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Recovery from COVID-19 depends on the ability of the host to effectively neutralize virions and infected cells, a process largely driven by antibody-mediated immunity. However, with the newly emerging variants that evade Spike-targeting antibodies, re-infections and breakthrough infections are increasingly common. A full characterization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mechanisms counteracting antibody-mediated immunity is therefore needed. Here, we report that ORF8 is a virally encoded SARS-CoV-2 factor that controls cellular Spike antigen levels. We show that ORF8 limits the availability of mature Spike by inhibiting host protein synthesis and retaining Spike at the endoplasmic reticulum, reducing cell-surface Spike levels and recognition by anti-SARS-CoV-2 antibodies. In conditions of limited Spike availability, we found ORF8 restricts Spike incorporation during viral assembly, reducing Spike levels in virions. Cell entry of these virions then leaves fewer Spike molecules at the cell surface, limiting antibody recognition of infected cells. Based on these findings, we propose that SARS-CoV-2 variants may adopt an ORF8-dependent strategy that facilitates immune evasion of infected cells for extended viral production., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Protein disulfide isomerases (PDIs) constitute a family of oxidoreductases promoting redox protein folding and quality control in the endoplasmic reticulum. PDIs catalyze disulfide bond formation, isomerization, and reduction, operating in concert with molecular chaperones to fold secretory cargoes in addition to directing misfolded proteins to be refolded or degraded. Importantly, PDIs are emerging as key components of the proteostasis network, integrating protein folding status with central surveillance mechanisms to balance proteome stability according to cellular needs. Recent advances in the field driven by the generation of new mouse models, human genetic studies, and omics methodologies, in addition to interventions using small molecules and gene therapy, have revealed the significance of PDIs to the physiology of the nervous system. PDIs are also implicated in diverse pathologies, ranging from neurodevelopmental conditions to neurodegenerative diseases and traumatic injuries. Here, we review the principles of redox protein folding in the ER with a focus on current evidence linking genetic mutations and biochemical alterations to PDIs in the etiology of neurological conditions., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Skeletal muscle atrophy is a highly-prevalent and debilitating condition that remains poorly understood at the molecular level. Previous work found that aging, fasting, and immobilization promote skeletal muscle atrophy via expression of activating transcription factor 4 (ATF4) in skeletal muscle fibers. However, the direct biochemical mechanism by which ATF4 promotes muscle atrophy is unknown. ATF4 is a member of the basic leucine zipper transcription factor (bZIP) superfamily. Because bZIP transcription factors are obligate dimers, and because ATF4 is unable to form highly-stable homodimers, we hypothesized that ATF4 may promote muscle atrophy by forming a heterodimer with another bZIP family member. To test this hypothesis, we biochemically isolated skeletal muscle proteins that associate with the dimerization- and DNA-binding domain of ATF4 (the bZIP domain) in mouse skeletal muscle fibers in vivo Interestingly, we found that ATF4 forms at least five distinct heterodimeric bZIP transcription factors in skeletal muscle fibers. Furthermore, one of these heterodimers, composed of ATF4 and CCAAT enhancer-binding protein β (C/EBPβ), mediates muscle atrophy. Within skeletal muscle fibers, the ATF4-C/EBPβ heterodimer interacts with a previously unrecognized and evolutionarily conserved ATF-C/EBP composite site in exon 4 of the Gadd45a gene. This three-way interaction between ATF4, C/EBPβ, and the ATF-C/EBP composite site activates the Gadd45a gene, which encodes a critical mediator of muscle atrophy. Together, these results identify a biochemical mechanism by which ATF4 induces skeletal muscle atrophy, providing molecular-level insights into the etiology of skeletal muscle atrophy., (© 2020 Ebert et al.)

