Milan Žižić

The interactions of vanadium with Phycomyces blakesleeanus mycelium: enzymatic reduction, transport and metabolic effects

The biological and chemical basis of vanadium action and transport in fungi is relatively poorly understood. In this study we investigated the interactions of vanadium in physiologically-relevant redox states: vanadate (+5) and vanadyl (+4), with mycelium of fungus Phycomyces blakesleeanus using EPR and (31)P NMR spectroscopy and biochemical assays. We determined that P. blakesleeanus reduces V(5+) to V(4+) in the extracellular compartment by the means of cell surface enzyme with ferricyanide reductase activity, which contains molybdenum-molybdopterin as a cofactor. Both, V(5+) and V(4+) bind to cell wall. They enter the cytoplasm via phosphate transporter and cation channels, respectively, and exhibit different metabolic effects. Vanadate provokes increased biomass production, the effects being inverted to toxic at higher V(5+) concentrations. In addition, V(5+) activates the synthesis of sugar phosphates and oligophosphates. On the other hand, V(4+) exhibits toxic effects even at low concentrations. The V(4+) detoxification route involves binding to vacuolar polyphosphates. Altogether our results imply that the mechanism of interaction of vanadium with P. blakesleeanus involves three major steps: extracellular enzymatic V(5+)/V(4+) reduction, V(4+) influx, and vacuolar storage, with an additional step - V(5+) import occurring at higher vanadate concentrations.

Oxygen regulation of alternative respiration in fungus Phycomyces blakesleeanus: connection with phosphate metabolism

Environmental changes can often result in oxygen deficiency which influences cellular energy metabolism, but such effects have been insufficiently studied in fungi. The effects of oxygen deprivation on respiration and phosphate metabolites in Phycomyces blakesleeanus were investigated by oxygen electrode and (31)P NMR spectroscopy. Mycelium was incubated in hypoxic and anoxic conditions for 1.5, 3 and 5 h and then reoxygenated. Participation of alternative oxidase (AOX) in total respiration increased gradually in both treatments and after 5 h of anoxia exceeded a value 50% higher than in control. Shortly after reintroduction of oxygen into the system AOX level decreased close to the control level. Oxygen deprivation also caused a reversible decrease of polyphosphate/inorganic phosphate ratio (PPc/Pi), which was strongly correlated with the increase of AOX participation in total respiration. Unexpectedly, ATP content remained almost constant, probably due to the ability of PolyP to sustain energy and phosphate homeostasis of the cell under stress conditions. This was further substantiated by the effects of azide, a cytochrome c oxidase inhibitor, which also decreased PPc/Pi ratio, but to a smaller extent in oxygen deprived than control and reoxygenated specimens.

Vanadate Influence on Metabolism of Sugar Phosphates in Fungus Phycomyces blakesleeanus

The biological and chemical basis of vanadium action in fungi is relatively poorly understood. In the present study, we investigate the influence of vanadate (V5+) on phosphate metabolism of Phycomyces blakesleeanus. Addition of V5+ caused increase of sugar phosphates signal intensities in 31P NMR spectra in vivo. HPLC analysis of mycelial phosphate extracts demonstrated increased concentrations of glucose 6 phosphate, fructose 6 phosphate, fructose 1, 6 phosphate and glucose 1 phosphate after V5+ treatment. Influence of V5+ on the levels of fructose 2, 6 phosphate, glucosamine 6 phosphate and glucose 1, 6 phosphate (HPLC), and polyphosphates, UDPG and ATP (31P NMR) was also established. Increase of sugar phosphates content was not observed after addition of vanadyl (V4+), indicating that only vanadate influences its metabolism. Obtained results from in vivo experiments indicate catalytic/inhibitory vanadate action on enzymes involved in reactions of glycolysis and glycogenesis i.e., phosphoglucomutase, phosphofructokinase and glycogen phosphorylase in filamentous fungi.

Coordinate and redox interactions of epinephrine with ferric and ferrous iron at physiological pH

Coordinate and redox interactions of epinephrine (Epi) with iron at physiological pH are essential for understanding two very different phenomena – the detrimental effects of chronic stress on the cardiovascular system and the cross-linking of catecholamine-rich biopolymers and frameworks. Here we show that Epi and Fe3+ form stable high-spin complexes in the 1:1 or 3:1 stoichiometry, depending on the Epi/Fe3+ concentration ratio (low or high). Oxygen atoms on the catechol ring represent the sites of coordinate bond formation within physiologically relevant bidentate 1:1 complex. Redox properties of Epi are slightly impacted by Fe3+. On the other hand, Epi and Fe2+ form a complex that acts as a strong reducing agent, which leads to the production of hydrogen peroxide via O2 reduction, and to a facilitated formation of the Epi–Fe3+ complexes. Epi is not oxidized in this process, i.e. Fe2+ is not an electron shuttle, but the electron donor. Epi-catalyzed oxidation of Fe2+ represents a plausible chemical basis of stress-related damage to heart cells. In addition, our results support the previous findings on the interactions of catecholamine moieties in polymers with iron and provide a novel strategy for improving the efficiency of cross-linking.

