Pathways linked by hydrogen bonds with redox-dependent breaks implicated in electron transfer in human cytochrome c protein

Pathways of hydrogen-bond-linked peptide units, polar side chains of the amino acid residues and buried water molecules have been traced in human cytochrome c protein. These connect heme-Fe to the surface through axially coordinated Met80-S and His18-N on the two sides of the heme plate. Oxygen atoms of the heme-propionate side chain and of the internal invariant water molecules form hydrogen bonds in connecting these pathways. With 28 out of the 37 amino acid residues being in the conserved list, these pathways are likely to be common in the highly conserved cytochrome proteins. Selective breaks appear in hydrogen bonds on the His18 side in the oxidized form and on the Met80 side in the reduced form consequent to the accompanying structural changes consistent with a regulatory role. These changes are defined by φ, ψ angles of the backbone and dihedral angles of the side chains, between the redox states. The pathways are identical in both the redox forms. They are suitable for intramolecular atom-to-atom electron transfer with hydrogen bond now experimentally found to transfer electrons better than covalent σ-bond, hitherto used for making the paths.

Dioxygen reduction, reduced oxygen species, oxygen toxicity and antioxidants — A commentary.

Molecular oxygen, a diradical, needs intervention of redox metal ions or other radicals to receive electrons for its reduction. The oxygen radicals thus produced are responsible for oxygen toxicity and oxidative stress. But, autoxidation, relevant in ischemia-reperfusion injury, is absent in any discussion on oxygen toxicity. Naturally occurring compounds which prevent formation or action of the reactive oxygen species (ROS) are generally referred as antioxidants. The reduced oxygen species, superoxide, peroxide and hydroxyl radicals, are formed in a variety of systems in the cell and are useful in selective oxidations. Currently, the popular method for assaying ROS with fluorescence of dichlorofluorescein actually measures a hemeprotein-Fe-oxo complex. The Fe-oxyl radicals are the likely oxidants in damaging proteins, nucleic acids and lipids. Such major lesions are normally repaired or replaced in the cells. The antioxidants counter the damaging oxidant actions. Among these, occurring in large concentration, are glutathione and ubiquinol, synthesized in the body and ascorbic acid and α-tocopherol, drawn from the food. A large number of plant-derived phenolic compounds, especially the flavonoid variety, are also absorbed, albeit poorly, from the food. At the natural low concentrations, these compounds show wide ranging biological effects. Increased benefit on increasing them in circulating blood needs individual verification. The polyphenolic compounds demonstrated powerful antioxidant effects in laboratory experiments. But the clinical studies did not support the consequent expectations of countering the oxidative stress, the purported crucial factor in pathology in several diseases. Antioxidant action against ROS causing oxygen toxicity needs to be reassessed. This commentary is a reappraisal of formation and reactivity of ROS in different cells, the active cellular oxidant forms, products of oxidant action on proteins, nucleic acids and lipids as marker of oxidant injury, bulk antioxidants of endogenous and exogenous origin, limited absorption occurrence and functions of polyphenolic classes.

A glucose-centric perspective of hyperglycemia.

