A Quinol Oxidase, Encoded by \u3cem\u3ecyoABCD\u3c/em\u3e, Is Utilized to Adapt to Lower O\u3csub\u3e2\u3c/sub\u3e Concentrations in \u3cem\u3eRhizobium etli\u3c/em\u3e CFN42

Bacteria have branched aerobic respiratory chains that terminate at different terminal oxidases. These terminal oxidases have varying properties such as their affinity for oxygen, transcriptional regulation and proton pumping ability. The focus of this study was a quinol oxidase encoded by cyoABCD. Although this oxidase (Cyo) is widespread among bacteria, not much is known about its role in the cell, particularly in bacteria that contain both cytochrome c oxidases and quinol oxidases. Using Rhizobium etli CFN42 as a model organism, a cyo mutant was analysed for its ability to grow in batch cultures at high (21 % O2) and low (1 and 0.1 % O2) ambient oxygen concentrations. In comparison with other oxidase mutants, the cyo mutant had a significantly longer lag phase under low-oxygen conditions. Using a cyo :: lacZ transcriptional fusion, it was shown that cyo expression in the wild type peaks between 1 and 2.5 % O2. In addition, it was shown with quantitative reverse transcriptase PCR that cyoB is upregulated approximately fivefold in 1 % O2 compared with fully aerobic (21 % O2) conditions. Analysis of the cyo mutant during symbiosis with Phaseolous vulgaris indicated that Cyo is utilized during early development of the symbiosis. Although it is commonly thought that Cyo is utilized only at higher oxygen concentrations, the results from this study indicate that Cyo is important for adaptation to and sustained growth under low oxygen

Oxygen Activation by Mononuclear Mn, Co, and Ni Centers in Biology and Synthetic Complexes

The active sites of metalloenzymes that catalyze O2-dependent reactions generally contain iron or copper ions. However, several enzymes are capable of activating O2 at manganese or nickel centers instead, and a handful of dioxygenases exhibit activity when substituted with cobalt. This minireview summarizes the catalytic properties of oxygenases and oxidases with mononuclear Mn, Co, or Ni active sites, including oxalate-degrading oxidases, catechol dioxygenases, and quercetin dioxygenase. In addition, recent developments in the O2 reactivity of synthetic Mn, Co, or Ni complexes are described, with an emphasis on the nature of reactive intermediates featuring superoxo-, peroxo-, or oxo-ligands. Collectively, the biochemical and synthetic studies discussed herein reveal the possibilities and limitations of O2 activation at these three “overlooked” metals

The Arabidopsis NADPH oxidases RbohD and RbohF display differential expression patterns and contributions during plant immunity

Plant NADPH oxidases, also known as respiratory burst oxidase homologues (RBOHs), produce reactive oxygen species (ROS) that perform a wide range of functions. RbohD and RbohF, two of the 10 Rboh genes present in Arabidopsis, are pleiotropic and mediate diverse physiological processes including the response to pathogens. We hypothesized that the spatio-temporal control of RbohD and RbohF gene expression might be critical in determining their multiplicity of functions. Transgenic Arabidopsis plants with RbohD and RbohF promoter fusions to β-glucuronidase and Luciferase reporter genes were generated. Analysis of these plants revealed a differential expression pattern for RbohD and RbohF throughout plant development and during immune responses. RbohD and RbohF gene expression was differentially modulated by pathogen-associated molecular patterns. Histochemical stains and in vivo expression analysis showed a correlation between the level of RbohD and RbohF promoter activity, H2O2 accumulation and the amount of cell death in response to the pathogenic bacterium Pseudomonas syringae pv. tomato DC3000 and the necrotrophic fungus Plectosphaerella cucumerina. A promoter-swap strategy revealed that the promoter region of RbohD was required to drive production of ROS by this gene in response to pathogens. Moreover, RbohD promoter was activated during Arabidopsis interaction with a non-virulent P. cucumerina isolate, and susceptibility tests with the double mutant rbohD rbohF uncovered a new function for these oxidases in basal resistance. Altogether, our results suggest that differential spatio-temporal expression of the Rboh genes contributes to fine-tune RBOH/NADPH oxidase-dependent ROS production and signaling in Arabidopsis immunity

