Role In Electron Transfer Reactions Research Articles

Longer Ubiquinone Side Chains Contribute to Enhanced Farnesol Resistance in Yeasts.

Ubiquinones (UQ) are intrinsic lipid components of many membranes. Besides their role in electron-transfer reactions there is evidence for them acting as free radical scavengers, yet their other roles in biological systems have received little study. The dimorphic fungal pathogen Candida albicans secretes farnesol as both a virulence factor and a quorum-sensing molecule. Thus, we were intrigued by the presence of UQ9 isoprenologue in farnesol-producing Candida species while other members of this genera harbor UQ7 as their major electron carrier. We examined the effect of UQ side chain length in Saccharomyces cerevisiae and C. albicans with a view towards identifying the mechanisms by which C. albicans protects itself from the high levels of farnesol it secretes, levels that are toxic to many other fungi including S. cerevisiae. In this study, we identify UQ9 as the major UQ isoprenoid in C. albicans, regardless of growth conditions or cell morphology. A S. cerevisiae model yeast engineered to make UQ9 instead of UQ6 was 4–5 times more resistant to exogenous farnesol than the parent yeast and this resistance was accompanied by greatly reduced reactive oxygen species (ROS) production. The resistance provided by UQ9 is specific for farnesol in that it does not increase resistance to high salt (1M NaCl) or other oxidants (5 mM H2O2 or 1 mM menadione). Additionally, the protection provided by UQ9 appears to be structural rather than transcriptional; UQ9 does not alter key transcriptional responses to farnesol stress. Here, we propose a model in which the longer UQ side chains are more firmly embedded in the mitochondrial membrane making them harder to pry out, so that in the presence of farnesol they remain functional without producing excess ROS. C. albicans and Candida dubliniensis evolved to use UQ9 rather than UQ7 as in other Candida species or UQ6 as in S. cerevisiae. This adaptive mechanism highlights the significance of UQ side chains in farnesol production and resistance quite apart from being an electron carrier in the respiratory chain.

NAD Analogs in Aid of Chemical Biology and Medicinal Chemistry.

Nicotinamide adenine dinucleotide (NAD) serves as an essential redox co-factor and mediator of multiple biological processes. Besides its well-established role in electron transfer reactions, NAD serves as a substrate for other biotransformations, which, at the molecular level, can be classified as protein post-translational modifications (protein deacylation, mono-, and polyADP-ribosylation) and formation of signaling molecules (e.g., cyclic ADP ribose). These biochemical reactions control many crucial biological processes, such as cellular signaling and recognition, DNA repair and epigenetic modifications, stress response, immune response, aging and senescence, and many others. However, the links between the biological effects and underlying molecular processes are often poorly understood. Moreover, NAD has recently been found to tag the 5′-ends of some cellular RNAs, but the function of these NAD-capped RNAs remains largely unrevealed. Synthetic NAD analogs are invaluable molecular tools to detect, monitor, structurally investigate, and modulate activity of NAD-related enzymes and biological processes in order to aid their deeper understanding. Here, we review the recent advances in the design and development of NAD analogs as probes for various cellular NAD-related enzymes, enzymatic inhibitors with anticancer or antimicrobial therapeutic potential, and other NAD-related chemical biology tools. We focus on research papers published within the last 10 years.

Mutant L-chain ferritins that cause neuroferritinopathy alter ferritin functionality and iron permeability.

