Kinetics of haem binding to human albumin and haemopexin: nonspecific interactions of haem with proteins

The kinetics of haem binding to human serum albumin and haemopexin were studied by means of the stopped flow technique. The reaction could be divided into three kinetically clearly distinguished steps: (1) extremely fast reaction of haem with nonspecific binding sites on the surface of the apoprotein molecule; this type of haem binding site seems to exist in proteins in general; (2) by meaas of equilibrium with its monomer, haem is transferred to the specific binding site; this second order reaction takes about 1–2 s, the reaction rate constant amounts to ≈10⁶ l mol^?1 s^?1 both for albumin and haemopexin: (3) conformational changes of haemoprotein molecule, accompanied by changes of absorption spectra in the Soret region; this series of slow monomolecular reactions takes about 20 min. These results are discussed in connection with the mechanism of haem transport from blood to liver cells.     

A GAF domain in the hypoxia/NO-inducible Mycobacterium tuberculosis DosS protein binds haem

The majority of the Mycobacterium tuberculosis response to hypoxia and nitric oxide is through the DosRS (DevRS) two-component regulatory system. The N-terminal input domain of the DosS sensor contains two GAF domains. We demonstrate here that the proximal GAF domain binds haem, and identified histidine 149 of DosS as critical to haem-binding; the location of this histidine residue is similar to the cGMP-binding site in a crystal structure of cyclic nucleotide phosphodiesterase 2A. GAF domains are frequently involved in binding cyclic nucleotides, but this is the first GAF domain to be identified that binds haem. In contrast, PAS domains (similar to GAF domains in structure but not primary sequence) frequently use haem cofactors, and these findings further illustrate how the functions of these domains overlap. We propose that the activation of the DosS sensor is controlled through the haem binding of molecular oxygen or nitric oxide.     

Model systems for redox cofactor activity

Numerous model studies of organic redox cofactor activity have appeared in the latter half of 1998 and the first half of 1999. These investigations include the use of solution models to explore flavin-dependent, quinone-dependent and pyrroloquinone-dependent redox processes, the exploration of flavin and quinone redox events using organized interfaces, and the application of computational methods to increase the understanding of flavin-catalyzed, nicotinamide-catalyzed and quinone-catalyzed redox processes.     

A simple method to engineer a protein-derived redox cofactor for catalysis

The 6 ×-Histidine tag which is commonly used for purification of recombinant proteins was converted to a catalytic redox-active center by incorporation of Co^2 +. Two examples of the biological activity of this engineered protein-derived cofactor are presented. After inactivation of the natural diheme cofactor of MauG, it was shown that the Co^2 +-loaded 6 × His-tag could substitute for the hemes in the H₂O₂-driven catalysis of tryptophan tryptophylquinone biosynthesis. To further demonstrate that the Co^2 +-loaded 6 × His-tag could mediate long range electron transfer, it was shown that addition of H₂O₂ to the Co^2 +-loaded 6 × His-tagged Cu^1 + amicyanin oxidizes the copper site which is 20 Å away. These results provide proof of principle for this simple method by which to introduce a catalytic redox-active site into proteins for potential applications in research and biotechnology.     

A redox trap to augment the intein toolbox

The unregulated activity of inteins during expression and consequent side reactions during work‐up limits their widespread use in biotechnology and chemical biology. Therefore, we exploited a mechanism‐based approach to regulate intein autocatalysis for biotechnological application. The system, inspired by our previous structural studies, is based on reversible trapping of the intein's catalytic cysteine residue through a disulfide bond. Using standard mutagenesis, the disulfide trap can be implemented to impart redox control over different inteins and for a variety of applications both in vitro and in Escherichia coli. Thereby, we first enhanced the output for bioconjugation in intein‐mediated protein ligation, also referred to as expressed protein ligation, where precursor recovery and product yield were augmented fourfold to sixfold. Second, in bioseparation experiments, the redox trap boosted precursor recovery and product yield twofold. Finally, the disulfide‐trap intein technology stimulated development of a novel bacterial redox sensor. This sensor reliably identified hyperoxic E. coli harboring mutations that disrupt the reductive pathways for thioredoxin and glutathione, against a background of wild‐type cells. Biotechnol. Bioeng. 2013; 110: 1565–1573. © 2012 Wiley Periodicals, Inc.     

