Mitochondrial DNA and Electron Transport Chain Protein Levels Are Altered in Peripheral Nerve Tissues from Donors with HIV Sensory Neuropathy: A Pilot Study.

Distal sensory polyneuropathy (DSP) and distal neuropathic pain (DNP) remain significant challenges for older people with HIV (PWH), necessitating enhanced clinical attention. HIV and certain antiretroviral therapies (ARTs) can compromise mitochondrial function and impact mitochondrial DNA (mtDNA) replication, which is linked to DSP in ART-treated PWH. This study investigated mtDNA, mitochondrial fission and fusion proteins, and mitochondrial electron transport chain protein changes in the dorsal root ganglions (DRGs) and sural nerves (SuNs) of 11 autopsied PWH. In antemortem standardized assessments, six had no or one sign of DSP, while five exhibited two or more DSP signs. Digital droplet polymerase chain reaction was used to measure mtDNA quantity and the common deletions in isolated DNA. We found lower mtDNA copy numbers in DSP+ donors. SuNs exhibited a higher proportion of mtDNA common deletion than DRGs in both groups. Mitochondrial electron transport chain (ETC) proteins were altered in the DRGs of DSP+ compared to DSP- donors, particularly Complex I. These findings suggest that reduced mtDNA quantity and increased common deletion abundance may contribute to DSP in PWH, indicating diminished mitochondrial activity in the sensory neurons. Accumulated ETC proteins in the DRG imply impaired mitochondrial transport to the sensory neuron's distal portion. Identifying molecules to safeguard mitochondrial integrity could aid in treating or preventing HIV-associated peripheral neuropathy.

Membrane-Bound Redox Enzyme Cytochrome bd-I Promotes Carbon Monoxide-Resistant Escherichia coli Growth and Respiration.

The terminal oxidases of bacterial aerobic respiratory chains are redox-active electrogenic enzymes that catalyze the four-electron reduction of O2 to 2H2O taking out electrons from quinol or cytochrome c. Living bacteria often deal with carbon monoxide (CO) which can act as both a signaling molecule and a poison. Bacterial terminal oxidases contain hemes; therefore, they are potential targets for CO. However, our knowledge of this issue is limited and contradictory. Here, we investigated the effect of CO on the cell growth and aerobic respiration of three different Escherichia coli mutants, each expressing only one terminal quinol oxidase: cytochrome bd-I, cytochrome bd-II, or cytochrome bo3. We found that following the addition of CO to bd-I-only cells, a minimal effect on growth was observed, whereas the growth of both bd-II-only and bo3-only strains was severely impaired. Consistently, the degree of resistance of aerobic respiration of bd-I-only cells to CO is high, as opposed to high CO sensitivity displayed by bd-II-only and bo3-only cells consuming O2. Such a difference between the oxidases in sensitivity to CO was also observed with isolated membranes of the mutants. Accordingly, O2 consumption of wild-type cells showed relatively low CO sensitivity under conditions favoring the expression of a bd-type oxidase.

LncRNA FGD5-AS1 Alleviates Inflammation in Allergic Rhinitis through the miR-223-3p/COX11 Axis.