In mammals, the iron storage and detoxification protein ferritin is composed of two functionally and genetically distinct subunit types, H (heavy) and L (light). The two subunits co-assemble in various ratios, with a tissue specific distribution, to form shell-like protein structures of 24 subunits within which a mineralized iron core is stored. The H-subunits possess ferroxidase centers that catalyze the rapid oxidation of ferrous ions, whereas the L-subunit does not have such centers and is believed to play an important role in electron transfer reactions that occur during the uptake and release of iron. Pathogenic mutations on the L-chain lead to neuroferritinopathy, a neurodegenerative disease characterized by abnormal accumulation of ferritin inclusion bodies and iron in the central nervous system. Here, we have characterized the thermal stability, iron loading capacity, iron uptake, and iron release properties of ferritin heteropolymers carrying the three pathogenic L-ferritin mutants (L154fs, L167fs, and L148fs, which for simplicity we named Ln1, Ln2 and Ln3, respectively), and a non-pathogenic variant (L135P) bearing a single substitution on the 3-fold axes of L-subunits. The UV-Vis data show a similar iron loading capacity (ranging between 1800 to 2400 Fe(iii)/shell) for all ferritin samples examined in this study, with Ln2 holding the least amount of iron (i.e. 1800 Fe(iii)/shell). The three pathogenic L-ferritin mutants revealed higher rates of iron oxidation and iron release, suggesting that a few mutated L-chains on the heteropolymer have a significant effect on iron permeability through the ferritin shell. DSC thermograms showed a strong destabilization effect, the severity of which depends on the location of the frameshift mutations (i.e. wt heteropolymer ferritin ≅ homopolymer H-chain > L135P > Ln2 > Ln1 > Ln3). Variant L135P had only minor effects on the protein functionality and stability, suggesting that local melting of the 3-fold axes in this variant may not be responsible for neuroferritinopathy-like disorders. The data support the hypothesis that hereditary neuroferritinopathies are due to alterations of ferritin functionality and lower physical stability which correlate with the frameshifts introduced at the C-terminal sequence and explain the dominant transmission of the disorder.

Modulation of the ligand-field anisotropy in a series of ferric low-spin cytochrome c mutants derived from Pseudomonas aeruginosa cytochrome c-551 and Nitrosomonas europaea cytochrome c-552: a nuclear magnetic resonance and electron paramagnetic resonance study.

Cytochromes of the c type with histidine-methionine (His-Met) heme axial ligation play important roles in electron-transfer reactions and in enzymes. In this work, two series of cytochrome c mutants derived from Pseudomonas aeruginosa (Pa c-551) and from the ammonia-oxidizing bacterium Nitrosomonas europaea (Ne c-552) were engineered and overexpressed. In these proteins, point mutations were induced in a key residue (Asn64) near the Met axial ligand; these mutations have a considerable impact both on heme ligand-field strength and on the Met orientation and dynamics (fluxionality), as judged by low-temperature electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectra. Ne c-552 has a ferric low-spin (S = 1/2) EPR signal characterized by large g anisotropy with g(max) resonance at 3.34; a similar large g(max) value EPR signal is found in the mitochondrial complex III cytochrome c1. In Ne c-552, deletion of Asn64 (NeN64Delta) changes the heme ligand field from more axial to rhombic (small g anisotropy and g(max) at 3.13) and furthermore hinders the Met fluxionality present in the wild-type protein. In Pa c-551 (g(max) at 3.20), replacement of Asn64 with valine (PaN64V) induces a decrease in the axial strain (g(max) at 3.05) and changes the Met configuration. Another set of mutants prepared by insertion (ins) and/or deletion (Delta) of a valine residue adjacent to Asn64, resulting in modifications in the length of the axial Met-donating loop (NeV65Delta, NeG50N/V65Delta, PaN50G/V65ins), did not result in appreciable alterations of the originally weak (Ne c-552) or very weak (Pa c-551) axial field but had an impact on Met orientation, fluxionality, and relaxation dynamics. Comparison of the electronic fingerprints in the overexpressed proteins and their mutants reveals a linear relationship between axial strain and average paramagnetic heme methyl shifts, irrespective of Met orientation or dynamics. Thus, for these His-Met axially coordinated Fe(III), the large g(max) value EPR signal does not represent a special case as is observed for bis-His axially coordinated Fe(III) with the two His planes perpendicular to each other.