Mitochondrial production of superoxide and hydrogen peroxide is potentially important in cell signaling and disease. Eleven distinct mitochondrial sites that differ markedly in capacity are known to leak electrons to oxygen to produce O 2 ̇̄ and/or H 2 O 2 We discuss their contributions to O 2 ̇̄ /H 2 O 2 production under native conditions in mitochondria oxidizing different substrates and in conditions mimicking physical exercise and the changes in their capacities after caloric restriction. We review the use of S1QELs and S3QELs, suppressors of mitochondrial O 2 ̇̄ /H 2 O 2 generation that do not inhibit oxidative phosphorylation, as tools to characterize the contributions of specific sites in situ and in vivo ., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

SIRT5 is a lysine desuccinylase known to regulate mitochondrial fatty acid oxidation and the urea cycle. Here, SIRT5 was observed to bind to cardiolipin via an amphipathic helix on its N terminus. In vitro , succinyl-CoA was used to succinylate liver mitochondrial membrane proteins. SIRT5 largely reversed the succinyl-CoA-driven lysine succinylation. Quantitative mass spectrometry of SIRT5-treated membrane proteins pointed to the electron transport chain, particularly Complex I, as being highly targeted for desuccinylation by SIRT5. Correspondingly, SIRT5 -/- HEK293 cells showed defects in both Complex I- and Complex II-driven respiration. In mouse liver, SIRT5 expression was observed to localize strictly to the periportal hepatocytes. However, homogenates prepared from whole SIRT5 -/- liver did show reduced Complex II-driven respiration. The enzymatic activities of Complex II and ATP synthase were also significantly reduced. Three-dimensional modeling of Complex II suggested that several SIRT5-targeted lysine residues lie at the protein-lipid interface of succinate dehydrogenase subunit B. We postulate that succinylation at these sites may disrupt Complex II subunit-subunit interactions and electron transfer. Lastly, SIRT5 -/- mice, like humans with Complex II deficiency, were found to have mild lactic acidosis. Our findings suggest that SIRT5 is targeted to protein complexes on the inner mitochondrial membrane via affinity for cardiolipin to promote respiratory chain function., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Partitioning of ATP generation between glycolysis and oxidative phosphorylation is central to cellular bioenergetics but cumbersome to measure. We describe here how rates of ATP generation by each pathway can be calculated from simultaneous measurements of extracellular acidification and oxygen consumption. We update theoretical maximum ATP yields by mitochondria and cells catabolizing different substrates. Mitochondrial P/O ratios (mol of ATP generated per mol of [O] consumed) are 2.73 for oxidation of pyruvate plus malate and 1.64 for oxidation of succinate. Complete oxidation of glucose by cells yields up to 33.45 ATP/glucose with a maximum P/O of 2.79. We introduce novel indices to quantify bioenergetic phenotypes. The glycolytic index reports the proportion of ATP production from glycolysis and identifies cells as primarily glycolytic (glycolytic index > 50%) or primarily oxidative. The Warburg effect is a chronic increase in glycolytic index, quantified by the Warburg index. Additional indices quantify the acute flexibility of ATP supply. The Crabtree index and Pasteur index quantify the responses of oxidative and glycolytic ATP production to alterations in glycolysis and oxidative reactions, respectively; the supply flexibility index quantifies overall flexibility of ATP supply; and the bioenergetic capacity quantifies the maximum rate of total ATP production. We illustrate the determination of these indices using C2C12 myoblasts. Measurement of ATP use revealed no significant preference for glycolytic or oxidative ATP by specific ATP consumers. Overall, we demonstrate how extracellular fluxes quantitatively reflect intracellular ATP turnover and cellular bioenergetics. We provide a simple spreadsheet to calculate glycolytic and oxidative ATP production rates from raw extracellular acidification and respiration data., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Skeletal muscle atrophy is a serious and highly prevalent condition that remains poorly understood at the molecular level. Previous work found that skeletal muscle atrophy involves an increase in skeletal muscle Gadd45a expression, which is necessary and sufficient for skeletal muscle fiber atrophy. However, the direct mechanism by which Gadd45a promotes skeletal muscle atrophy was unknown. To address this question, we biochemically isolated skeletal muscle proteins that associate with Gadd45a as it induces atrophy in mouse skeletal muscle fibers in vivo We found that Gadd45a interacts with multiple proteins in skeletal muscle fibers, including, most prominently, MEKK4, a mitogen-activated protein kinase kinase kinase that was not previously known to play a role in skeletal muscle atrophy. Furthermore, we found that, by forming a complex with MEKK4 in skeletal muscle fibers, Gadd45a increases MEKK4 protein kinase activity, which is both sufficient to induce skeletal muscle fiber atrophy and required for Gadd45a-mediated skeletal muscle