The Profound Influence of Lipid Composition on the Catalysis of the Drug Target NADH Type II Oxidoreductase

Lipids play a pivotal role in cellular respiration, providing the natural environment in which an oxidoreductase interacts with the quinone pool. To date, it is generally accepted that negatively charged lipids play a major role in the activity of quinone oxidoreductases. By changing lipid compositions when assaying a type II NADH:quinone oxidoreductase, we demonstrate that phosphatidylethanolamine has an essential role in substrate binding and catalysis. We also reveal the importance of acyl chain composition, specifically c14:0, on membrane-bound quinone-mediated catalysis. This demonstrates that oxidoreductase lipid specificity is more diverse than originally thought and that the lipid environment plays an important role in the physiological catalysis of membrane-bound oxidoreductases.

Computationally modeling mammalian succinate dehydrogenase kinetics identifies the origins and primary determinants of ROS production

Succinate dehydrogenase (SDH) is an inner mitochondrial membrane protein complex that links the Krebs cycle to the electron transport system. It can produce significant amounts of superoxide (O2·¯) and hydrogen peroxide (H2O2); however, the precise mechanisms are unknown. This fact hinders the development of next-generation antioxidant therapies targeting mitochondria. To help address this problem, we developed a computational model to analyze and identify the kinetic mechanism of O2·¯ and H2O2 production by SDH. Our model includes the major redox centers in the complex, namely FAD, three iron-sulfur clusters, and a transiently bound semiquinone. Oxidation state transitions involve a one- or two-electron redox reaction, each being thermodynamically constrained. Model parameters were simultaneously fit to many data sets using a variety of succinate oxidation and free radical production data. In the absence of respiratory chain inhibitors, model analysis revealed the 3Fe-4S iron-sulfur cluster as the primary O2·¯ source. However, when the quinone reductase site is inhibited or the quinone pool is highly reduced, O2·¯ is generated primarily by the FAD. In addition, H2O2 production is only significant when the enzyme is fully reduced, and fumarate is absent. Our simulations also reveal that the redox state of the quinone pool is the primary determinant of free radical production by SDH. In this study, we showed the importance of analyzing enzyme kinetics and associated side reactions in a consistent, quantitative, and biophysically detailed manner using a diverse set of experimental data to interpret and explain experimental observations from a unified perspective.

Analysis of Mammalian Succinate Dehydrogenase Kinetics and Reactive Oxygen Species Production

Succinate dehydrogenase is an inner mitochondrial membrane protein complex that links the tricarboxylic acid cycle to the electron transport system. It catalyzes the reaction between succinate and ubiquinone to produce fumarate and ubiquinol. In addition, it can produce significant amounts of superoxide and hydrogen peroxide under the right conditions. While the flavin adenine dinucleotide (FAD) is the putative site of reactive oxygen species production, free radical production from other sites are less certain. Herein, we developed a computational model to analyze free radical production data from complex II and identify the mechanism of superoxide and hydrogen peroxide production. The model includes the major redox centers consisting of the FAD, three iron-sulfur clusters, and a transiently catalytic bound semi quinone. The model consists of five-states that represent oxidation status of the enzyme complex. Each step in the reaction scheme is thermodynamically constrained, and transitions between each state involve either one-electron or two-electron redox reactions. The model parameters were simultaneously fit using data consisting of enzyme kinetics and free radical production rates under a range of conditions. In the absence of respiratory chain inhibitors, model analysis revealed that the 3Fe-4S iron-sulfur cluster is the primary source of superoxide production followed by the FAD radical. However, when the quinone reductase site of complex II is inhibited or the quinone pool is highly reduced, superoxide production from the FAD site dominates at low succinate concentrations. In addition, hydrogen peroxide formation from the complex is only significant when these one of these conditions is met and the fumarate concentrations is in the low micromolar range. From the model simulations, the redox state of the quinone pool was found to be the primary determinant of free radical production from complex II. This study highlights the importance of evaluating enzyme kinetics and associated side-reactions in a consistent, quantitative and biophysical detailed manner. By incorporating the results from a diverse set of experiments, this computational approach can be used to interpret and explain key differences among the observations from a single, unified perspective.