Mechanisms of oxidative stress in human aortic aneurysms — association with clinical risk factors for atherosclerosis and disease severity

Aortic abdominal aneurysms (AAA) are important causes of cardiovascular morbidity and mortality. Oxidative stress may link multiple mechanisms of AAA including vascular inflammation and increased metalloproteinase activity. However, the mechanisms of vascular free radical production remain unknown. Accordingly, we aimed to determine sources and molecular regulation of vascular superoxide (O2radical dot−) production in human AAA. Methods and results: AAA segments and matched non-dilated aortic samples were obtained from 40 subjects undergoing AAA repair. MDA levels (determined by HPLC/MS) were greater in plasma of AAA subjects (n = 16) than in risk factor matched controls (n = 16). Similarly, superoxide production, measured by lucigenin chemiluminescence and dihydroethidium fluorescence, was increased in aneurysmatic segments compared to non-dilated aortic specimens. NADPH oxidases and iNOS are the primary sources of O2radical dot− in AAA. Xanthine oxidase, mitochondrial oxidases and cyclooxygenase inhibition had minor or no effect. Protein kinase C inhibition had no effect on superoxide production in AAA. NADPH oxidase subunit mRNA levels for p22phox, nox2 and nox5 were significantly increased in AAAs while nox4 mRNA expression was lower. Superoxide production was higher in subjects with increased AAA repair risk Vanzetto score and was significantly associated with smoking, hypercholesterolemia and presence of CAD in AAA cohort. Basal superoxide production and NADPH oxidase activity were correlated to aneurysm size. Conclusions: Increased expression and activity of NADPH oxidases are important mechanisms underlying oxidative stress in human aortic abdominal aneurysm. Uncoupled iNOS may link oxidative stress to inflammation in AAA. Oxidative stress is related to aneurysm size and major clinical risk factors in AAA patients

Quinol Oxidase Encoded by \u3cem\u3ecyoABCD\u3c/em\u3e in \u3cem\u3eRhizobium etli\u3c/em\u3e CFN42 is Regulated by ActSR and is Crucial for Growth at Low pH or Low Iron Conditions

Rhizobium etli aerobically respires with several terminal oxidases. The quinol oxidase (Cyo) encoded by cyoABCD is needed for efficient adaptation to low oxygen conditions and cyo transcription is upregulated at low oxygen. This study sought to determine how transcription of the cyo operon is regulated. The 5′ sequence upstream of cyo was analysed in silico and revealed putative binding sites for ActR of the ActSR two-component regulatory system. The expression of cyo was decreased in an actSR mutant regardless of the oxygen condition. As ActSR is known to be important for growth under low pH in another rhizobial species, the effect of growth medium pH on cyo expression was tested. As the pH of the media was incrementally decreased, cyo expression gradually increased in the WT, eventually reaching ∼10-fold higher levels at low pH (4.8) compared with neutral pH (7.0) conditions. This upregulation of cyo under decreasing pH conditions was eliminated in the actSR mutant. Both the actSR and cyo mutants had severe growth defects at low pH (4.8). Lastly, the actSR and cyo mutants had severe growth defects when grown in media treated with an iron chelator. Under these conditions, cyo was upregulated in the WT, whereas cyo was not induced in the actSR mutant. Altogether, the results indicated cyo expression is largely dependent on the ActSR two-component system. This study also demonstrated additional physiological roles for Cyo in R. etli CFN42, in which it is the preferred oxidase for growth under acidic and low iron conditions