fiber atrophy. Together, these results identify a direct biochemical mechanism by which Gadd45a induces skeletal muscle atrophy and provide new insight into the way that skeletal muscle atrophy occurs at the molecular level., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Synaptic mitochondria are thought to be critical in supporting neuronal energy requirements at the synapse, and bioenergetic failure at the synapse may impair neural transmission and contribute to neurodegeneration. However, little is known about the energy requirements of synaptic vesicle release or whether these energy requirements go unmet in disease, primarily due to a lack of appropriate tools and sensitive assays. To determine the dependence of synaptic vesicle cycling on mitochondrially derived ATP levels, we developed two complementary assays sensitive to mitochondrially derived ATP in individual, living hippocampal boutons. The first is a functional assay for mitochondrially derived ATP that uses the extent of synaptic vesicle cycling as a surrogate for ATP level. The second uses ATP FRET sensors to directly measure ATP at the synapse. Using these assays, we show that endocytosis has high ATP requirements and that vesicle reacidification and exocytosis require comparatively little energy. We then show that to meet these energy needs, mitochondrially derived ATP is rapidly dispersed in axons, thereby maintaining near normal levels of ATP even in boutons lacking mitochondria. As a result, the capacity for synaptic vesicle cycling is similar in boutons without mitochondria as in those with mitochondria. Finally, we show that loss of a key respiratory subunit implicated in Leigh disease markedly decreases mitochondrially derived ATP levels in axons, thus inhibiting synaptic vesicle cycling. This proves that mitochondria-based energy failure can occur and be detected in individual neurons that have a genetic mitochondrial defect., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The cascade of events that lead to cognitive decline, motor deficits, and psychiatric symptoms in patients with Huntington disease (HD) is triggered by a polyglutamine expansion in the N-terminal region of the huntingtin (HTT) protein. A significant mechanism in HD is the generation of mutant HTT fragments, which are generally more toxic than the full-length HTT. The protein fragments observed in human HD tissue and mouse models of HD are formed by proteolysis or aberrant splicing of HTT. To systematically investigate the relative contribution of the various HTT protein proteolysis events observed in vivo, we generated transgenic mouse models of HD representing five distinct proteolysis fragments ending at amino acids 171, 463, 536, 552, and 586 with a polyglutamine length of 148. All lines contain a single integration at the ROSA26 locus, with expression of the fragments driven by the chicken β-actin promoter at nearly identical levels. The transgenic mice N171-Q148 and N552-Q148 display significantly accelerated phenotypes and a shortened life span when compared with N463-Q148, N536-Q148, and N586-Q148 transgenic mice. We hypothesized that the accelerated phenotype was due to altered HTT protein interactions/complexes that accumulate with age. We found evidence for altered HTT complexes in caspase-2 fragment transgenic mice (N552-Q148) and a stronger interaction with the endogenous HTT protein. These findings correlate with an altered HTT molecular complex and distinct proteins in the HTT interactome set identified by mass spectrometry. In particular, we identified HSP90AA1 (HSP86) as a potential modulator of the distinct neurotoxicity of the caspase-2 fragment mice (N552-Q148) when compared with the caspase-6 transgenic mice (N586-Q148)., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The sites and rates of mitochondrial production of superoxide and H2O2 in vivo are not yet defined. At least 10 different mitochondrial sites can generate these species. Each site has a different maximum capacity (e.g. the outer quinol site in complex III (site IIIQo) has a very high capacity in rat skeletal muscle mitochondria, whereas the flavin site in complex I (site IF) has a very low capacity). The maximum capacities can greatly exceed the actual rates observed in the absence of electron transport chain inhibitors, so maximum capacities are a poor guide to actual rates. Here, we use new approaches to measure the rates at which different mitochondrial sites produce superoxide/H2O2 using isolated muscle mitochondria incubated in media mimicking the cytoplasmic substrate and effector mix of skeletal muscle during rest and exercise. We find that four or five sites dominate during rest in this ex vivo system. Remarkably, the quinol site in complex I (site IQ) and the flavin site in complex II (site IIF) each account for about a quarter of the total measured rate of H2O2 production. Site IF, site IIIQo, and perhaps site EF in the β-oxidation pathway account for most of the remainder. Under conditions mimicking mild and intense aerobic exercise, total production is much less, and the low capacity site IF dominates. These results give novel insights into which mitochondrial sites may produce superoxide/H2O2 in vivo., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The