Digestion of food in the intestines converts the compacted storage carbohydrates, starch and glycogen, to glucose. After each meal, a flux of glucose (>200 g) passes through the blood pool (4-6 g) in a short period of 2 h, keeping its concentration ideally in the range of 80-120 mg/100 mL. Tissue-specific glucose transporters (GLUTs) aid in the distribution of glucose to all tissues. The balance glucose after meeting the immediate energy needs is converted into glycogen and stored in liver (up to 100 g) and skeletal muscle (up to 300 g) for later use. High blood glucose gives the signal for increased release of insulin from pancreas. Insulin binds to insulin receptor on the plasma membrane and activates its autophosphorylation. This initiates the post-insulin-receptor signal cascade that accelerates synthesis of glycogen and triglyceride. Parallel control by phos-dephos and redox regulation of proteins exists for some of these steps. A major action of insulin is to inhibit gluconeogensis in the liver decreasing glucose output into blood. Cases with failed control of blood glucose have alarmingly increased since 1960 coinciding with changed life-styles and large scale food processing. Many of these turned out to be resistant to insulin, usually accompanied by dysfunctional glycogen storage. Glucose has an extended stay in blood at 8 mM and above and then indiscriminately adds on to surface protein-amino groups. Fructose in common sugar is 10-fold more active. This random glycation process interferes with the functions of many proteins (e.g., hemoglobin, eye lens proteins) and causes progressive damage to heart, kidneys, eyes and nerves. Some compounds are known to act as insulin mimics. Vanadium-peroxide complexes act at post-receptor level but are toxic. The fungus-derived 2,5-dihydroxybenzoquinone derivative is the first one known to act on the insulin receptor. The safe herbal products in use for centuries for glucose control have multiple active principles and targets. Some are effective in slowing formation of glucose in intestines by inhibiting α–glucosidases (e.g., salacia/saptarangi). Knowledge gained from French lilac on active guanidine group helped developing Metformin (1,1-dimethylbiguanide) one of the popular drugs in use. One strategy of keeping sugar content in diets in check is to use artificial sweeteners with no calories, no glucose or fructose and no effect on blood glucose (e.g., steviol, erythrytol). However, the three commonly used non-caloric artificial sweeteners, saccharin, sucralose and aspartame later developed glucose intolerance, the very condition they are expected to evade. Ideal way of keeping blood glucose under 6 mM and HbA1c, the glycation marker of hemoglobin, under 7% in blood is to correct the defects in signals that allow glucose flow into glycogen, still a difficult task with drugs and diets.

Emergence of oxyl radicals as selective oxidants.

Hydroxyl radicals (HO·) are derived in Fenton reaction with ferrous salt and H2O2 in acid medium, and at neutral pH, metal-oxyl radicals (M-O·) predominate. Evidence is accumulating that M-O· radicals are also active in oxidation reactions, in addition to metal-oxo (M=O) now shown in many publications. Reactivity of these radicals gives selective oxidized products useful in cellular activities, in contrast to purported indiscriminate cell damage by hydroxyl radicals. Reactions with vanadium compounds, such as diperoxovanadate, peroxo-bridged mixed valency divanadate, vanadium-oxyl radical, tetravalent vanadyl and decavanadate illustrates selective gain in oxidative capacity of oxo- and oxyl- species. Occurrence of ESR signals typical of hydroxyl radicals is demonstrated in cell homogenates and tissue perfusates treated with spin trap agents. It is known for a long time lipid peroxides are formed in tissue microsomal systems exclusively in presence of salts of iron, among many metals tested. Oxygen and a reducing agent, ascorbate (non-enzymic) or NADPH (enzymic) are required to produce 'ferryl', the chelated Fe=O active form (possibly Fe-O· and Fe-O-O-Fe ?) for the crucial step of H-atom abstraction. Yet literature is replete with unsupported affirmations that hydroxyl radicals initiate lipid peroxidation, an unexplained fixation of mindset. The best-known ·OH generator, a mixture of ferrous salt and H2O2, does not promote lipid peroxidation, nor do the many hydroxyl radical quenching agents stop it. The availability of oxo and oxyl-radical forms with transition metals, and also with non metals, P, S, N and V, calls for expansion of vision beyond superoxide and hydroxyl radicals and explore functions of multiple oxygen radicals for their biological relevance.

Reinstate hydrogen peroxide as the product of alternative oxidase of plant mitochondria.

Chill treatment of potato tubers for 8 days induced mitochondrial O2 consumption by cyanide-insensitive alternative oxidase (AOX). About half of the total O2 consumption in such mitochondria was found to be sensitive to salicylhydroxamate (SHAM), a known inhibitor of AOX activity. Addition of catalase to the reaction mixture of AOX during the reaction decreased the rate of SHAM-sensitive O2 consumption by nearly half, and addition at the end of the reaction released half of the O2 consumed by AOX, both typical of catalase action on H2O2. This reaffirmed that the product of reduction of O2 by plant AOX was H2O2 as found earlier and not H2O as reported in some recent reviews.

A perspective of biological supramolecular electron transfer.