INTRODUCTION: Long noncoding RNAs (lncRNAs) have been implicated in the pathogenesis of allergic rhinitis (AR). The current investigation is focused on elucidating the functional impact of a specific lncRNA, FGD5 antisense RNA 1 (FGD5-AS1), on the development and progression of AR through its interaction with miR-223-3p. METHODS: An experimental framework for AR was constructed in both cellular and animal models. Quantitative assessment of FGD5-AS1, miR-223-3p, and COX11 mRNA expression was conducted using real-time quantitative reverse transcription PCR. The expression of inflammatory factors, immunoglobulin E, LTC4, and ECP, was examined using ELISA. Apoptosis in human nasal epithelial cells was assessed by the flow cytometry method. The protein expression of COX11 was examined using Western blotting. Nasal mucosal function was further evaluated by hematoxylin and eosin staining. Furthermore, bioinformatics evaluations, dual-luciferase reporter assays, and a series of experimental procedures unveiled a putative competitive endogenous RNA regulatory mechanism. RESULTS: We found the expression of lncRNA FGD5-AS1 was decreased in AR. In vitro lncRNA FGD5-AS1 attenuated the production of inflammatory cytokines in nasal epithelial cells. Furthermore, elevated FGD5-AS1 expression significantly alleviated AR symptoms by reducing nasal epithelial apoptosis and inflammation. MiR-223-3p was identified as a direct target of FGD5-AS1. Moreover, miRNA-223-3p directly downregulated the expression of COX11 mRNA. Subsequent experiments confirmed that FGD5-AS1 regulated AR through the miR-223-3p/COX11 axis, thereby inhibiting inflammation. CONCLUSION: The FGD5-AS1/miR-223-3p/COX11 axis plays a pivotal role in the pathogenesis of AR, suggesting that FGD5-AS1 could serve as a potential diagnostic biomarker and therapeutic target for AR.

Understanding differential aspects of microdiffusion (channeling) in the Coenzyme Q and Cytochrome c regions of the mitochondrial respiratory system.

Over the past decades, models of the organization of mitochondrial respiratory system have been controversial. The goal of this perspective is to assess this "conflict of models" by focusing on specific kinetic evidence in the two distinct segments of Coenzyme Q- and Cytochrome c-mediated electron transfer. Respiratory supercomplexes provide kinetic advantage by allowing a restricted diffusion of Coenzyme Q and Cytochrome c, and short-range interaction with their partner enzymes. In particular, electron transfer from NADH is compartmentalized by channeling of Coenzyme Q within supercomplexes, whereas succinate oxidation proceeds separately using the free Coenzyme Q pool. Previous evidence favoring Coenzyme Q random diffusion in the NADH-dependent electron transfer is due to downstream flux interference and misinterpretation of results. Indeed, electron transfer by complexes III and IV via Cytochrome c is less strictly dependent on substrate channeling in mammalian mitochondria. We briefly describe these differences and their physiological implications.

Novel COX11 Mutations Associated with Mitochondrial Disorder: Functional Characterization in Patient Fibroblasts and Saccharomyces cerevisiae.

Genetic defects in the nuclear encoded subunits and assembly factors of cytochrome c oxidase (mitochondrial complex IV) are very rare and are associated with a wide variety of phenotypes. Biallelic pathogenic variants in the COX11 protein were previously identified in two unrelated children with infantile-onset mitochondrial encephalopathies. Through comprehensive clinical, genetic and functional analyses, here we report on a new patient harboring novel heterozygous variants in COX11, presenting with Leigh-like features, and provide additional experimental evidence for a direct correlation between COX11 protein expression and sensitivity to oxidative stress. To sort out the contribution of the single mutations to the phenotype, we employed a multi-faceted approach using Saccharomyces cerevisiae as a genetically manipulable system, and in silico structure-based analysis of human COX11. Our results reveal differential effects of the two novel COX11 mutations on yeast growth, respiration, and cellular redox status, as well as their potential impact on human protein stability and function. Strikingly, the functional deficits observed in patient fibroblasts are recapitulated in yeast models, validating the conservation of COX11's role in mitochondrial integrity across evolutionarily distant organisms. This study not only expands the mutational landscape of COX11-associated mitochondrial disorders but also underscores the continued translational relevance of yeast models in dissecting complex molecular pathways.

[Cytochrome bd as Antioxidant Redox Enzyme].

One of the main functions of enzyme complexes that constitute electron transport (respiratory) chains of organisms is to maintain cellular redox homeostasis by oxidizing reducing equivalents, NADH and quinol. Cytochrome bd is a unique terminal oxidase of the chains of many bacteria including pathogenic species. This redox enzyme couples the oxidation of ubiquinol or menaquinol by molecular oxygen to the generation of proton motive force, a universal energy currency. The latter is used by the organism to produce ATP, another cellular energy currency, via oxidative phosphorylation. Escherichia coli contains two bd-type oxidases, bd-I and bd-II, encoded by the cydAB and appCB operons, respectively. Surprisingly, both bd enzymes make a further contribution to molecular mechanisms of maintaining the appropriate redox balance in the bacterial cell by means of elimination of reactive oxygen species, such as hydrogen peroxide. This review summarizes recent data on the redox-modulated H2O2-scavenging activities of cytochromes bd-I and bd-II from E. coli. The possibility of such antioxidant properties in cytochromes bd from other bacteria is also discussed.