Low‐Temperature EPR and Mössbauer Spectroscopy of Two Cytochromes with His–Met Axial Coordination Exhibiting HALS Signals

C-type cytochromes with histidine-methionine (His-Met) iron coordination play important roles in electron-transfer reactions and in enzymes. Low-temperature electron paramagnetic resonance (EPR) spectra of low-spin ferric cytochromes c can be divided into two groups, depending on the spread of g values: the normal rhombic ones with small g anisotropy and g(max) below 3.2, and those featuring large g anisotropy with g(max) between 3.3 and 3.8, also denoted as highly axial low spin (HALS) species. Herein we present the detailed magnetic properties of cytochrome c(553) from Bacillus pasteurii (g(max) 3.36) and cytochrome c(552) from Nitrosomonas europaea (g(max) 3.34) over the pH range 6.2 to 8.2. Besides being structurally very similar, cytochrome c(553) shows the presence of a minor rhombic species at pH 6.2 (6 %), whereas cytochrome c(552) has about 25 % rhombic species over pH 7.5. The detailed Mössbauer analysis of cytochrome c(552) confirms the presence of these two low-spin ferric species (HALS and rhombic) together with an 8 % ferrous form with parameters comparable to the horse cytochrome c. Both EPR and Mössbauer data of axial cytochromes c with His-Met iron coordination are consistent with an electronic (d(xy))(2) (d(xz))(2) (d(yz))(1) ground state, which is typical for Type I model hemes.

Exposing Solvent's Roles in Electron Transfer Reactions: Tunneling Pathway and Solvation

We describe studies of electron transfer in donor−spacer−acceptor molecules for which the highly curved spacer topology imparts a vacant cleft along the “line-of-sight” between the electron donor and electron acceptor moieties. The electron transfer kinetics in nondipolar and weakly polar solvents allow experimental determination of the reaction free energy as a function of solvent structure and temperature. These data were used to parametrize a molecular solvation model developed by Matyushov. The model provides reasonable estimates of reaction free energy in solvents that are too polar for its direct measurement and provides reasonable values of the solvent reorganization energy in all solvents. Successful modeling of the solvation enables quantitative study of the factors that control electron tunneling through molecules located in the solute's cleft, i.e., the electronic coupling. Electron tunneling in these systems is mediated by the unoccupied orbitals of the solvent (“electron-mediated superexchang...

A kinetic study on the interaction of deprotonated purine radical cations with amino acids and model peptides

By use of pulse radiolysis techniques, the radical cations of purine nucleotides have been successfully produced by the SO 4 − ion oxidation. Time-resolved spectroscopic evidence is provided that the one-electron-oxidized radicals of dAMP and dGMP can be efficiently repaired by aromatic amino acids (including tyrosine and tryptophan) via electron transfer reaction. As a model peptide, Arg-Tyr-AcOH was also investigated with regard to its interaction with deprotonated purine radical cations. The rate constants of the electron transfer reactions were determined to be (1∼5)×10 8 dm 3 mol −1 s −1. These results suggest that the aromatic amino acids in DNA-associated proteins may play some role in electron transfer reactions through DNA.

On the electronic structure of the primary electron donor in bacterial photosynthesis – the bacteriochlorophyll dimer as viewed by EPR/ENDOR methods

The primary electron donor (D) plays a prominent role in electron transfer reactions in the primary processes of photosynthesis. In purple photosynthetic bacteria D is a dimer of bacteriochlorophyll molecules. Investigations on its electronic structure using EPR and ENDOR (electron nuclear double resonance) methods are summarized, focussing on results obtained in the last six years. These encompass studies on the cation radical (D+·) of mutants in which the immediate environment of D is modified through mutagenesis, particularly hydrogen bond and heterodimer mutants. Models using these results to describe the electronic interaction of the dimer halves are discussed. Also, High-Field (95 GHz) EPR to obtain the G-tensor of D+· is addressed. Furthermore, ENDOR on the photoexcited triplet state of D (DT), which in some aspects could serve as a model for the excited singlet state, are discussed. Different approaches towards correlating the electronic structure with function, in particular with the rates of electron transfer reactions, are described.