Electron transfer is an essential activity in biological systems. The migrating electron originates from water-oxygen in photosynthesis and reverts to dioxygen in respiration. In this cycle two metal porphyrin complexes possessing circular conjugated system and macrocyclic pi-clouds, chlorophyll and heme, play a decisive role in mobilising electrons for travel over biological structures as extraneous electrons. Transport of electrons within proteins (as in cytochromes) and within DNA (during oxidative damage and repair) is known to occur. Initial evaluations did not favour formation of semiconducting pathways of delocalized electrons of the peptide bonds in proteins and of the bases in nucleic acids. Direct measurement of conductivity of bulk material and quantum chemical calculations of their polymeric structures also did not support electron transfer in both proteins and nucleic acids. New experimental approaches have revived interest in the process of charge transfer through DNA duplex. The fluorescence on photo-excitation of Ru-complex was found to be quenched by Rh-complex, when both were tethered to DNA and intercalated in the base stack. Similar experiments showed that damage to G-bases and repair of T-T dimers in DNA can occur by possible long range electron transfer through the base stack. The novelty of this phenomenon prompted the apt name, "chemistry at a distance". Based on experiments with ruthenium modified proteins, intramolecular electron transfer in proteins is now proposed to use pathways that include C-C sigma-bonds and surprisingly hydrogen bonds which remained out of favour for a long time. In support of this, some experimental evidence is now available showing that hydrogen bond-bridges facilitate transfer of electrons between metal-porphyrin complexes. By molecular orbital calculations over 20 years ago we found that "delocalization of an extraneous electron is pronounced when it enters low-lying virtual orbitals of the electronic structures of peptide units linked by hydrogen bonds". This review focuses on supramolecular electron transfer pathways that can emerge on interlinking by hydrogen bonds and metal coordination of some unnoticed structures with pi-clouds in proteins and nucleic acids, potentially useful in catalysis and energy missions.

A perspective of smooth muscle contractility through actions of vanadium compounds.

Smooth muscle contraction has a characteristic step-response with successive additions of stimulating compounds, and instant reversal on withdrawing the stimulus, indicative of an equilibrium situation wherein continuous, rapid reactions are occurring. Vanadium compounds, ortho- and meta-vanadates, decavanadate and peroxovanadate, were found to contract a variety of smooth muscles. Their actions were analyzed with respect to activation of receptors, increase in the intracellular calcium concentration, and increase in calmodulin-dependent myosin light chain phosphorylation leading to contraction. A new perspective of smooth muscle contractility has emerged from the studies with vanadium compounds suggesting control mechanisms involving phosphorylation for contraction and redox for relaxation.

Transmembrane domains participate in functions of integral membrane proteins.

Integral membrane proteins have one or more transmembrane alpha-helical domains and carry out a variety of functions such as enzyme catalysis, transport across membranes, transducing signals as receptors of hormones and growth factors, and energy transfer in ATP synthesis. These transmembrane domains are not mere structural units anchoring the protein to the lipid bilayer but seem to-contribute in the overall activity. Recent findings in support of this are described using some typical examples-LDL receptor, growth factor receptor tyrosine kinase, HMG-CoA reductase, F0-ATPase and adrenergic receptors. The trends in research indicate that these transmembrane domains participate in a variety of ways such as a linker, a transducer or an exchanger in the overall functions of these proteins in transfer of materials, energy and signals.

Functions of cytochrome c in regulation of electron transfer and protein folding.

Cytochrome c, a "mobile electron carrier" of the mitochondrial respiratory chain, also occurs in detectable amounts in the cytosol, and can receive electrons from cytochromes present in endoplasmic reticulum and plasma membranes as well as from superoxide and ascorbate. The pigment was found to dissociate from mitochondrial membranes in liver and kidney when rats were subjected to heat exposure and starvation, respectively. Treating cytochrome c with hydroxylamine gives a partially deaminated product with altered redox properties; decreased stimulation of respiration by deficient mitochondria, increased reduction by superoxide, and complete loss of reducibility by plasma membranes. Mitochondria isolated from brown adipose tissue of cold-exposed rats are found to be sub-saturated with cytochrome c. The ability of cytochrome c to reactivate reduced ribonuclease is now reinterpreted as a molecular chaperone role for the hemoprotein.

