Mitochondrial Oxidation of the Cytoplasmic Reducing Equivalents at the Onset of Oxidant Stress in the Isoproterenol-Induced Rat Myocardial Infarction

We have developed and characterized a model of isoproterenol (ISO)-induced myocardial necrosis, identifying three stages of cardiac damage: a pre-infarction (0–12 h), infarction (24 h), and post-infarction period (48–96 h). Using this model, we have previously found alterations in calcium homeostasis and their relationship with oxidant stress in mitochondria, which showed deficient oxygen consumption and coupled ATP synthesis. Therefore, the present study was aimed at assessing the mitochondrial ability to transport and oxidize cytoplasmic reducing equivalents (NADH), correlating the kinetic parameters of the malate-aspartate shuttle, oxidant stress, and mitochondrial functionality. Our results showed only discreet effects during the cardiotoxic ISO action on the endogenous malate-aspartate shuttle activity, suggesting that endogenous mitochondrial NADH oxidation capacity (Nohl dehydrogenase) was not affected by the cellular stress. On the contrary, the reconstituted system showed significant enhancement in maximal capacity of the malate-aspartate shuttle activity only at later times (post-infarction period), probably as a compensatory part of cardiomyocytes’ response to the metabolic and functional consequences of the infarcted tissue. Therefore, these findings support the notion that heart damage associated with myocardial infarction suffers a set of sequential biochemical and metabolic modifications within cardiomyocytes, where mitochondrial activity, controlling the redox state, could play a relevant role.

A Balance between the Activities of Chloroplasts and Mitochondria Is Crucial for Optimal Plant Growth

Energy metabolism in plant cells requires a balance between the activities of chloroplasts and mitochondria, as they are the producers and consumers of carbohydrates and reducing equivalents, respectively. Recently, we showed that the overexpression of Arabidopsis thaliana purple acid phosphatase 2 (AtPAP2), a phosphatase dually anchored on the outer membranes of chloroplasts and mitochondria, can boost the plant growth and seed yield of Arabidopsis thaliana by coordinating the activities of both organelles. However, when AtPAP2 is solely overexpressed in chloroplasts, the growth-promoting effects are less optimal, indicating that active mitochondria are required for dissipating excess reducing equivalents from chloroplasts to maintain the optimal growth of plants. It is even more detrimental to plant productivity when AtPAP2 is solely overexpressed in mitochondria. Although these lines contain high level of adenosine triphosphate (ATP), they exhibit low leaf sucrose, low seed yield, and early senescence. These transgenic lines can be useful tools for studying how hyperactive chloroplasts or mitochondria affect the physiology of their counterparts and how they modify cellular metabolism and plant physiology.

The {FeO2}8 Intermediate of the TxtE Nitration Pathway Resists Reduction, Facilitating Its Reaction with Nitric Oxide

<p>TxtE is a cytochome P450 (CYP) homolog that mediates a nitric oxide (NO)-dependent direct nitration of l-tryptophan (l-Trp) to form 4-nitrotryptophan (4-NO₂-l-Trp). This nitrated product is a precursor for thaxtomin A, a virulence factor produced by plant-pathogenic bacteria that causes the disease potato scab. A recent study provided the first characterization of intermediates along the TxtE nitration pathway.¹ The authors’ accumulated evidence supported a mechanism in which O₂ binds to Fe^II TxtE to form an {FeO₂}⁸ intermediate, which subsequently reacted with NO to ultimately form Fe^III TxtE and 4-NO₂-l-Trp. Typical CYP mechanisms reduce and protonate the {FeO₂}⁸ intermediate to form a ferric-hydroperoxo species (Fe^III–OOH) en route to formation of the active oxidant compound I. The previously reported lack of hydroxylated tryptophan resulting from TxtE turnover suggests that the TxtE cycle must stall at the {FeO₂}⁸ intermediate to avoid hydroxylation. Here we present LC-MS experiments showing suggesting that TxtE can hydroxylate l-Trp by the peroxide shunt but not via reduction of the {FeO₂}⁸ intermediate. Comparison of stopped-flow time courses in the presence and absence of excess reducing equivalents and common CYP electron transfer partners shown no spectral or kinetic evidence for reduction of the {FeO₂}⁸ intermediate. Furthermore, the electron coupling efficiency of TB14—a self-sufficient TxtE variant with C-terminal reductase domain—to form 4-NO₂-l-Trp exhibits a 3% electron coupling efficiency when it is loaded with one reducing equivalent. This efficiency <i>increases</i> by 2-fold when TB14 is loaded with two or four reducing equivalents. This observation provides further evidence for our key conclusion that the TxtE {FeO₂}⁸ intermediate resists reduction. The resistance of the {FeO₂}⁸ intermediate to reduction is a key feature of TxtE, enabling reaction with NO and efficient nitration turnover.<b></b></p>

