Inactivation of Rhodospirillum rubrum coupling factor by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Modification of a tyrosine protected by phosphate

Chemical modification of Rhodospirillum rubrum chromatophores by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) results in inactivation of photophosphorylation, Mg²⁺-ATPase, oxidative phosphorylation and ATP-driven transhydrogenase, with apparent first-order kinetics. Other energy-linked reactions such as light-driven transhydrogenase and light-dependent proton uptake were insensitive to NBD-Cl. The Ca²⁺-ATPase activity of the soluble coupling factor from chromatophores (R. rubrum F₁) was inactivated by NBD-Cl with kinetics resembling those described for Mg²⁺-ATPase and photophosphorylation activities of chromatophores. Both NBD-chromatophores and NBD-R. rubrum F₁ fully recovered their activities when subjected to thiolysis by dithioerythritol. Phosphoryl transfer reactions of chromatophores and Ca²⁺-ATPase activity of R. rubrum F₁ were fully protected by 5 mM P_i against modification by NBD-Cl. ADP or ATP afforded partial protection. Analysis of the protection of Ca²⁺-ATPase activity by P_i indicated that NBD-Cl and P_i are mutually exclusive ligands. Spectroscopic studies revealed that tyrosine and sulfhydryl residues in R. rubrum F₁ underwent modification by NBD-Cl. However, the inactivation was only related to the modification of tyrosine groups.     

Protein sulfenic acid formation: from cellular damage to redox regulation

Protein sulfenic acid formation has long been regarded as unwanted damage caused by reactive oxygen species (ROS). However, over the past 10 years, accumulating evidence has shown that the reversible oxidation of cysteine thiol groups to sulfenic acid functions as a redox-based signal transduction mechanism. Here, we review the mechanisms of sulfenic acid formation by ROS. We present some of the most important roles played by sulfenic acids in living cells as well as the pathways that regulate sulfenic acid formation. We highlight the experimental tools that have been developed to study the cellular sulfenome and show how computational approaches might help to better understand the mechanisms of sulfenic acid formation.     

Structural and Biochemical Characterization of Yeast Monothiol Glutaredoxin Grx6

Glutaredoxins (Grxs) are a ubiquitous family of proteins that reduce disulfide bonds in substrate proteins using electrons from reduced glutathione (GSH). The yeast Saccharomyces cerevisiae Grx6 is a monothiol Grx that is localized in the endoplasmic reticulum and Golgi compartments. Grx6 consists of three segments, a putative signal peptide (M1-I36), an N-terminal domain (K37-T110), and a C-terminal Grx domain (K111-N231, designated Grx6C). Compared to the classic dithiol glutaredoxin Grx1, Grx6 has a lower glutathione disulfide reductase activity but a higher glutathione S-transferase activity. In addition, similar to human Grx2, Grx6 binds GSH via an iron-sulfur cluster in vitro. The N-terminal domain is essential for noncovalent dimerization, but not required for either of the above activities. The crystal structure of Grx6C at 1.5 Å resolution revealed a novel two-strand antiparallel β-sheet opposite the GSH binding groove. This extra β-sheet might also exist in yeast Grx7 and in a group of putative Grxs in lower organisms, suggesting that Grx6 might represent the first member of a novel Grx subfamily.     

Association of the eukaryotic V1VO ATPase subunits a with d and d with A

Owing to the complex nature of V₁V_O ATPases, identification of neighboring subunits is essential for mechanistic understanding of this enzyme. Here, we describe the links between the V₁ headpiece and the V_O-domain of the yeast V₁V_O ATPase via subunit A and d as well as the V_O subunits a and d using surface plasmon resonance and fluorescence correlation spectroscopy. Binding constants of about 60 and 200 nM have been determined for the a-d and d-A assembly, respectively. The data are discussed in light of subunit a and d forming a peripheral stalk, connecting the catalytic A₃B₃ hexamer with V_O.