mammalian target of rapamycin (mTOR) pathway regulates stem cell regeneration and differentiation in response to growth factors, nutrients, cellular energetics, and various extrinsic stressors. Inhibition of mTOR activity has been shown to enhance the regenerative potential of pluripotent stem cells. DEPTOR is the only known endogenous inhibitor of all known cellular mTOR functions. We show that DEPTOR plays a key role in maintaining stem cell pluripotency by limiting mTOR activity in undifferentiated embryonic stem cells (ESCs). DEPTOR levels dramatically decrease with differentiation of mouse ESCs, and knockdown of DEPTOR is sufficient to promote ESC differentiation. A strong decrease in DEPTOR expression is also observed during human ESCs differentiation. Furthermore, reduction in DEPTOR level during differentiation is accompanied by a corresponding increase in mTOR complex 1 activity in mouse ESCs. Our data provide evidence that DEPTOR is a novel stemness factor that promotes pluripotency and self-renewal in ESCs by inhibiting mTOR signaling., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Several flavin-dependent enzymes of the mitochondrial matrix utilize NAD(+) or NADH at about the same operating redox potential as the NADH/NAD(+) pool and comprise the NADH/NAD(+) isopotential enzyme group. Complex I (specifically the flavin, site IF) is often regarded as the major source of matrix superoxide/H2O2 production at this redox potential. However, the 2-oxoglutarate dehydrogenase (OGDH), branched-chain 2-oxoacid dehydrogenase (BCKDH), and pyruvate dehydrogenase (PDH) complexes are also capable of considerable superoxide/H2O2 production. To differentiate the superoxide/H2O2-producing capacities of these different mitochondrial sites in situ, we compared the observed rates of H2O2 production over a range of different NAD(P)H reduction levels in isolated skeletal muscle mitochondria under conditions that favored superoxide/H2O2 production from complex I, the OGDH complex, the BCKDH complex, or the PDH complex. The rates from all four complexes increased at higher NAD(P)H/NAD(P)(+) ratios, although the 2-oxoacid dehydrogenase complexes produced superoxide/H2O2 at high rates only when oxidizing their specific 2-oxoacid substrates and not in the reverse reaction from NADH. At optimal conditions for each system, superoxide/H2O2 was produced by the OGDH complex at about twice the rate from the PDH complex, four times the rate from the BCKDH complex, and eight times the rate from site IF of complex I. Depending on the substrates present, the dominant sites of superoxide/H2O2 production at the level of NADH may be the OGDH and PDH complexes, but these activities may often be misattributed to complex I.

Huntington disease (HD) is an inherited neurodegenerative disease caused by a CAG expansion in the HTT gene. Using yeast two-hybrid methods, we identified a large set of proteins that interact with huntingtin (HTT)-interacting proteins. This network, composed of HTT-interacting proteins (HIPs) and proteins interacting with these primary nodes, contains 3235 interactions among 2141 highly interconnected proteins. Analysis of functional annotations of these proteins indicates that primary and secondary HIPs are enriched in pathways implicated in HD, including mammalian target of rapamycin, Rho GTPase signaling, and oxidative stress response. To validate roles for HIPs in mutant HTT toxicity, we show that the Rho GTPase signaling components, BAIAP2, EZR, PIK3R1, PAK2, and RAC1, are modifiers of mutant HTT toxicity. We also demonstrate that Htt co-localizes with BAIAP2 in filopodia and that mutant HTT interferes with filopodial dynamics. These data indicate that HTT is involved directly in membrane dynamics, cell attachment, and motility. Furthermore, they implicate dysregulation in these pathways as pathological mechanisms in HD.

Long-chain acyl-CoA dehydrogenase (LCAD) is a key mitochondrial fatty acid oxidation enzyme. We previously demonstrated increased LCAD lysine acetylation in SIRT3 knockout mice concomitant with reduced LCAD activity and reduced fatty acid oxidation. To study the effects of acetylation on LCAD and determine sirtuin 3 (SIRT3) target sites, we chemically acetylated recombinant LCAD. Acetylation impeded substrate binding and reduced catalytic efficiency. Deacetylation with recombinant SIRT3 partially restored activity. Residues Lys-318 and Lys-322 were identified as SIRT3-targeted lysines. Arginine substitutions at Lys-318 and Lys-322 prevented the acetylation-induced activity loss. Lys-318 and Lys-322 flank residues Arg-317 and Phe-320, which are conserved among all acyl-CoA dehydrogenases and coordinate the enzyme-bound FAD cofactor in the active site. We propose that acetylation at Lys-318/Lys-322 causes a conformational change which reduces hydride transfer from substrate to FAD. Medium-chain acyl-CoA dehydrogenase and acyl-CoA dehydrogenase 9, two related enzymes with lysines at positions equivalent to Lys-318/Lys-322, were also efficiently deacetylated by SIRT3 following chemical acetylation. These results suggest that acetylation/deacetylation at Lys-318/Lys-322 is a mode of regulating fatty acid oxidation. The same mechanism may regulate other acyl-CoA dehydrogenases.