The {FeO2}8 Intermediate of the TxtE Nitration Pathway Resists Reduction, Facilitating Its Reaction with Nitric Oxide

<p>TxtE is a cytochome P450 (CYP) homolog that mediates a nitric oxide (NO)-dependent direct nitration of l-tryptophan (l-Trp) to form 4-nitrotryptophan (4-NO₂-l-Trp). This nitrated product is a precursor for thaxtomin A, a virulence factor produced by plant-pathogenic bacteria that causes the disease potato scab. A recent study provided the first characterization of intermediates along the TxtE nitration pathway.¹ The authors’ accumulated evidence supported a mechanism in which O₂ binds to Fe^II TxtE to form an {FeO₂}⁸ intermediate, which subsequently reacted with NO to ultimately form Fe^III TxtE and 4-NO₂-l-Trp. Typical CYP mechanisms reduce and protonate the {FeO₂}⁸ intermediate to form a ferric-hydroperoxo species (Fe^III–OOH) en route to formation of the active oxidant compound I. The previously reported lack of hydroxylated tryptophan resulting from TxtE turnover suggests that the TxtE cycle must stall at the {FeO₂}⁸ intermediate to avoid hydroxylation. Here we present LC-MS experiments showing suggesting that TxtE can hydroxylate l-Trp by the peroxide shunt but not via reduction of the {FeO₂}⁸ intermediate. Comparison of stopped-flow time courses in the presence and absence of excess reducing equivalents and common CYP electron transfer partners shown no spectral or kinetic evidence for reduction of the {FeO₂}⁸ intermediate. Furthermore, the electron coupling efficiency of TB14—a self-sufficient TxtE variant with C-terminal reductase domain—to form 4-NO₂-l-Trp exhibits a 3% electron coupling efficiency when it is loaded with one reducing equivalent. This efficiency <i>increases</i> by 2-fold when TB14 is loaded with two or four reducing equivalents. This observation provides further evidence for our key conclusion that the TxtE {FeO₂}⁸ intermediate resists reduction. The resistance of the {FeO₂}⁸ intermediate to reduction is a key feature of TxtE, enabling reaction with NO and efficient nitration turnover.<b></b></p>

A 35-fold enhancement in photocurrent generation of Synechocystis sp. PCC 6803 by outer membrane deprivation

Biophotovoltaics (BPV) is an emerging technology developed to utilize reducing equivalents produced by photosynthetic organisms. It generates electrical power by exploiting a phenomenon called extracellular electron transfer (EET), where reducing equivalents are transferred extracellularly to exogenous electron acceptors. Although cyanobacteria have been extensively studied in BPV because of their high photosynthetic activity and ease of handling, their extremely low EET activity remains a limitation. In this study, we achieved a 35-fold enhancement in photocurrent generation of the cyanobacterium Synechocystis sp. PCC 6803 by deprivation of the outer membrane, where electrons are suggested to stem from NADPH; this, along with a significantly higher rate of exogenous ferricyanide reduction, verified that low permeability of the outer membrane contributues to low cyanobacterial EET activity. Moreover, outer membrane deprivation enhanced extracellular derivation of reducing equivalents serving as respiratory substrates for other heterotrophic bacteria, making it promising and useful for effective derivation of reducing equivalents.