Structured summary

MINT-7012054: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by fluorescence correlation spectroscopy (MI:0052)MINT-7012041: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by surface plasmon resonance (MI:0107)MINT-7012028: d (uniprotkb:P32366) binds (MI:0407) to a (uniprotkb:P32563) by surface plasmon resonance (MI:0107)     

Catalytic properties of glutathione-binding residues in a tau class glutathione transferase (PtGSTU1) from Pinus tabulaeformis

Glutathione transferases (GSTs) play important roles in stress tolerance and detoxification in plants. However, there is extremely little information on the molecular characteristics of GSTs in gymnosperms. In a previous study, we cloned a tau class GST (PtGSTU1) from a gymnosperm (Pinus tabulaeformis) for the first time. Based on the N-terminal amino acid sequence identity to the available crystal structures of plant tau GSTs, Ser13, Lys40, Ile54, Glu66 and Ser67 of PtGSTU1 were proposed as glutathione-binding (G-site) residues. The importance of Ser13 as a G-site residue was investigated previously. The functions of Lys40, Ile54, Glu66 and Ser67 of PtGSTU1 are examined in this study through site-directed mutagenesis. Enzyme assays and thermal stability measurements on the purified recombinant PtGSTU1 showed that substitution at each of these sites significantly affects the enzyme's substrate specificity and affinity for GSH, and these residues are essential for maintaining the stability of PtGSTU1. The results of protein expression and refolding analyses suggest that Ile54 is involved in the protein folding process. The findings demonstrate that the aforementioned residues are critical components of active sites that contribute to the enzyme's catalytic activity and structural stability.     

Application of 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole in analysis: Fluorescent dyes and unexpected reaction with tertiary amines

4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is widely applied as a fluorescent tagging reagent in biochemistry, as a derivatization agent in analytical chemistry, and as a component for design of fluorescent nanoparticles. Four new 7-nitrobenzo-2-oxa-1,3-diazole (NBD)-tagged polyamines containing two to four amine moieties were synthesized and used as an effective tool for staining of siliceous frustules of the diatom algae and spicules of the siliceous sponges, including fossilized samples. An unexpected reaction between NBD-Cl and tertiary amine groups was found, giving rise to NBD-tagged amines with elimination of an alkyl group. The reaction proceeds through the Meisenheimer complex and quaternary salt, which transform to the product by Hofmann reaction (alkene elimination) or nucleophilic substitution (halogenated compound formation). In the case of polyamines, NBD-Cl causes chain scissoring, giving a set of NBD-tagged amines. The found NBD-Cl reaction with tertiary amines must be taken into account when using NBD-Cl and similar activated aromatic systems for amine derivatization in analytical and biochemistry applications. The reaction with polyamines opens the way to libraries of NBD-tagged compounds.     

Selective and reversible inhibition of the ATPase of Micrococcus denitrificans by 7-chloro-4-nitrobenzo-2-oxa-1,3 diazole

The covalent inhibitor of the beef heart mitochondrial ATPase 7-chloro-4-nitrobenzo-2-oxa-1,3 diazole inhibits the ATPase of phosphorylating particles prepared from Micrococcus denitrificans. Inhibition of both ATP synthesis and ATP hydrolysis occurs at similar rates, with a similar pH dependence, and in each case the inhibition is relieved by treatment with dithiothreitol. These results are compared with those previously obtained with the mitochondrial ATPase.     

Involvement of ATP synthase residues alphaArg-376, betaArg-182, and betaLys-155 in Pi binding

alphaArg-376, betaLys-155, and betaArg-182 are catalytically important ATP synthase residues that were proposed to be involved in substrate Pi binding and subsequent steps of ATP synthesis [Senior, A.E., Nadanaciva, S. and Weber, J. (2002) Biochim. Biophys. Acta 1553, 188-211]. Here, it was shown using purified Escherichia coli F(1)-ATPase that whereas Pi protected wild-type from reaction with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, mutations betaK155Q, betaR182Q, betaR182K, and alphaR376Q abolished protection. Therefore, in ATP synthesis initial binding of substrate Pi in open catalytic site betaE is supported by each of these three residues.