The Quantum Biology of Reactive Oxygen Species Partitioning Impacts Cellular Bioenergetics.

Quantum biology is the study of quantum effects on biochemical mechanisms and biological function. We show that the biological production of reactive oxygen species (ROS) in live cells can be influenced by coherent electron spin dynamics, providing a new example of quantum biology in cellular regulation. ROS partitioning appears to be mediated during the activation of molecular oxygen (O2) by reduced flavoenzymes, forming spin-correlated radical pairs (RPs). We find that oscillating magnetic fields at Zeeman resonance alter relative yields of cellular superoxide (O2•-) and hydrogen peroxide (H2O2) ROS products, indicating coherent singlet-triplet mixing at the point of ROS formation. Furthermore, the orientation-dependence of magnetic stimulation, which leads to specific changes in ROS levels, increases either mitochondrial respiration and glycolysis rates. Our results reveal quantum effects in live cell cultures that bridge atomic and cellular levels by connecting ROS partitioning to cellular bioenergetics.

Inhibition of cellular proliferation and enhancement of hydrogen peroxide production in fibrosarcoma cell line by weak radio frequency magnetic fields.

This study presents experimental data for the effects of weak radio frequency (RF) magnetic fields on hydrogen peroxide (H2O2) production and cellular growth rates of fibrosarcoma HT1080 cells in vitro. Cells were exposed either to 45 µT static magnetic fields (SMFs)-oriented vertical to the plane of growth or to SMFs combined with weak 5 and 10 MHz RF magnetic fields of 10 µTRMS intensity perpendicular to the static field. Cell numbers were reduced up to 30% on Day 2 for the cells exposed to the combination of SMF and a 10 MHz RF magnetic field compared with the SMF control cells. In addition, cells exposed to 10 MHz RF magnetic fields for 8 h increased H2O2 production by 55%. The results demonstrate an overall magnetic field-induced biological effect that shows elevated H2O2 levels with accompanying decrease in cellular growth rates.

Modulation of hydrogen peroxide production in cellular systems by low level magnetic fields.

Increased generation of reactive oxygen species (ROS) and an altered redox status have long been observed in cancer cells, suggesting that ROS might be involved in the development of these cells. However, recent studies suggest that inducing an excess of ROS in cancer cells can be exploited for therapeutic benefits. Cancer cells in advanced stage tumors frequently exhibit multiple genetic alterations and high oxidative stress, suggesting that it might be possible to preferentially modulate the development of these cells by controlling their ROS production. Low levels of ROS are also important for the development and survival of normal cells. In this manuscript, we present data on the influence of the suppression of the Earth's magnetic field (low level magnetic fields or LLF) which magnitudes range from 0.2 µT to 2 µT on the modulation of hydrogen peroxide (H(2)O(2)) in human fibrosarcoma cancer cell line HT1080, pancreatic AsPC-1 cancer cell line, and bovine pulmonary artery endothelial cells (PAEC) exposed to geomagnetic field (control; 45 µT-60 µT). Reduction of the Earth's magnetic field suppressed H(2)O(2) production in cancer cells and PAEC. The addition of catalase and superoxide dismutase (SOD) mimetic MnTBAP inhibited the magnetic field effect. Modulating ROS production by magnetic fields may open new venues of biomedical research and therapeutic strategies.

Identification of potential calorie restriction-mimicking yeast mutants with increased mitochondrial respiratory chain and nitric oxide levels.

Calorie restriction (CR) induces a metabolic shift towards mitochondrial respiration; however, molecular mechanisms underlying CR remain unclear. Recent studies suggest that CR-induced mitochondrial activity is associated with nitric oxide (NO) production. To understand the role of mitochondria in CR, we identify and study Saccharomyces cerevisiae mutants with increased NO levels as potential CR mimics. Analysis of the top 17 mutants demonstrates a correlation between increased NO, mitochondrial respiration, and longevity. Interestingly, treating yeast with NO donors such as GSNO (S-nitrosoglutathione) is sufficient to partially mimic CR to extend lifespan. CR-increased NO is largely dependent on mitochondrial electron transport and cytochrome c oxidase (COX). Although COX normally produces NO under hypoxic conditions, CR-treated yeast cells are able to produce NO under normoxic conditions. Our results suggest that CR may derepress some hypoxic genes for mitochondrial proteins that function to promote the production of NO and the extension of lifespan.