Cytotoxic and cytoprotective effects of tryptamine-4,5-dione on neuronal cells: a double-edged sword

Serotonin (5-hydroxytryptamine) is a putative substrate for myeloperoxidase, which may convert it into the reactive quinone tryptamine-4,5-dione (TD). In this study, we found that the viability of human SH-SY5Y neuroblastoma cells treated with 25?μM TD was increased to approximately 117%. On the other hand, the cell viability was significantly decreased by exposure to TD (150–200?μM), with an increase in intracellular reactive oxygen species (ROS). Interestingly, pre-treatment of SH-SY5Y cells with 100?μM TD prevented cell death and suppressed intracellular ROS generation evoked by the addition of hydrogen peroxide (H₂O₂). Expression of the phase-II antioxidant enzyme NAD(P)H: quinone oxidoreductase 1 and haem oxygenase 1 were upregulated by TD at a concentration of 50–100?μM. Nuclear factor erythroid 2-related factor 2 (Nrf2), the regulator of these enzyme, was translocated from the cytosol to the nucleus by 100?μM TD. In summary, moderate concentrations of TD may increase the self-defence capacity of neuronal cells against oxidative stress.     

Resonance Raman quantification of the redox state of cytochromes b and c in‐vivo and in‐vitro

We observe the redox state changes with respiration of cytochromes b and c in mitochondria in a living Saccharomyces cerevisiae cell as well as in isolated mitochondria with the very use of Raman microspectroscopy. The possibility of monitoring the respiration activity of mitochondria in vivo and in vitro by Raman microspectroscopic quantification of the cytochrome redox states is suggested. It will lead to a new means to assess mitochondrial respiration activity in vivo and in vitro without using any labelling or genetic manipulation. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Thiol/disulfide redox states in signaling and sensing

Abstract

Rapid advances in redox systems biology are creating new opportunities to understand complexities of human disease and contributions of environmental exposures. New understanding of thiol–disulfide systems have occurred during the past decade as a consequence of the discoveries that thiol and disulfide systems are maintained in kinetically controlled steady states displaced from thermodynamic equilibrium, that a widely distributed family of NADPH oxidases produces oxidants that function in cell signaling and that a family of peroxiredoxins utilize thioredoxin as a reductant to complement the well-studied glutathione antioxidant system for peroxide elimination and redox regulation. This review focuses on thiol/disulfide redox state in biologic systems and the knowledge base available to support development of integrated redox systems biology models to better understand the function and dysfunction of thiol–disulfide redox systems. In particular, central principles have emerged concerning redox compartmentalization and utility of thiol/disulfide redox measures as indicators of physiologic function. Advances in redox proteomics show that, in addition to functioning in protein active sites and cell signaling, cysteine residues also serve as redox sensors to integrate biologic functions. These advances provide a framework for translation of redox systems biology concepts to practical use in understanding and treating human disease. Biological responses to cadmium, a widespread environmental agent, are used to illustrate the utility of these advances to the understanding of complex pleiotropic toxicities.     

Quantitative analysis of the redox states of cytochromes in a living L929 (NCTC) cell by resonance Raman microspectroscopy

Raman spectra and images of a living L929 (NCTC) cell have been measured with 532 nm excitation. Both reduced and oxidized forms of cytochromes b and c (cyt b and cyt c) have been observed in situ without any pretreatment. The redox states of cyts b and c have been assessed quantitatively with a spectral analysis. It has been found that reduced cyt c is more abundant than oxidized cyt c, while oxidized cyt b is slightly more abundant than reduced cyt b in a living cell. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Design of a cytochrome P450BM3 reaction system linked by two‐step cofactor regeneration catalyzed by a soluble transhydrogenase and glycerol dehydrogenase

A cytochrome P450BM3‐catalyzed reaction system linked by a two‐step cofactor regeneration was investigated in a cell‐free system. The two‐step cofactor regeneration of redox cofactors, NADH and NADPH, was constructed by NAD⁺‐dependent bacterial glycerol dehydrogenase (GLD) and bacterial soluble transhydrogenase (STH) both from Escherichia coli. In the present system, the reduced cofactor (NADH) was regenerated by GLD from the oxidized cofactor (NAD⁺) using glycerol as a sacrificial cosubstrate. The reducing equivalents were subsequently transferred to NADP⁺ by STH as a cycling catalyst. The resultant regenerated NADPH was used for the substrate oxidation catalyzed by cytochrome P450BM3. The initial rate of the P450BM3‐catalyzed reaction linked by the two‐step cofactor regeneration showed a slight increase (approximately twice) when increasing the GLD units 10‐fold under initial reaction conditions. In contrast, a 10‐fold increase in STH units resulted in about a 9‐fold increase in the initial reaction rate, implying that transhydrogenation catalyzed by STH was the rate‐determining step. In the system lacking the two‐step cofactor regeneration, 34% conversion of 50 μM of a model substrate (p‐nitrophenoxydecanoic acid) was attained using 50 μM NADPH. In contrast, with the two‐step cofactor regeneration, the same amount of substrate was completely converted using 5 μM of oxidized cofactors (NAD⁺ and NADP⁺) within 1 h. Furthermore, a 10‐fold dilution of the oxidized cofactors still led to approximately 20% conversion in 1 h. These results indicate the potential of the combination of GLD and STH for use in redox cofactor recycling with catalytic quantities of NAD⁺ and NADP⁺. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