Generation of Membrane Potential by Cytochrome bd.

An overview of current notions on the mechanism of generation of a transmembrane electric potential difference (Δψ) during the catalytic cycle of a bd-type triheme terminal quinol oxidase is presented in this work. It is suggested that the main contribution to Δψ formation is made by the movement of H+ across the membrane along the intra-protein hydrophilic proton-conducting pathway from the cytoplasm to the active site for oxygen reduction of this bacterial enzyme.

The terminal oxidase cytochrome bd-I confers carbon monoxide resistance to Escherichia coli cells.

Carbon monoxide (CO) plays a multifaceted role in the physiology of organisms, from poison to signaling molecule. Heme proteins, including terminal oxidases, are plausible CO targets. Three quinol oxidases terminate the branched aerobic respiratory chain of Escherichia coli. These are the heme­copper cytochrome bo3 and two copper-lacking bd-type cytochromes, bd-I and bd-II. All three enzymes generate a proton motive force during the four-electron oxygen reduction reaction that is used for ATP production. The bd-type oxidases also contribute to mechanisms of bacterial defense against various types of stresses. Here we report that in E. coli cells, at the enzyme concentrations tested, cytochrome bd-I is much more resistant to inhibition by CO than cytochrome bd-II and cytochrome bo3. The apparent half-maximal inhibitory concentration values, IC50, for inhibition of O2 consumption of the membrane-bound bd-II and bo3 oxidases by CO at ~150 µM O2 were estimated to be 187.1 ± 11.1 and 183.3 ± 13.5 µM CO, respectively. Under the same conditions, the maximum inhibition observed with the membrane-bound cytochrome bd-I was 20 ± 10% at ~200 µM CO.

QM Calculations Revealed that Outer-Sphere Electron Transfer Boosted O-O Bond Cleavage in the Multiheme-Dependent Cytochrome bd Oxygen Reductase.

The cytochrome bd oxygen reductase catalyzes the four-electron reduction of dioxygen to two water molecules. The structure of this enzyme reveals three heme molecules in the active site, which differs from that of heme-copper cytochrome c oxidase. The quantum chemical cluster approach was used to uncover the reaction mechanism of this intriguing metalloenzyme. The calculations suggested that a proton-coupled electron transfer reduction occurs first to generate a ferrous heme b595. This is followed by the dioxygen binding at the heme d center coupled with an outer-sphere electron transfer from the ferrous heme b595 to the dioxygen moiety, affording a ferric ion superoxide intermediate. A second proton-coupled electron transfer produces a heme d ferric hydroperoxide, which undergoes efficient O-O bond cleavage facilitated by an outer-sphere electron transfer from the ferrous heme b595 to the O-O σ* orbital and an inner-sphere proton transfer from the heme d hydroxyl group to the leaving hydroxide. The synergistic benefits of the two types of hemes rationalize the highly efficient oxygen reduction repertoire for the multi-heme-dependent cytochrome bd oxygen reductase family.

Mutating the environment of heme b595 of E. coli cytochrome bd-I oxidase shifts its redox potential by 200 mV without inactivating the enzyme.

Cytochrome bd-I catalyzes the reduction of oxygen to water with the aid of hemes b558, b595 and d. Here, effects of a mutation of E445, a ligand of heme b595 and of R448, hydrogen bonded to E445 are studied electrochemically in the E. coli enzyme. The equilibrium potential of the three hemes are shifted by up to 200 mV in these mutants. Strikingly the E445D and the R448N mutants show a turnover of 41 ± 2 % and 20 ± 4 %, respectively. Electrocatalytic studies confirm that the mutants react with oxygen and bind and release NO. These results point towards the ability of cytochrome bd to react even if the electron transfer is less favorable.

pH-dependent kinetics of NO release from E. coli bd-I and bd-II oxidase reveals involvement of Asp/Glu58B.