Investigation of the free energy functions for electron transfer reactions

The free energy functions for electron transfer reactions in solution are explored using a previously developed microscopic simulation approach that provides a clear definition of these functions and the variable (the reaction coordinate) used as their argument. The issue of whether the curvatures of the two functions (which correspond to states with nonpolar and polar solutes) are different is given special attention. It is found, in contrast to some previous suggestions, that the curvatures of the two functions are quite similar, even when one would expect differences due to dielectric saturation effects, and that Marcus’ approximation (and, in fact, the linear response theory inherent in this approximation) provides a valid description of the solvent’s role in electron transfer reactions over a wide range of conditions. The present study demonstrates that direct simulations of the reactant and product states do not provide the data needed for determination of the free energy functions in high energy regions. On the other hand, a free energy perturbation approach developed in our early studies does allow one to obtain statistically significant data for these regions of high free energy. Inclusion of the solvent electronic polarizabilities in the potential results in a marked lowering of the solvent reorganization energy, but does not change the ratio of the curvatures of the free energy functions for the nonpolar and polar states. Finally, the effect of solvent tunneling is evaluated with the dispersed polaron method and shown to be rather small, and it is argued that the observed deviations from the classical Marcus theory in the inverted region are due to quantum mechanical tunneling associated with the solute vibrations, rather than to differences in the curvatures of the solvent free energy functions.

Antioxidant action of ubiquinol homologues with different isoprenoid chain length in biomembranes

Ubiquinones (CoQn) are intrinsic lipid components of many membranes. Besides their role in electron-transfer reactions they may act as free radical scavengers, yet their antioxidant function has received relatively little study. The efficiency of ubiquinols of varying isoprenoid chain length (from Q 0 to Q 10) in preventing (Fe 2+ + ascorbate)-dependent or (Fe 2+ + NADPH)-dependent lipid peroxidation was investigated in rat liver microsomes and brain synaptosomes and mitochondria. Ubiquinols, the reduced forms of CoQ n , possess much greater antioxidant activity than the oxidized ubiquinone forms. In homogenous solution the radical scavenging activity of ubiquinol homologues does not depend on the length of their isoprenoid chain. However in membranes ubiquinols with short isoprenoid chains (Q 1–Q 4) are much more potent inhibitors of lipid peroxidation than the longer chain homologues (Q 5–Q 10). It is found that: i) the inhibitory action, that is, antioxidant efficiency of short-chain ubiquinols decreases in order Q 1 > Q 2 > Q 3 > Q 4; ii) the antioxidant efficiency of long-chain ubiquinols is only slightly dependent on their concentrations in the order Q 5 > Q 6 > Q 7 > Q 8 > Q 9 > Q 10 and iii) the antioxidant efficiency of Q 0 is markedly less than that of other homologues. Interaction of ubiquinols with oxygen radicals was followed by their effects on luminol-activated chemiluminescence. Ubiquinols Q 1–Q 4 at 0.1 mM completely inhibit the luminol-activated NADPH-dependent chemiluminescent response of microsomes, while homologues Q 6–Q 10 exert no effect. In contrast to ubiquinol Q 10 (ubiquinone Q 10) ubiquinone Q 1 synergistically enhances NADPH-dependent regeneration of endogenous vitamin E in microsomes thus providing for higher antioxidant protection against lipid peroxidation. The differences in the antioxidant potency of ubiquinols in membranes are suggested to result from differences in partitioning into membranes, intramembrane mobility and non-uniform distribution of ubiquinols resulting in differing efficiency of interaction with oxygen and lipid radicals as well as different efficiency of ubiquinols in regeneration of endogenous vitamin E.

Gene synthesis aids study of cytochrome

In an effort to better understand characteristics of the heme protein cytochrome b 5 , including how amino acid side chains determine the properties of the heme iron, researchers have synthesized the gene that encodes the protein and have expressed it in bacteria. A number of features make cytochrome b 5 an ideal target for site-directed mutagenesis experiments, says Stephen G. Sligar, biochemistry professor at the University of Illinois, Urbana. It plays an important role in electron transfer reactions, including reduction of cytochrome P-450 in the liver and regeneration of ferrous hemoglobin in red blood cells. The protein's amino acid sequence is highly conserved among species, and a high-resolution crystal structure has been determined for bovine cytochrome b 5 . The problem, however, is that efforts to express native mammalian cytochromes at high levels in a bacterial system have not been successful. Why they have not been isn't entirely clear, Sligar says. To circumvent this problem and developed ...