Osmotic swelling activates a novel anionic current with VRAC-like properties in a cytoplasmic droplet membrane from Phycomyces blakesleeanus sporangiophores.

We describe here whole-cell currents of droplets prepared from the apical region of growing Phycomyces blakesleeanus sporangiophores. Whole-cell current recordings revealed the osmotically activated, outwardly rectifying, fast inactivating instantaneous current (ORIC) with biophysical properties closely resembling volume-regulated anionic current (VRAC). ORIC is activated under conditions of osmotically induced swelling and shows strong selectivity for anions over cations. In addition, ORIC shows voltage and time-dependent inactivation at positive potentials and recovery from inactivation at negative potentials. ORIC is blocked by anthracene-9-carboxylic acid, an anion channel blocker, in a voltage-dependent manner. This is the first report of the presence of VRAC-like current in an organism outside the chordate lineage.

The conformation of epinephrine in polar solvents: an NMR study

Epinephrine (Epi) is a physiologically important catecholamine. Molecular conformation of Epi controls the interactions with other molecules and its biological effects. There have been a number of theoretical studies addressing conformation and hydrogen bonding of Epi in different solvents, but experimental data are scarce. Herein, we applied 1H NMR, 1H-1H COSY, 1H-15N HSQC, and NOESY to examine and compare the conformation of Epi in polar solvents—dimethyl sulfoxide (DMSO) and water. The main differences were observed for NH2 and CH2 groups. Both showed chemical nonequivalence of protons in DMSO that was not present in water. The analysis of the effects of increasing temperature and solvent substitution on NMR signals showed that one of the protons in amine group forms a strong intramolecular hydrogen bond with aliphatic OH group, which is H-donor in another hydrogen bond with DMSO. NOESY provided data on the spatial positions of protons in the side chain, allowing for 3D model of the structure of Epi in DMSO to be built. In close, Epi molecule forms an additional 5-membered ring that encompasses bifurcate intra-/intermolecular hydrogen bonds, and acquires conformation that resembles the shape of a “scorpion”—the catechol ring representing the body and the side chain being a forward-curved tail. The conformation of Epi in water lacks the intramolecular hydrogen bond and most likely largely depends on hydrogen bonds with water molecules.

Effects of vanadate on the mycelium of edible fungus Coprinus comatus

Vanadate is proposed to play a pivotal role in application of edible fungus Coprinus comatus for medical purposes. In this study the concentration of extracellular vanadate acceptable for the submerged cultivation of C. comatus mycelium was established. The mycelium could grow, and overcome vanadate toxic effects, up to the concentration of 3.3 mM. Moreover, in this condition, at the end of the exponential phase of growth, biomass yield was almost identical to that in the control. 31P NMR spectroscopy showed that addition of 10 mM vanadate to the mycelium in the exponential phase of growth provoked instantaneous increase of a sugar phosphates level which could be related to changes in activities of glycolytic enzymes. Exposure to higher vanadate concentration was toxic for the cell. 51V NMR measurements revealed that monomer of vanadate is present in the cytoplasm causing the metabolic changes. C. comatus has also capacity for vanadate reduction, as shown by EPR measurements, but vanadyl uptake is significantly less comparing to vanadate.

Growth inhibition of fungus Phycomyces blakesleeanus by anion channel inhibitors anthracene-9-carboxylic and niflumic acid attained through decrease in cellular respiration and energy metabolites.

Increasing resistance of fungal strains to known fungicides has prompted identification of new candidates for fungicides among substances previously used for other purposes. We have tested the effects of known anion channel inhibitors anthracene-9-carboxylic acid (A9C) and niflumic acid (NFA) on growth, energy metabolism and anionic current of mycelium of fungus Phycomyces blakesleeanus. Both inhibitors significantly decreased growth and respiration of mycelium, but complete inhibition was only achieved by 100 and 500 µM NFA for growth and respiration, respectively. A9C had no effect on respiration of human NCI-H460 cell line and very little effect on cucumber root sprout clippings, which nominates this inhibitor for further investigation as a potential new fungicide. Effects of A9C and NFA on respiration of isolated mitochondria of P. blakesleeanus were significantly smaller, which indicates that their inhibitory effect on respiration of mycelium is indirect. NMR spectroscopy showed that both A9C and NFA decrease the levels of ATP and polyphosphates in the mycelium of P. blakesleeanus, but only A9C caused intracellular acidification. Outwardly rectifying, fast inactivating instantaneous anionic current (ORIC) was also reduced to 33±5 and 21±3 % of its pre-treatment size by A9C and NFA, respectively, but only in the absence of ATP. It can be assumed from our results that the regulation of ORIC is tightly linked to cellular energy metabolism in P. blakesleeanus, and the decrease in ATP and polyphosphate levels could be a direct cause of growth inhibition.