Escherichia coli contains two cytochrome bd oxidases, bd-I and bd-II. The structure of both enzymes is highly similar, but they exhibit subtle differences such as the accessibility of the active site through a putative proton channel. Here, we demonstrate that the duroquinol:dioxygen oxidoreductase activity of bd-I increased with alkaline pH, whereas bd-II showed a broad activity maximum around pH 7. Likewise, the pH dependence of NO release from the reduced active site, an essential property of bd oxidases, differed between the two oxidases as detected by UV/vis spectroscopy. Both findings may be attributed to differences in the proton channel leading to the active site heme d. The channel comprises a titratable residue (Asp58B in bd-I and Glu58B in bd-II). Conservative mutations at this position drastically altered NO release demonstrating its contribution to the process.

Preparations of Terminal Oxidase Cytochrome bd-II Isolated from Escherichia coli Reveal Significant Hydrogen Peroxide Scavenging Activity.

Cytochrome bd-II is one of the three terminal quinol oxidases of the aerobic respiratory chain of Escherichia coli. Preparations of the detergent-solubilized untagged bd-II oxidase isolated from the bacterium were shown to scavenge hydrogen peroxide (H2O2) with high rate producing molecular oxygen (O2). Addition of H2O2 to the same buffer that does not contain enzyme or contains thermally denatured cytochrome bd-II does not lead to any O2 production. The latter observation rules out involvement of adventitious transition metals bound to the protein. The H2O2-induced O2 production is not susceptible to inhibition by N-ethylmaleimide (the sulfhydryl binding compound), antimycin A (the compound that binds specifically to a quinol binding site), and CO (diatomic gas that binds specifically to the reduced heme d). However, O2 formation is inhibited by cyanide (IC50 = 4.5 ± 0.5 µM) and azide. Addition of H2O2 in the presence of dithiothreitol and ubiquinone-1 does not inactivate cytochrome bd-II and apparently does not affect the O2 reductase activity of the enzyme. The ability of cytochrome bd-II to detoxify H2O2 could play a role in bacterial physiology by conferring resistance to the peroxide-mediated stress.

Biallelic pathogenic variants in COX11 are associated with an infantile-onset mitochondrial encephalopathy.

Primary mitochondrial diseases are a group of genetically and clinically heterogeneous disorders resulting from oxidative phosphorylation (OXPHOS) defects. COX11 encodes a copper chaperone that participates in the assembly of complex IV and has not been previously linked to human disease. In a previous study, we identified that COX11 knockdown decreased cellular adenosine triphosphate (ATP) derived from respiration, and that ATP levels could be restored with coenzyme Q10 (CoQ10 ) supplementation. This finding is surprising since COX11 has no known role in CoQ10 biosynthesis. Here, we report a novel gene-disease association by identifying biallelic pathogenic variants in COX11 associated with infantile-onset mitochondrial encephalopathies in two unrelated families using trio genome and exome sequencing. Functional studies showed that mutant COX11 fibroblasts had decreased ATP levels which could be rescued by CoQ10 . These results not only suggest that COX11 variants cause defects in energy production but reveal a potential metabolic therapeutic strategy for patients with COX11 variants.

E. coli cytochrome bd-I requires Asp58 in the CydB subunit for catalytic activity.

The reduction of oxygen to water is crucial to life and a central metabolic process. To fulfil this task, prokaryotes use among other enzymes cytochrome bd oxidases (Cyt bds) that also play an important role in bacterial virulence and antibiotic resistance. To fight microbial infections by pathogens, an in-depth understanding of the enzyme mechanism is required. Here, we combine bioinformatics, mutagenesis, enzyme kinetics and FTIR spectroscopy to demonstrate that proton delivery to the active site contributes to the rate limiting steps in Cyt bd-I and involves Asp58 of subunit CydB. Our findings reveal a previously unknown catalytic function of subunit CydB in the reaction of Cyt bd-I.