H2O2 has a role in cellular regulation.

H2O2, in addition to producing highly reactive molecules through hydroxyl radicals or peroxidase action, can exert a number of direct effects on cells, organelles and enzymes. The stimulations include glucose transport, glucose incorporation into glycogen, HMP shunt pathway, lipid synthesis, release of calcium from mitochondria and of arachidonate from phospholipids, poly ADP ribosylation, and insulin receptor tyrosine kinase and pyruvate dehydrogenase activities. The inactivations include glycolysis, lipolysis, reacylation of lysophospholipids, ATP synthesis, superoxide dismutase and protein kinase C. Damages to DNA and proteoglycan and general cytotoxicity possibly through oxygen radicals were also observed. A whole new range of effects will be opened by the finding that H2O2 can act as a signal transducer in oxidative stress by oxidizing a dithiol protein to disulphide form which then activates transcription of the stress inducible genes. Many of these direct effects seem to be obtained by dithiol-disulphide modification of proteins and their active sites, as part of adaptive responses in oxidative stress.

Polyvanadate acts at the level of plasma membranes through aadrenergic receptor and affects cellular calcium distribution and some oxidation activities.

The activities of calcium-stimulated respiration, calcium uptake, a-glycerophosphate dehydrogenase and rates of oxidation in state 3 and of H2O2 generation, were found to increase and that of pyruvate dehydrogenase decrease in mitochondria isolated from livers of rats administered intraperitoneally or perfused with polyvanadate. Phenoxybenzamine, an antagonist of a−adrenergic receptor, effectively prevented these changes. It was also found that perfusion of the liver with polyvanadate reproduced one of the best characterized events of a-adrenergic activation-stimulation of protein kinase C in plasma membrane accompanied by its decrease in cytosol. These experiments indicate for the first time the α-adrenergic mimetic action of polyvanadate.

Changes in thyroid hormone and insulin status of the brown adipose tissue of cold acclimated rats on short term exposure to heat.

Acclimation of rats to cold caused 45% increase in the concentration of triidothyronine (T3) and 35% increase in the concentration of thyroxine (T4) in serum. Exposure of cold-acclimated rats to heat (12 hr, 37 degrees C) failed to decrease the concentrations of thyroid hormones in circulation. The concentration of T3 in brown adipose tissue (BAT) increased almost 10-fold on cold acclimation. Iodothyronine deiodinase activity also registered 3-fold increase. Exposure of cold-acclimated animals to heat caused decrease in the concentration of T3 in BAT without appreciably affecting T4 concentration. In liver tissue, the changes in hormone concentrations were quite small compared to those in BAT. On thyroidectomy or when fed with propyl thiouracil, rats could not survive exposure to the cold. The concentration of insulin in circulation showed small increase, while that in the tissues showed significant decrease on acclimation of rats to the cold. The concentration of the hormone in BAT registered significant increase on exposure of cold-acclimated animals to heat (12 hr, 37 degrees C). The increase in liver was marginal. The temperature-dependent response of T3 indicates an important role for this hormone in rapid physiological response in BAT.

Increase in alpha-glycerophosphate dehydrogenase and other oxidoreductase activities of hepatic mitochondria on administration of vanadate to the rat.

Liver mitochondria isolated from vanadate-administered rats showed increased (20-25%) rates of oxidation of both NAD(+)-linked substrates and succinate. Respiratory control index and ADP/O were unaffected by the treatment. Dormant and uncoupler-stimulated ATPase activity also was not affected by vanadate administration. Membrane-bound, electron-transport-linked dehydrogenase activities (both NAD(+)- and succinate-dependent) increased by 15-20% on vanadate treatment. Mitochondrial alpha-glycerophosphate dehydrogenase activity increased by 50% on vanadate administration. The above effects of vanadate on oxidoreductase activities could be prevented by the prior administration of antagonists to alpha-adrenergic receptors. Substrate-dependent H2O2 generation by mitochondria also showed an increase on vanadate administration.