A Cajal body-independent pathway for telomerase trafficking in mice

The intranuclear trafficking of human telomerase involves a dynamic interplay between multiple nuclear sites, most notably Cajal bodies and telomeres. Cajal bodies are proposed to serve as sites of telomerase maturation, storage, and assembly, as well as to function in the cell cycle-regulated delivery of telomerase to telomeres in human cells. Here, we find that telomerase RNA does not localize to Cajal bodies in mouse cells, and instead resides in separate nuclear foci throughout much of the cell cycle. However, as in humans, mouse telomerase RNA (mTR) localizes to subsets of telomeres specifically during S phase. The localization of mTR to telomeres in mouse cells does not require coilin-containing Cajal bodies, as mTR is found at telomeres at similar frequencies in cells from wild-type and coilin knockout mice. At the same time, we find that human TR localizes to Cajal bodies (as well as telomeres) in mouse cells, indicating that the distinct trafficking of mTR is attributable to an intrinsic property of the RNA (rather than a difference in the mouse cell environment such as the properties of mouse Cajal bodies). We also find that during S phase, mTR foci coalesce into short chains, with at least one of the conjoined mTR foci co-localizing with a telomere. These findings point to a novel, Cajal body-independent pathway for telomerase biogenesis and trafficking in mice.     

A ba3 oxygen reductase from the thermohalophilic bacterium Rhodothermus marinus

The aerobic respiratory chain of the thermohalophilic bacterium Rhodothermus marinus has been extensively studied. In this study the isolation and characterization of a third oxygen reductase expressed in this organism are described. This newly isolated enzyme is a typical member of the type B family of haem-copper oxygen reductases, showing 43% amino acid sequence identity and 63% similarity with the ba3 oxygen reductase from Thermus thermophilus. It constitutes two subunits with apparent molecular masses of 42 and 38 kDa. It contains a low-spin B-type haem and a high-spin A-type haem. A stoichiometry of 1B: 1A haem per protein was obtained by spectral integration of UV-visible spectra. Metal analysis showed the presence of two iron and three copper ions, which is in agreement with the existence of a CuA centre. Taking advantage of having two spectroscopically distinct haems, the redox behaviour of the ba3 oxygen reductase was analysed and discussed in the framework of a model with interacting centres. Both haems, B and A, present two transitions, have unusually low reduction potentials of -65 mV and an interaction potential of -52.5 mV.     

A conserved archaeal pathway for tail-anchored membrane protein insertion

Eukaryotic tail‐anchored (TA) membrane proteins are inserted into the endoplasmic reticulum by a post‐translational TRC40 pathway, but no comparable pathway is known in other domains of life. The crystal structure of an archaebacterial TRC40 sequence homolog bound to ADP?AlF₄^? reveals characteristic features of eukaryotic TRC40, including a zinc‐mediated dimer and a large hydrophobic groove. Moreover, archaeal TRC40 interacts with the transmembrane domain of TA substrates and directs their membrane insertion. Thus, the TRC40 pathway is more broadly conserved than previously recognized.     

Measurement and meaning of cellular thiol:disufhide redox status

The functional group of cysteine is a thiol group (SH) that, due to its chemical reactivity, is able to undergo a wide array of modifications each with the potential to confer a different property or function to the molecule harboring this residue. Most of these modifications involve the reversible oxidation of the thiol to sulfenic acid (SOH), and disulfide, including intra- and intermolecular disulfides between polypeptides and glutathione (glutathionylation). The reversibility of these oxidations allows thiol groups to serve as versatile chemical and structural transducing elements in several low molecular mass metabolites and proteins. A plethora of cellular functions such as DNA and protein synthesis, protein secretion, cytoskeleton architecture, differentiation, apoptosis, and anti-oxidant defense, are recognized to be modulated, at certain stage, by thiol–disulfide exchange mechanisms of redox active thiol groups. All organisms are equipped with enzymatic systems composed by NADPH-dependent reductases, redoxins, and peroxidases that provide kinetic control of global thiol-redox homeostasis as well as target selectivity. These redox systems are distributed in different subcellular compartments and are not in equilibrium with each other. In consequence, measuring cellular thiol–disulfide status represents a challenge for studies aimed to obtain dynamic and spatio-temporal resolution. This review provides a summary of the methods and tools available to quantify the thiol redox status of cells.     