Low intensity light stimulates nitrite-dependent nitric oxide synthesis but not oxygen consumption by cytochrome c oxidase: Implications for phototherapy.

Cytochrome c oxidase (Cco) has been reported to be a receptor for some of the beneficial effects of low intensity visible and near-infrared light on cells and tissues. Here, we have explored the role of low intensity light in affecting a newly described function of Cco, its ability to catalyze nitrite-dependent nitric oxide (NO) synthesis (Cco/NO). Using a new assay for Cco/NO we have found that both yeast and mouse brain mitochondrial Cco produce NO over a wide range of oxygen concentrations and that the rate of NO synthesis increases as the oxygen concentration decreases, becoming optimal under hypoxic conditions. Low intensity broad-spectrum light increases Cco/NO activity in an intensity-dependent fashion but has no effect on oxygen consumption by Cco. By using a series of bandpass filters and light emitting devices (LEDs) we have determined that maximal stimulation of Cco/NO activity is achieved by exposure to light whose central wavelength is 590 ± 14 nm. This wavelength of light stimulates Cco/NO synthesis at physiological nitrite concentrations. These findings raise the interesting possibility that low intensity light exerts a beneficial effect on cells and tissues by increasing NO synthesis catalyzed by Cco and offer a new explanation for the increase in NO bioavailability experienced by tissue exposed to light.

Ice-induced partial unfolding and aggregation of an integral membrane protein.

Although the deleterious effects of ice on water-soluble proteins are well established, little is known about the freeze stability of membrane proteins. Here we explore this issue through a combined kinetic and spectroscopic approach using micellar-purified plasma membrane calcium pump as a model. The ATPase activity of this protein significantly diminished after freezing using a slow-cooling procedure, with the decrease in the activity being an exponential function of the storage time at 253K, with t(½)=3.9±0.6h. On the contrary, no significant changes on enzyme activity were detected when a fast cooling procedure was performed. Regardless of the cooling rate, successive freeze-thaw cycles produced an exponential decrease in the Ca(2+)-ATPase activity, with the number of cycles at which the activity was reduced to half being 9.2±0.3 (fast cooling) and 3.7±0.2 (slow cooling). PAGE analysis showed that neither degradation nor formation of SDS-stable aggregates of the protein takes place during protein inactivation. Instead, the inactivation process was found to be associated with the irreversible partial unfolding of the polypeptide chain, as assessed by Trp fluorescence, far UV circular dichroism, and 1-anilino-naphtalene-8-sulfonate binding. This inactive protein undergoes, in a later stage, a further irreversible transformation leading to large aggregates.

Mitochondria and hypoxic signaling: a new view.

Eukaryotic cells respond to low oxygen concentrations by upregulating hypoxic and downregulating aerobic nuclear genes (hypoxic signaling). Most of the oxygen-regulated genes in yeast require the mitochondrial respiratory chain for their up- or downregulation when cells experience hypoxia. Although it was shown previously that the mitochondrial respiratory chain is required for the upregulation of some hypoxic genes in both yeast and mammalian cells, its underlying role in this process has been unclear. Recently, we have reported that mitochondria produce nitric oxide (NO(*)) when oxygen becomes limiting. This NO(*) production is nitrite (NO(2) (-))-dependent, requires an electron donor, and is carried out by cytochrome c oxidase in a pH-dependent fashion. We call this activity Cco/NO(*) and incorporate it into a new model for hypoxic signaling. In addition, we have found that some of the NO(*) produced by Cco/NO(*) is released from cells, raising the possibility that mitochondrially generated NO(*) also functions in extracellular hypoxic signaling pathways.

Mitochondrial generation of free radicals and hypoxic signaling.

Most reactive oxygen species (ROS) are generated in cells by the mitochondrial respiratory chain. Mitochondrial ROS production is modulated largely by the rate of electron flow through respiratory chain complexes. Recently, it has become clear that under hypoxic conditions, the mitochondrial respiratory chain also produces nitric oxide (NO), which can generate other reactive nitrogen species (RNS). Although excess ROS and RNS can lead to oxidative and nitrosative stress, moderate to low levels of both function in cellular signaling pathways. Especially important are the roles of these mitochondrially generated free radicals in hypoxic signaling pathways, which have important implications for cancer, inflammation and a variety of other diseases.