A broad distribution of the alternative oxidase in microsporidian parasites

Microsporidia are a group of obligate intracellular parasitic eukaryotes that were considered to be amitochondriate until the recent discovery of highly reduced mitochondrial organelles called mitosomes. Analysis of the complete genome of Encephalitozoon cuniculi revealed a highly reduced set of proteins in the organelle, mostly related to the assembly of ironsulphur clusters. Oxidative phosphorylation and the Krebs cycle proteins were absent, in keeping with the notion that the microsporidia and their mitosomes are anaerobic, as is the case for other mitosome bearing eukaryotes, such as Giardia. Here we provide evidence opening the possibility that mitosomes in a number of microsporidian lineages are not completely anaerobic. Specifically, we have identified and characterized a gene encoding the alternative oxidase (AOX), a typically mitochondrial terminal oxidase in eukaryotes, in the genomes of several distantly related microsporidian species, even though this gene is absent from the complete genome of E. cuniculi. In order to confirm that these genes encode functional proteins, AOX genes from both A. locustae and T. hominis were over-expressed in E. coli and AOX activity measured spectrophotometrically using ubiquinol-1 (UQ-1) as substrate. Both A. locustae and T. hominis AOX proteins reduced UQ-1 in a cyanide and antimycin-resistant manner that was sensitive to ascofuranone, a potent inhibitor of the trypanosomal AOX. The physiological role of AOX microsporidia may be to reoxidise reducing equivalents produced by glycolysis, in a manner comparable to that observed in trypanosome

Redox signaling, Nox5 and vascular remodeling in hypertension

Purpose of review: Extensive data indicate a role for reactive oxygen species (ROS) and redox signaling in vascular damage in hypertension. However, molecular mechanisms underlying these processes remain unclear, but oxidative post-translational modification of vascular proteins is critical. This review discusses how proteins are oxidatively modified and how redox signaling influences vascular smooth muscle cell growth and vascular remodeling in hypertension. We also highlight Nox5 as a novel vascular ROS-generating oxidase. Recent findings: Oxidative stress in hypertension leads to oxidative imbalance that affects vascular cell function through redox signaling. Many Nox isoforms produce ROS in the vascular wall, and recent findings show that Nox5 may be important in humans. ROS regulate signaling by numerous processes including cysteine oxidative post-translational modification such as S-nitrosylation, S-glutathionylation and sulfydration. In vascular smooth muscle cells, this influences cellular responses to oxidative stimuli promoting changes from a contractile to a proliferative phenotype. Summary: In hypertension, Nox-induced ROS production is increased, leading to perturbed redox signaling through oxidative modifications of vascular proteins. This influences mitogenic signaling and cell cycle regulation, leading to altered cell growth and vascular remodeling in hypertension

Molecular biology of amitraz resistance in cattle ticks of the genus Rhipicephalus

Amitraz is an important product for the control of cattle ticks around the world. In comparison with other products for the control of ticks, it is quite affordable and it has a rapid knock-down effect. It binds with and activates adrenergic neuro-receptors of animals and it inhibits the action of monoamine oxidases (MAO). Resistance to amitraz has been documented in Rhipicephalus microplus, R. decoloratus and R. appendiculatus. Four mechanisms of resistance have been proposed, each of which is supported by evidence but none of which has been definitively confirmed as the cause of resistance in the field. The proposed mechanisms include genetic target site insensitivity in two G protein-coupled receptors, the beta-adrenergic octopamine receptor (BAOR) and the octopamine/tyramine receptor (OCT/Tyr), increased expression or activity of monoamine oxidases and increased expression or activity of the ATP binding cassette transporter

The relationship between redox enzyme activity and electrochemical potential—cellular and mechanistic implications from protein film electrochemistry

In protein film electrochemistry a redox protein of interest is studied as an electroactive film adsorbed on an electrode surface. For redox enzymes this configuration allows quantification of the relationship between catalytic activity and electrochemical potential. Considered as a function of enzyme environment, i.e., pH, substrate concentration etc., the activity–potential relationship provides a fingerprint of activity unique to a given enzyme. Here we consider the nature of the activity–potential relationship in terms of both its cellular impact and its origin in the structure and catalytic mechanism of the enzyme. We propose that the activity–potential relationship of a redox enzyme is tuned to facilitate cellular function and highlight opportunities to test this hypothesis through computational, structural, biochemical and cellular studies