Microsomal redox systems in brown adipose tissue: high lipid peroxidation,low cholesterol biosynthesis and no detectable cytochrome P-450

The presence of redox systems in microsomes of brown adipose tissue (BAT) in cold exposed rats was investigated and compared with liver. BAT microsomes showed high activity of lipid peroxidation measured both by the formation of malondialdehyde (MDA) and by oxygen uptake. NADH and NADPH dependent cytochrome c reductase activity were present in both BAT and liver microsomes. Aminopyrine demethylase and aniline hydroxylase activities, the characteristic detoxification enzymes in liver microsomes could not be detected in BAT microsomes. BAT minces showed very poor incorporation of [1-¹⁴C]acetate and [2-¹⁴C]-mevalonate in unsaponifiable lipid fraction compared to liver. Biosynthesis of cholesterol and ubiquinone, but not fatty acids, and the activity of 3-hydroxy-3-methyl glutaryl CoA reductase appear to be very low in BAT. Examination of difference spectra showed the presence of only cytochrome b ₅ in BAT microsomes. In addition to the inability to detect the enzyme activities dependent on cytochrome P-450, a protein with the characteristic spectrum, molecular size in SDS-PAGE and interaction with antibodies in double diffusion test, also could not be detected in BAT microsomes. The high activity of lipid peroxidation in microsomes, being associated with large oxygen uptake and oxidation of NADPH, will also contribute to the energy dissipation as heat in BAT, considered important in thermogenesis.Abbreviations BAT Brown Adipose Tissue - MDA malondialdehyde     

RALES, EPHESUS and redox

In RALES, low doses of the mineralocorticoid receptor (MR) antagonist spironolactone, added to standard of care for severe heart failure, improved survival by 30% and lowered hospitalization by 35%. Animal studies with the selective MR antagonist eplerenone have similarly shown MR blockade to prevent the cerebral, renal and coronary vascular inflammatory response to elevated aldosterone levels. There is now general acceptance that aldosterone concentrations inappropriate for salt status have major deleterious effects on the cardiovascular system.

In many instances, however (e.g. Randomized Aldactone Evaluation Study (RALES), EPHESUS) aldosterone levels are normal and salt status unremarkable and yet MR blockade has unquestioned benefits. In these instances, there is increasing evidence that coronary and cardiac MR are activated by normal circulating cortisol levels, in the cellular context of generation of reactive oxygen species (ROS) and/or alteration in intracellular redox status.

MR in VSMC and cardiomyocytes are normally predominantly occupied by cortisol in tonic inhibitory mode. Blockade of 11β hydroxysteroid dehydrogenase type II (11βHSD2) or ROS generation both serve to activate cortisol–MR complexes, thus mimicking the effects of mineralocorticoid/salt imbalance on blood vessels and the heart. In RALES and EPHESUS, it is likely that the antagonists are blocking normal levels of cortisol, not aldosterone, from activating MR in the context of tissue damage and ROS generation. If this is the case, MR antagonists may be of wide therapeutic potential in cardiovascular disease and not confined to those characterized by aldosterone/salt excess. Finally, the pathophysiologic roles of always-occupied MR in ‘unprotected’ tissues such as cardiomyocytes or neurons in response to altered intracellular redox status remain to be explored.     

Phosphoinositide regulation of integrin trafficking required for muscle attachment and maintenance

Muscles must maintain cell compartmentalization when remodeled during development and use. How spatially restricted adhesions are regulated with muscle remodeling is largely unexplored. We show that the myotubularin (mtm) phosphoinositide phosphatase is required for integrin-mediated myofiber attachments in Drosophila melanogaster, and that mtm-depleted myofibers exhibit hallmarks of human XLMTM myopathy. Depletion of mtm leads to increased integrin turnover at the sarcolemma and an accumulation of integrin with PI(3)P on endosomal-related membrane inclusions, indicating a role for Mtm phosphatase activity in endocytic trafficking. The depletion of Class II, but not Class III, PI3-kinase rescued mtm-dependent defects, identifying an important pathway that regulates integrin recycling. Importantly, similar integrin localization defects found in human XLMTM myofibers signify conserved MTM1 function in muscle membrane trafficking. Our results indicate that regulation of distinct phosphoinositide pools plays a central role in maintaining cell compartmentalization and attachments during muscle remodeling, and they suggest involvement of Class II PI3-kinase in MTM-related disease.