Modulating the activities of chloroplasts and mitochondria promotes adenosine triphosphate production and plant growth

Efficient photosynthesis requires a balance of ATP and NADPH production/consumption in chloroplasts, and the exportation of reducing equivalents from chloroplasts is important for balancing stromal ATP/NADPH ratio. Here, we showed that the overexpression of purple acid phosphatase 2 on the outer membranes of chloroplasts and mitochondria can streamline the production and consumption of reducing equivalents in these two organelles, respectively. A higher capacity of consumption of reducing equivalents in mitochondria can indirectly help chloroplasts to balance the ATP/NADPH ratio in stroma and recycle NADP+, the electron acceptors of the linear electron flow (LEF). A higher rate of ATP and NADPH production from the LEF, a higher capacity of carbon fixation by the Calvin–Benson–Bassham (CBB) cycle and a greater consumption of NADH in mitochondria enhance photosynthesis in the chloroplasts, ATP production in the mitochondria and sucrose synthesis in the cytosol and eventually boost plant growth and seed yields in the overexpression lines.

Metabolites potentiate nitrofurans in non-growing Escherichia coli

Nitrofurantoin (NIT) is a broad-spectrum bactericidal antibiotic used in the treatment of urinary tract infections. It is a pro-drug that once activated by nitroreductases goes on to inhibit bacterial DNA, RNA, cell wall, and protein synthesis. Previous work has suggested that NIT retains considerable activity against non-growing bacteria. Here we have found that Escherichia coli (E. coli) grown to stationary phase in minimal or artificial urine media are not susceptible to NIT. Supplementation with glucose under conditions where cells remained non-growing (other essential nutrients were absent) sensitized cultures to NIT. We conceptualized NIT sensitivity as a multi-input AND gate, and lack of susceptibility as an insufficiency in one or more of those inputs. The inputs considered were an activating enzyme, cytoplasmic abundance of NIT, and reducing equivalents required for NIT activation. We systematically assessed the contribution of each of these inputs and found that NIT import and the level of activating enzyme were not contributing factors to the lack of susceptibility. Rather, evidence suggested that the low abundance of reducing equivalents is why stationary-phase E. coli are not killed by NIT, and catabolites can re-sensitize those cells. We found that this phenomenon also occurred when using nitrofurazone, which established generality to the nitrofuran antibiotic class. In addition, we observed that NIT activity against stationary-phase uropathogenic E. coli (UPEC) could also be potentiated through metabolite supplementation. These findings suggest that the combination of nitrofurans with specific metabolites could improve the outcome of uncomplicated urinary tract infections (UTIs).

Efficient cooperation of chloroplasts and mitochondria enhances ATP and sucrose production

ABSTRACTEfficient photosynthesis requires a balance of ATP and NADPH production/consumption in chloroplasts as the linear electron flow generates a higher NADPH/ATP ratio than that is consumed by the Calvin-Benson-Bassham cycle. Recent works suggested that ATP importation into mature chloroplasts of Arabidopsis thaliana is negligible, and therefore the exportation of reducing equivalents from chloroplasts is important for balancing stromal ATP/NADPH ratio. Here we showed that the overexpression of purple acid phosphatase 2 on the outer membranes of chloroplasts and mitochondria can streamline the production and consumption of reducing equivalents in these two organelles, respectively. A higher capacity of consumption of reducing equivalents in mitochondria can indirectly help chloroplasts to balance the ATP/NADPH ratio in stroma and recycle NADP+, the electron acceptors of the linear electron flow. A higher rate of ATP and NADPH production from the linear electron flow, a higher capacity of carbon fixation by the Calvin-Benson-Bassham cycle and a greater consumption of NADH in mitochondria, enhance photosynthesis in the chloroplasts, ATP production in the mitochondria, sucrose synthesis in the cytosol, and eventually boosting plant growth and seed yields in the overexpression lines.Significance StatementThis study demonstrates the importance of chloroplast-mitochondria cooperation in redox balance and illustrates that an optimized function of mitochondria can enhance the efficiency of photosynthesis.