Effects of phosphatidylethanolamine glycation on lipid-protein interactions and membrane protein thermal stability.

Non-enzymatic glycation of biomolecules has been implicated in the pathophysiology of aging and diabetes. Among the potential targets for glycation are biological membranes, characterized by a complex organization of lipids and proteins interacting and forming domains of different size and stability. In the present study, we analyse the effects of glycation on the interactions between membrane proteins and lipids. The phospholipid affinity for the transmembrane surface of the PMCA (plasma-membrane Ca(2+)-ATPase) was determined after incubating the protein or the phospholipids with glucose. Results show that the affinity between PMCA and the surrounding phospholipids decreases significantly after phosphospholipid glycation, but remains unmodified after glycation of the protein. Furthermore, phosphatidylethanolamine glycation decreases by approximately 30% the stability of PMCA against thermal denaturation, suggesting that glycated aminophospholipids induce a structural rearrangement in the protein that makes it more sensitive to thermal unfolding. We also verified that lipid glycation decreases the affinity of lipids for two other membrane proteins, suggesting that this effect might be common to membrane proteins. Extending these results to the in vivo situation, we can hypothesize that, under hyperglycaemic conditions, glycation of membrane lipids may cause a significant change in the structure and stability of membrane proteins, which may affect the normal functioning of membranes and therefore of cells.

Oxygen-regulated isoforms of cytochrome c oxidase have differential effects on its nitric oxide production and on hypoxic signaling.

Recently, it has been reported that mitochondria possess a novel pathway for nitric oxide (NO) synthesis. This pathway is induced when cells experience hypoxia, is nitrite (NO(2)(-))-dependent, is independent of NO synthases, and is catalyzed by cytochrome c oxidase (Cco). It has been proposed that this mitochondrially produced NO is a component of hypoxic signaling and the induction of nuclear hypoxic genes. In this study, we examine the NO(2)(-)-dependent NO production in yeast engineered to contain alternative isoforms, Va or Vb, of Cco subunit V. Previous studies have shown that these isoforms have differential effects on oxygen reduction by Cco, and that their genes (COX5a and COX5b, respectively) are inversely regulated by oxygen. Here, we find that the Vb isozyme has a higher turnover rate for NO production than the Va isozyme and that the Vb isozyme produces NO at much higher oxygen concentrations than the Va isozyme. We have also found that the hypoxic genes CYC7 and OLE1 are induced to higher levels in a strain carrying the Vb isozyme than in a strain carrying the Va isozyme. Together, these results demonstrate that the subunit V isoforms have differential effects on NO(2)(-)-dependent NO production by Cco and provide further support for a role of Cco in hypoxic signaling. These findings also suggest a positive feedback mechanism in which mitochondrially produced NO induces expression of COX5b, whose protein product then functions to enhance the ability of Cco to produce NO in hypoxic/anoxic cells.

Inhibition of mitochondrial hydrogen peroxide production by lipophilic metalloporphyrins.

Many studies have established a role for oxidative stress and mitochondrial dysfunction as an important mechanism in the pathogenesis of neuronal disorders. Metalloporphyrins are a class of catalytic antioxidants that are capable of detoxifying a wide range of reactive oxygen species. The AEOL112 series of glyoxylate metalloporphyrins were designed with increased lipid solubility for better oral bioavailability and penetration of the blood-brain barrier. The goal of this study was to develop an in vitro assay using rat brain mitochondria to reliably detect endogenously released hydrogen peroxide (H(2)O(2)) and identify glyoxylate metalloporphyrins based on rank order of potency for removal of physiologically relevant H(2)O(2). A polarographic method was established for the sensitive, accurate, and reproducible detection of low levels of H(2)O(2). The assay identified several potent glyoxylate metalloporphyrins with H(2)O(2) scavenging potencies (IC(50)) in the nanomolar range. These results provide a simplified in vitro model system to detect physiologically generated mitochondrial H(2)O(2) as a screening tool to predict the biological efficacy of potential therapeutic entities.

Mitochondria are a major source of paraquat-induced reactive oxygen species production in the brain.

