NMR studies of electron carrier proteins from sulphate reducing bacteria.

The sulphate-reducing bacteria have a complex electron transfer system which leads to the reduction of sulphate by oxidation of either organic substrates or molecular hydrogen. These bacteria can either produce or consume molecular hydrogen. The central part of this electron pathway for Desulovibrio gigas is constituted by hydrogenase (3 X (4Fe-4S)). cytochrome c3 (4 haems with different redox potentials) and a one (4Fe-4S) cluster ferredoxin. This ferredoxin is isolated in different oligomeric forms, which stabilize different oxidation states and have different physiological roles; the trimer FdI being involved in the production of H2 and the tetramer FdII being more efficient for the consumption of H2. The presence of intrinsic probes (the iron ions) in these proteins is particularly helpful for structural studies using NMR spectroscopy. These studies allowed a characterization of the oxidation states used by the different oligomers of the ferredoxin and obtaintion of structural information on multi-haem cytochromes (c3 and c7). NMR is also suitable to study protein-protein interaction. The study of the complex formed between FdII and cytochrome c3 has shown that there is an alteration of the kinetics of electron transfer upon complexation.     

Electron transfer properties of melanin.

Melanin synthesized from mushroom tyrosinase and 3,4-dihydroxyphenylalanine has been shown to oxidize NADH and NADPH, reduce ferricyanide, oxidized forms of cytochrome c and dichlorophenolindophenol, and catalyze the coupled oxidation of NADH and reduction of ferricyanide. Kinetic studies involving the determination of initial velocity at various concentrations of substrates and product inhibition measurements have been carried out on the NADH-ferricyanide-melanin reaction. The results are consistent with a ping-pong mechanism in which one product is formed prior to the reaction of melanin with the second substrate involving the reversible oxidation and reduction of melanin during the reaction. It may be concluded that melanin is capable of acting as an electron transfer agent in several reduction-oxidation systems.     

A fluorescently labeled undecapeptide derived from a protein in royal jelly of the honeybee—royalisin—for specific detection of oxidized low‐density lipoprotein

The probes for detection of oxidized low‐density lipoprotein (ox‐LDL) in plasma and in atherosclerotic plaques are expected to facilitate the diagnosis, prevention, and treatment of atherosclerosis. Recently, we have reported that a heptapeptide (Lys‐Trp‐Tyr‐Lys‐Asp‐Gly‐Asp, KP6) coupled through the ε‐amino group of N‐terminal Lys to fluorescein isothiocyanate (FITC), (FITC)KP6, can be useful as a fluorescent probe for specific detection of ox‐LDL. In the present study, to develop a novel fluorescent peptide for specific detection of ox‐LDL, we investigated the interaction (with ox‐LDL) of an undecapeptide corresponding to positions 41 to 51 of a potent antimicrobial protein (royalisin, which consists of 51 residues; from royal jelly of honeybees), conjugated at the N‐terminus to FITC in the presence of 6‐amino‐n‐caproic acid (AC) linker, (FITC‐AC)‐royalisin P11, which contains both sequences, Phe‐Lys‐Asp and Asp‐Lys‐Tyr, similar to Tyr‐Lys‐Asp in (FITC)KP6. The (FITC‐AC)‐royalisin P11 bound with high specificity to ox‐LDL in a dose‐dependent manner, through the binding to major lipid components in ox‐LDL (lysophosphatidylcholine and oxidized phosphatidylcholine). In contrast, a (FITC‐AC)‐shuffled royalisin P11 peptide, in which sequences Phe‐Lys‐Asp and Asp‐Lys‐Tyr were modified to Lys‐Phe‐Asp and Asp‐Tyr‐Lys, respectively, hardly bound to LDL and ox‐LDL. These findings strongly suggest that (FITC‐AC)‐royalisin P11 may be an effective fluorescent probe for specific detection of ox‐LDL and that royalisin from the royal jelly of honeybees may play a role in the treatment of atherosclerosis through the specific binding of the region at positions 41 to 51 to ox‐LDL.     

Mercury's neurotoxicity is characterized by its disruption of selenium biochemistry

Background

Methylmercury (CH₃Hg⁺) toxicity is characterized by challenging conundrums: 1) “selenium (Se)-protective” effects, 2) undefined biochemical mechanism/s of toxicity, 3) brain-specific oxidative damage, 4) fetal vulnerability, and 5) its latency effect. The “protective effects of Se” against CH₃Hg⁺ toxicity were first recognized >50?years ago, but awareness of Se's vital functions in the brain has transformed understanding of CH₃Hg⁺ biochemical mechanisms. Mercury's affinity for Se is ~1 million times greater than its affinity for sulfur, revealing it as the primary target of CH₃Hg⁺ toxicity.

The Effect of the Phenolic Antioxidant Ferulic Acid on the Oxidation of Low Density Lipoprotein Depends on the Pro-oxidant Used

The action of ferulic acid during the oxidation of LDL has been investigated using both copper ions and the haem protein metmyoglobin as pro-oxidants. The results demonstrate the ability of ferulic acid to act as a pro-oxidant when LDL oxidation is induced by copper at concentrations of the phenolic acid which are protective when the LDL oxidation is mediated by metmyoglobin. The suggested mechanism involves the reduction of Cu²⁺ to Cu⁺ by ferulic acid resulting in the production of the ferulic phenoxyl radical.     

Control of the NADPH supply and GSH recycling for oxidative stress management in hepatoma and liver mitochondria

To unveil what controls mitochondrial ROS detoxification, the NADPH supply and GSH/GSSG recycling for oxidative stress management were analyzed in cancer and non-cancer mitochondria. Therefore, proteomic and kinetomic analyses were carried out of the mitochondrial (i) NADPH producing and (ii) GSH/GSSG recycling enzymes associated to oxidative stress management. The protein contents of the eight enzymes analyzed were similar or even higher in AS-30D rat hepatoma mitochondria (HepM) than in rat liver (RLM) and rat heart (RHM) mitochondria, suggesting that the NADPH/GSH/ROS pathway was fully functional in cancer mitochondria.The Vmax values of IDH-2 were much greater than those of GDH, TH and ME, suggesting that IDH-2 is the predominant NADPH producer in the three mitochondrial types; in fact, the GDH reverse reaction was favored. The Vmax values of GR and GPx were lower in HepM than in RLM, suggesting that the oxidative stress management is compromised in cancer mitochondria. The Km values of IDH-2, GR and GPx were all similar among the different mitochondrial types.Kinetic modeling revealed that the oxidative stress management was mainly controlled by GR, GPx and IDH. Modeling and experimentation also revealed that, due to their higher IDH-2 activity and lower GPx activity presumably by acetylation, HepM (i) showed higher steady-state NADPH levels; (ii) required greater peroxide concentrations to achieve reliable steady-state fluxes and metabolite concentration; and (iii) endured higher peroxide concentrations without collapsing their GSH/GSSG ratios. Then, to specifically prompt lower GSH/GSSG ratios under oxidative stress thus compromising cancer mitochondria functioning, GPx should be re-activated.