Recent Advances in Structural Studies of Cytochrome bd and Its Potential Application as a Drug Target.

Cytochrome bd is a triheme copper-free terminal oxidase in membrane respiratory chains of prokaryotes. This unique molecular machine couples electron transfer from quinol to O2 with the generation of a proton motive force without proton pumping. Apart from energy conservation, the bd enzyme plays an additional key role in the microbial cell, being involved in the response to different environmental stressors. Cytochrome bd promotes virulence in a number of pathogenic species that makes it a suitable molecular drug target candidate. This review focuses on recent advances in understanding the structure of cytochrome bd and the development of its selective inhibitors.

Mitochondrial respiratory chain dysfunction alters ER sterol sensing and mevalonate pathway activity.

Mitochondrial dysfunction induces a strong adaptive retrograde signaling response; however, many of the downstream effectors of this response remain to be discovered. Here, we studied the shared transcriptional responses to three different mitochondrial respiratory chain inhibitors in human primary skin fibroblasts using QuantSeq 3'-RNA-sequencing. We found that genes involved in the mevalonate pathway were concurrently downregulated, irrespective of the respiratory chain complex affected. Targeted metabolomics demonstrated that impaired mitochondrial respiration at any of the three affected complexes also had functional consequences on the mevalonate pathway, reducing levels of cholesterol precursor metabolites. A deeper study of complex I inhibition showed a reduced activity of endoplasmic reticulum-bound sterol-sensing enzymes through impaired processing of the transcription factor Sterol Regulatory Element-Binding Protein 2 and accelerated degradation of the endoplasmic reticulum cholesterol-sensors squalene epoxidase and HMG-CoA reductase. These adaptations of mevalonate pathway activity affected neither total intracellular cholesterol levels nor the cellular free (nonesterified) cholesterol pool. Finally, measurement of intracellular cholesterol using the fluorescent cholesterol binding dye filipin revealed that complex I inhibition elevated cholesterol on intracellular compartments. Taken together, our study shows that mitochondrial respiratory chain dysfunction elevates intracellular free cholesterol levels and therefore attenuates the expression of mevalonate pathway enzymes, which lowers endogenous cholesterol biosynthesis, disrupting the metabolic output of the mevalonate pathway. We conclude that intracellular disturbances in cholesterol homeostasis may alter systemic cholesterol management in diseases associated with declining mitochondrial function.

A Structural Perspective on the RNA Editing of Plant Respiratory Complexes.

The last steps of respiration, a core energy-harvesting process, are carried out by a chain of multi-subunit complexes in the inner mitochondrial membrane. Several essential subunits of the respiratory complexes are RNA-edited in plants, frequently leading to changes in the encoded amino acids. While the impact of RNA editing is clear at the sequence and phenotypic levels, the underlying biochemical explanations for these effects have remained obscure. Here, we used the structures of plant respiratory complex I, complex III2 and complex IV to analyze the impact of the amino acid changes of RNA editing in terms of their location and biochemical features. Through specific examples, we demonstrate how the structural information can explain the phenotypes of RNA-editing mutants. This work shows how the structural perspective can bridge the gap between sequence and phenotype and provides a framework for the continued analysis of RNA-editing mutants in plant mitochondria and, by extension, in chloroplasts.

A mitochondrial ADXR-ADX-P450 electron transport chain is essential for maternal gametophytic control of embryogenesis in Arabidopsis.