Paraquat (PQ(2+)) is a prototypic toxin known to exert injurious effects through oxidative stress and bears a structural similarity to the Parkinson disease toxicant, 1-methyl-4-pheynlpyridinium. The cellular sources of PQ(2+)-induced reactive oxygen species (ROS) production, specifically in neuronal tissue, remain to be identified. The goal of this study was to determine the involvement of brain mitochondria in PQ(2+)-induced ROS production. Highly purified rat brain mitochondria were obtained using a Percoll density gradient method. PQ(2+)-induced hydrogen peroxide (H(2)O(2)) production was measured by fluorometric and polarographic methods. The production of H(2)O(2) was evaluated in the presence of inhibitors and modulators of the mitochondrial respiratory chain. The results presented here suggest that in the rat brain, (a) mitochondria are a principal cellular site of PQ(2+)-induced H(2)O(2) production, (b) PQ(2+)-induced H(2)O(2) production requires the presence of respiratory substrates, (c) complex III of the electron transport chain is centrally involved in H(2)O(2) production by PQ(2+), and (d) the mechanism by which PQ(2+) generates H(2)O(2) depends on the mitochondrial inner transmembrane potential. These observations were further confirmed by measuring PQ(2+)-induced H(2)O(2) production in primary neuronal cells derived from the midbrain. These findings shed light on the mechanism through which mitochondria may contribute to ROS production by other environmental and endogenous redox cycling agents implicated in Parkinson's disease.

Mitochondrial cytochrome oxidase produces nitric oxide under hypoxic conditions: implications for oxygen sensing and hypoxic signaling in eukaryotes.

Eukaryotic cells respond to low-oxygen concentrations by upregulating hypoxic nuclear genes (hypoxic signaling). Although it has been shown previously that the mitochondrial respiratory chain is required for hypoxic signaling, its underlying role in this process has been unclear. Here, we find that yeast and rat liver mitochondria produce nitric oxide (NO) at dissolved oxygen concentrations below 20 microM. This NO production is nitrite (NO2-) dependent, requires an electron donor, and is carried out by cytochrome c oxidase in a pH-dependent fashion. Mitochondrial NO production in yeast is influenced by the YHb flavohemoglobin NO oxidoreductase, stimulates expression of the hypoxic nuclear gene CYC7, and is accompanied by an increase in protein tyrosine nitration. These findings demonstrate an alternative role for the mitochondrial respiratory chain under hypoxic or anoxic conditions and suggest that mitochondrially produced NO is involved in hypoxic signaling, possibly via a pathway that involves protein tyrosine nitration.

Quantitative analysis of membrane protein-amphiphile interactions using resonance energy transfer.

This work describes a simple method for determining the association constant of amphiphiles to membrane proteins. The method uses a fluorescent phospholipid probe, which senses the competition among unlabeled amphiphiles for positions on the transmembrane surface of the protein. The contact between the probe and the protein surface is detected through resonance energy transfer. We have analyzed theoretically this process deriving a general equation for the dependence of the energy transfer efficiency on the composition of the micelles/bilayers in which the protein is inserted. This equation includes an exchange constant for each amphiphile, which gives a measure of its affinity for the protein with respect to that of an amphiphile set as the reference. We applied this method to determine the exchange constant of different phospholipids for the plasma membrane calcium pump.

Structural significance of the plasma membrane calcium pump oligomerization.

The oligomerization of the plasma membrane calcium pump (PMCA) in phospholipid/detergent micelles was evaluated using a combined spectroscopic and kinetic approach and related to the enzyme stability. Energy transfer between fluorescein-5'-isothiocyanate and eosin-5'-isothiocyanate attached to different PMCA molecules was used to determine the dissociation constant of dimeric PMCA (140 +/- 50 nM at 25 degrees C) and characterize the time course of dimerization. The enzyme thermal stability at different dimer/monomer ratios was evaluated, quantifying the kinetic coefficient of thermal inactivation. This coefficient decreases with PMCA concentration, becoming approximately constant beyond 300 nM. Thermal treatment leads to the formation of inactive monomers that associate only with native monomers. These mixed dimers are formed with a kinetic coefficient that is half that determined for the native dimers. We proposed a model for PMCA thermal inactivation that considers the equilibria among dimers, monomers, and mixed dimers, and the inactivation of the last two species through irreversible steps. The numerical resolution of the differential equations describing this model fitted to the experimental data allowed the determination of the model coefficients. This analysis shows that thermal inactivation occurs through the denaturation of the monomer, which lifetime is 25 min at 44 degrees C. The obtained results suggest that PMCA dimerization constitutes a mechanism of self protection against spontaneous denaturation.