Modeling the Interplay between Photosynthesis, CO2 Fixation, and the Quinone Pool in a Purple Non-Sulfur Bacterium

Rhodopseudomonas palustris CGA009 is a purple non-sulfur bacterium (PNSB) that can fix CO2 and nitrogen or break down organic compounds for its carbon and nitrogen requirements. Light, inorganic, and organic compounds can all be used for its source of energy. Excess electrons produced during its metabolic processes can be exploited to produce hydrogen gas or biodegradable polyesters (polyhydroxybutyrate). A genome-scale metabolic model of the bacterium was reconstructed to study the interactions between photosynthesis, carbon dioxide fixation, and the redox state of the quinone pool. A comparison of model-predicted flux values with published in vivo MFA fluxes resulted in predicted errors of 5-19% across four different growth substrates. The model predicted the presence of an unidentified sink responsible for the oxidation of excess quinols generated by the TCA cycle. Furthermore, light-dependent energy production was found to be highly dependent on the rate of quinol oxidation. Finally, the extent of CO2 fixation was predicted to be dependent on the amount of ATP generated through the electron transport cycle, with excess ATP going toward the energy-demanding CBB pathway. Based on this analysis, it is hypothesized that the quinone redox state acts as a feed-forward controller of the CBB pathway, signaling the amount of ATP available.

Escherichia coli cytochrome c peroxidase is a respiratory oxidase that enables the use of hydrogen peroxide as a terminal electron acceptor

Microbial cytochrome c peroxidases (Ccp) have been studied for 75 years, but their physiological roles are unclear. Ccps are located in the periplasms of bacteria and the mitochondrial intermembrane spaces of fungi. In this study, Ccp is demonstrated to be a significant degrader of hydrogen peroxide in anoxic Escherichia coli. Intriguingly, ccp transcription requires both the presence of H2O2 and the absence of O2. Experiments show that Ccp lacks enough activity to shield the cytoplasm from exogenous H2O2. However, it receives electrons from the quinone pool, and its flux rate approximates flow to other anaerobic electron acceptors. Indeed, Ccp enabled E. coli to grow on a nonfermentable carbon source when H2O2 was supplied. Salmonella behaved similarly. This role rationalizes ccp repression in oxic environments. We speculate that micromolar H2O2 is created both biologically and abiotically at natural oxic/anoxic interfaces. The OxyR response appears to exploit this H2O2 as a terminal oxidant while simultaneously defending the cell against its toxicity.

Physiology and Bioenergetics of [NiFe]-Hydrogenase 2-Catalyzed H2-Consuming and H2-Producing Reactions in Escherichia coli

Escherichia coliuptake hydrogenase 2 (Hyd-2) catalyzes the reversible oxidation of H2to protons and electrons. Hyd-2 synthesis is strongly upregulated during growth on glycerol or on glycerol-fumarate. Membrane-associated Hyd-2 is an unusual heterotetrameric [NiFe]-hydrogenase that lacks a typical cytochromebmembrane anchor subunit, which transfers electrons to the quinone pool. Instead, Hyd-2 has an additional electron transfer subunit, termed HybA, with four predicted iron-sulfur clusters. Here, we examined the physiological role of the HybA subunit. During respiratory growth with glycerol and fumarate, Hyd-2 used menaquinone/demethylmenaquinone (MQ/DMQ) to couple hydrogen oxidation to fumarate reduction. HybA was essential for electron transfer from Hyd-2 to MQ/DMQ. H2evolution catalyzed by Hyd-2 during fermentation of glycerol in the presence of Casamino Acids or in a fumarate reductase-negative strain growing with glycerol-fumarate was also shown to be dependent on both HybA and MQ/DMQ. The uncoupler carbonyl cyanidem-chlorophenylhydrazone (CCCP) inhibited Hyd-2-dependent H2evolution from glycerol, indicating the requirement for a proton gradient. In contrast, CCCP failed to inhibit H2-coupled fumarate reduction. Although a Hyd-2 enzyme lacking HybA could not catalyze Hyd-2-dependent H2oxidation or H2evolution in whole cells, reversible H2-dependent reduction of viologen dyes still occurred. Finally, hydrogen-dependent dye reduction by Hyd-2 was reversibly inhibited in extracts derived from cells grown in H2evolution mode. Our findings suggest that Hyd-2 switches between H2-consuming and H2-producing modes in response to the redox status of the quinone pool. Hyd-2-dependent H2evolution from glycerol requires reverse electron transport.