Mitochondrial adrenodoxins (ADXs) are small iron-sulfur proteins with electron transfer properties. In animals, ADXs transfer electrons between an adrenodoxin reductase (ADXR) and mitochondrial P450s, which is crucial for steroidogenesis. Here we show that a plant mitochondrial steroidogenic pathway, dependent on an ADXR-ADX-P450 shuttle, is essential for female gametogenesis and early embryogenesis through a maternal effect. The steroid profile of maternal and gametophytic tissues of wild-type (WT) and adxr ovules revealed that homocastasterone is the main steroid present in WT gametophytes and that its levels are reduced in the mutant ovules. The application of exogenous homocastasterone partially rescued adxr and P450 mutant phenotypes, indicating that gametophytic homocastasterone biosynthesis is affected in the mutants and that a deficiency of this hormone causes the phenotypic alterations observed. These findings also suggest not only a remarkable similarity between steroid biosynthetic pathways in plants and animals but also a common function during sexual reproduction.

Maternal Fructose Intake Causes Developmental Reprogramming of Hepatic Mitochondrial Catalytic Activity and Lipid Metabolism in Weanling and Young Adult Offspring.

Excess dietary fructose is a major public health concern, yet little is known about its influence on offspring development and later-life disease when consumed in excess during pregnancy. To determine whether increased maternal fructose intake could have long-term consequences on offspring health, we investigated the effects of 10% w/v fructose water intake during preconception and pregnancy in guinea pigs. Female Dunkin Hartley guinea pigs were fed a control diet (CD) or fructose diet (FD; providing 16% of total daily caloric intake) ad libitum 60 days prior to mating and throughout gestation. Dietary interventions ceased at day of delivery. Offspring were culled at day 21 (D21) (weaning) and at 4 months (4 M) (young adult). Fetal exposure to excess maternal fructose intake significantly increased male and female triglycerides at D21 and 4 M and circulating palmitoleic acid and total omega-7 through day 0 (D0) to 4 M. Proteomic and functional analysis of significantly differentially expressed proteins revealed that FD offspring (D21 and 4 M) had significantly increased mitochondrial metabolic activities of ß-oxidation, electron transport chain (ETC) and oxidative phosphorylation and reactive oxygen species production compared to the CD offspring. Western blotting analysis of both FD offspring validated the increased protein abundances of mitochondrial ETC complex II and IV, SREBP-1c and FAS, whereas VDAC1 expression was higher at D21 but lower at 4 M. We provide evidence demonstrating offspring programmed hepatic mitochondrial metabolism and de novo lipogenesis following excess maternal fructose exposure. These underlying asymptomatic programmed pathways may lead to a predisposition to metabolic dysfunction later in life.

Alternative oxidase (AOX) 1a and 1d limit proline-induced oxidative stress and aid salinity recovery in Arabidopsis.

Proline (Pro) catabolism and reactive oxygen species production have been linked in mammals and Caenorhabditis elegans, while increases in leaf respiration rate follow Pro exposure in plants. Here, we investigated how alternative oxidases (AOXs) of the mitochondrial electron transport chain accommodate the large, atypical flux resulting from Pro catabolism and limit oxidative stress during Pro breakdown in mature Arabidopsis (Arabidopsis thaliana) leaves. Following Pro treatment, AOX1a and AOX1d accumulate at transcript and protein levels, with AOX1d approaching the level of the typically dominant AOX1a isoform. We therefore sought to determine the function of both AOX isoforms under Pro respiring conditions. Oxygen consumption rate measurements in aox1a and aox1d leaves suggested these AOXs can functionally compensate for each other to establish enhanced AOX catalytic capacity in response to Pro. Generation of aox1a.aox1d lines showed complete loss of AOX proteins and activity upon Pro treatment, yet full respiratory induction in response to Pro remained possible via the cytochrome pathway. However, aox1a.aox1d leaves displayed symptoms of elevated oxidative stress and suffered increased oxidative damage during Pro metabolism compared to the wild-type (WT) or the single mutants. During recovery from salt stress, when relatively high rates of Pro catabolism occur naturally, photosynthetic rates in aox1a.aox1d recovered slower than in the WT or the single aox lines, showing that both AOX1a and AOX1d are beneficial for cellular metabolism during Pro drawdown following osmotic stress. This work provides physiological evidence of a beneficial role for AOX1a but also the less studied AOX1d isoform in allowing safe catabolism of alternative respiratory substrates like Pro.