our previous work. Considering that many systems for natural enzyme-mediated DNA cleavage contain two or more metal active sites, two new trinuclear complexes [Cu(NiL^3a)₂(dca)₂]·2CH₃OH (abbreviated as Cu(NiL^3a)₂) and [Ag(NiL^3a)₂(NO₃)]·2CH₃OH·0.5H₂O (abbreviated as Ag(NiL^3a)₂) were synthesized in this work, where dca is the dicyanamide. The complexes were structurally characterized by single crystal X-ray analysis. The central Cu(II) or Ag(I) atom is linked to two [NiL^3a] ligands by oxamido bridges forming a trinuclear structure. In Cu(NiL^3a)₂, the central Cu(II) ion is in an octahedral coordination geometry. Whereas in Ag(NiL^3a)₂, the central Ag(I) ion is in a rarely reported trigonal-prismatic coordination geometry. The DNA cleavage behavior of the complexes in the presence of H₂O₂ was studied in detail. Comparing with the NiL^3a, the trinuclear complex Ag(NiL^3a)₂ nearly has no ability to cleave DNA, whereas Cu(NiL^3a)₂ is a much better DNA cleavage agent. Cu(NiL^3a)₂ can efficiently convert supercoiled DNA to nicked DNA with a rate constant of 0.074 ± 0.002 min⁻¹ when 40 μM Cu(NiL^3a)₂ and 0.6 mM H₂O₂ are used. The cleavage mechanism between the complex Cu(NiL^3a)₂ and plasmid DNA is likely to involve singlet oxygen as reactive oxygen species. Circular dichroism (CD) and fluorescence spectroscopy indicate that both Cu(NiL^3a)₂ and NiL^3a bind to DNA by a groove binding mode, and the binding between Cu(NiL^3a)₂ and DNA is much stronger than that between NiL^3a and DNA. The present results may provide some information for the design of efficient multinuclear artificial nucleases.     

On the interaction of compounds of chromium(VI) with hydrogen peroxide. A study of chromium(VI) and (V) peroxides in the acid-basic pH range

A computational study of chromium(VI) and (V) peroxides, which exhibit important genotoxic and mutagenic activity, is reported. Energies and equilibrium geometries for [Cr^VI(O)(O₂)₂(OH)]⁻, [Cr^VI(O)(O₂)₂(OH₂)], [Cr^VI(O)(O₂)₂(py)], [Cr^VI(OH)(O₂)₂(OH₂)]⁺, [Cr^V(O)(O₂)₂(OH₂)]⁻ and species were calculated using molecular mechanics calculations (MMFF94 and MM⁺), quantum calculations with semi-empirical methods (RHF and UHF/PM3) and density functional theory (pBP86/DN* or pBP/DN* and B3LYP/6-31G(d). Equilibrium geometries for the compounds [Cr^V(O₂)₃(OH)]²⁻ and [Cr^V(O₂)₄]³⁻ were determined by molecular mechanics. Vibrational frequencies, standard thermodynamic quantities and electronic spectra were calculated using B3LYP/6-31G(d). The structural relationship between all these species and an explanation of the formation of peroxo species in the acid-basic pH range are given. An experimental study of peroxo species in basic medium was also performed (synthesis, X-ray powder diffraction patterns and infrared spectra of the peroxo complexes isolated) but did not confirm the existence of a tri-peroxo complex in the solid phase.     

Chloromethyltetramethylrosamine (Mitotracker Orange) induces the mitochondrial permeability transition and inhibits respiratory complex I. Implications for the mechanism of cytochrome c release.

We have investigated the interactions with isolated mitochondria and intact cells of chloromethyltetramethylrosamine (CMTMRos), a probe (Mitotracker Orange) that is increasingly used to monitor the mitochondrial membrane potential (Deltapsi(m)) in situ. CMTMRos binds to isolated mitochondria and undergoes a large fluorescence quenching. Most of the binding is energy-independent and can be substantially reduced by sulfhydryl reagents. A smaller fraction of the probe is able to redistribute across the inner membrane in response to a membrane potential, with further fluorescence quenching. Within minutes, however, this energy-dependent fluorescence quenching spontaneously reverts to the same level obtained by treating mitochondria with the uncoupler carbonylcyanide-p-trifluoromethoxyphenyl hydrazone. We show that this event depends on inhibition of the mitochondrial respiratory chain at complex I and on induction of the permeability transition pore by CMTMRos, with concomitant depolarization, swelling, and release of cytochrome c. After staining cells with CMTMRos, depolarization of mitochondria in situ with protonophores is accompanied by changes of CMTMRos fluorescence that range between small and undetectable, depending on the probe concentration. A lasting decrease of cellular CMTMRos fluorescence associated with mitochondria only results from treatment with thiol reagents, suggesting that CMTMRos binding to mitochondria in living cells largely occurs at SH groups via the probe chloromethyl moiety irrespective of the magnitude of Deltapsi(m). Induction of the permeability transition precludes the use of CMTMRos as a reliable probe of Deltapsi(m) in situ and demands a reassessment of the conclusion that cytochrome c release can occur without membrane depolarization and/or onset of the permeability transition.     

Expression of the cellular retinoic acid binding proteins,type II and type I,in mature rat olfactory epithelium

Cellular retinoic acid binding proteins, types I and II (CRABP I and II), are cytosolic proteins that exhibit a binding preference for all-trans retinoic acid. As part of a larger study to determine whether retinoic acid plays a role in neurogenesis in vivo, we questioned whether CRABP II is present in rat postnatal olfactory epithelium (OE), a sensory tissue that continually replaces neurons throughout adult life. We have determined that both CRABP II and CRABP I proteins and the mRNAs that encode them are present in postnatal rat OE. Immunoreactivity with CRABP II and CRABP I antibodies was not observed in the nasal respiratory epithelium. Double immunolabeling experiments, conducted with antibodies showing specificity for each antigen, indicate that CRABP II and CRABP I are found in different cell types within the olfactory neuroepithelium. We also asked whether CRABP II is expressed in the postnatal rat retina, a neural tissue that is not known to show neuron replacement during adult life. CRABP type II immunoreactivity was not observed in the mature rat retina. The presence of CRABP II in postnatal OE and its absence from mature retina is consistent with previous reports indicating that the distribution of CRABP II in adult mammals is restricted to tissue systems that exhibit ongoing growth and differentiation throughout life.