Randomly selected suppressor mutations in genes for NADH : quinone oxidoreductase-1, which rescue motility of a Salmonella ubiquinone-biosynthesis mutant strain

The primary mobile electron-carrier in the aerobic respiratory chain of Salmonella is ubiquinone. Demethylmenaquinone and menaquinone are alternative electron-carriers involved in anaerobic respiration. Ubiquinone biosynthesis was disrupted in strains bearing deletions of the ubiA or ubiE genes. In soft tryptone agar both mutant strains swam poorly. However, the ubiA deletion mutant strain produced suppressor mutant strains with somewhat rescued motility and growth. Six independent suppressor mutants were purified and comparative genome sequence analysis revealed that they each bore a single new missense mutation, which localized to genes for subunits of NADH : quinone oxidoreductase-1. Four mutants bore an identical nuoG(Q297K) mutation, one mutant bore a nuoM(A254S) mutation and one mutant bore a nuoN(A444E) mutation. The NuoG subunit is part of the hydrophilic domain of NADH : quinone oxidoreductase-1 and the NuoM and NuoN subunits are part of the hydrophobic membrane-embedded domain. Respiration was rescued and the suppressed mutant strains grew better in Luria–Bertani broth medium and could use l-malate as a sole carbon source. The quinone pool of the cytoplasmic membrane was characterized by reversed-phase HPLC. Wild-type cells made ubiquinone and menaquinone. Strains with a ubiA deletion mutation made demethylmenaquinone and menaquinone and the ubiE deletion mutant strain made demethylmenaquinone and 2-octaprenyl-6-methoxy-1,4-benzoquinone; the total quinone pool was reduced. Immunoblotting found increased NADH : quinone oxidoreductase-1 levels for ubiquinone-biosynthesis mutant strains and enzyme assays measured electron transfer from NADH to demethylmenaquinone or menaquinone. Under certain growth conditions the suppressor mutations improved electron flow activity of NADH : quinone oxidoreductase-1 for cells bearing a ubiA deletion mutation.

A Significant Loss in Photosynthetic Activity Associated with the Yellow Vine Syndrome of Cranberry

Numerous observations of yellow vine syndrome of cranberry have been reported from commercial cranberry growers. The molecular mechanism resulting in yellow vine syndrome is unknown. We have previously reported on the shading effect as an approach to explore the mechanisms of yellow vine formation and proposed photoinhibition as a possible cause. To compare the photosynthetic performance of yellow vine-affected and normal cranberry leaves, we conducted chlorophyll fluorescence analyses over 1 period of 1 day and 3 weeks, respectively. Both experimental data sets indicated that the maximum quantum efficiency of photosystem II, the size of the quinone pool, the numbers of reaction centers (RCs) per chlorophyll absorption, and the photosynthesis performance index of the yellow vine samples are substantially lower than those of normal cranberry leaves. These results are in line with the data of yellow vine leaves, having 26% to 28% less in chlorophyll than the normal leaves as measured by spectrometric and high-performance liquid chromatography analysis. We concluded that yellow vine syndrome is associated with poor photosynthetic activity and is likely becoming a threat for the long-term growth and crop production of cranberries.