The oxidation of sn-glycerol 3-phosphate by mitochondrial sn-glycerol 3-phosphate dehydrogenase (mGPDH) is a major pathway for transfer of cytosolic reducing equivalents to the mitochondrial electron transport chain. It is known to generate H(2)O(2) at a range of rates and from multiple sites within the chain. The rates and sites depend upon tissue source, concentrations of glycerol 3-phosphate and calcium, and the presence of different electron transport chain inhibitors. We report a detailed examination of H(2)O(2) production during glycerol 3-phosphate oxidation by skeletal muscle, brown fat, brain, and heart mitochondria with an emphasis on conditions under which mGPDH itself is the source of superoxide and H(2)O(2). Importantly, we demonstrate that a substantial portion of H(2)O(2) production commonly attributed to mGPDH originates instead from electron flow through the ubiquinone pool into complex II. When complex II is inhibited and mGPDH is the sole superoxide producer, the rate of superoxide production depends on the concentrations of glycerol 3-phosphate and calcium and correlates positively with the predicted reduction state of the ubiquinone pool. mGPDH-specific superoxide production plateaus at a rate comparable with the other major sites of superoxide production in mitochondria, the superoxide-producing center shows no sign of being overreducible, and the maximum superoxide production rate correlates with mGPDH activity in four different tissues. mGPDH produces superoxide approximately equally toward each side of the mitochondrial inner membrane, suggesting that the Q-binding pocket of mGPDH is the major site of superoxide generation. These results clarify the maximum rate and mechanism of superoxide production by mGPDH.

Respiratory complex II oxidizes succinate to fumarate as part of the Krebs cycle and reduces ubiquinone in the electron transport chain. Previous experimental evidence suggested that complex II is not a significant contributor to the production of reactive oxygen species (ROS) in isolated mitochondria or intact cells unless mutated. However, we find that when complex I and complex III are inhibited and succinate concentration is low, complex II in rat skeletal muscle mitochondria can generate superoxide or H(2)O(2) at high rates. These rates approach or exceed the maximum rates achieved by complex I or complex III. Complex II generates these ROS in both the forward reaction, with electrons supplied by succinate, and the reverse reaction, with electrons supplied from the reduced ubiquinone pool. ROS production in the reverse reaction is prevented by inhibition of complex II at either the ubiquinone-binding site (by atpenin A5) or the flavin (by malonate), whereas ROS production in the forward reaction is prevented by malonate but not by atpenin A5, showing that the ROS from complex II arises only from the flavin site (site II(F)). We propose a mechanism for ROS production by complex II that relies upon the occupancy of the substrate oxidation site and the reduction state of the enzyme. We suggest that complex II may be an important contributor to physiological and pathological ROS production.

The mammalian target of rapamycin (mTOR) is an atypical serine/threonine kinase that responds to extracellular environment to regulate a number of cellular processes. These include cell growth, proliferation, and differentiation. Although both kinase-dependent and -independent functions of mTOR are known to be critical modulators of muscle cell differentiation and regeneration, the signaling mechanisms regulating mTOR activity during differentiation are still unclear. In this study we identify a novel mTOR interacting protein, the ubiquitin-specific protease USP9X, which acts as a negative regulator of mTOR activity and muscle differentiation. USP9X can co-immunoprecipitate mTOR with both Raptor and Rictor, components of mTOR complexes 1 and 2 (mTORC1 and -2), respectively, suggesting that it is present in both mTOR complexes. Knockdown of USP9X leads to increased mTORC1 activity in response to growth factor stimulation. Interestingly, upon initiation of differentiation of C2C12 mouse skeletal myoblasts, knockdown of USP9X increases mTORC2 activity. This increase in mTORC2 activity is accompanied by accelerated differentiation of myoblasts into myotubes. Taken together, our data describe the identification of the deubiquitinase USP9X as a novel mTORC1 and -2 binding partner that negatively regulates mTOR activity and skeletal muscle differentiation.

Huntington disease (HD) is a dominantly inherited neurodegenerative disease caused by a polyglutamine expansion in the protein huntingtin (Htt). Striatal and cortical neuronal loss are prominent features of this disease. No disease-modifying treatments have been discovered for HD. To identify new therapeutic targets in HD, we screened a kinase inhibitor library for molecules that block mutant Htt cellular toxicity in a mouse HD striatal cell model, Hdh(111Q/111Q) cells. We found that diacylglycerol kinase (DGK) inhibitor II (R59949) decreased caspase-3/7 activity after serum withdrawal in striatal Hdh(111Q/111Q) cells. In addition, R59949 decreased the accumulation of a 513-amino acid N-terminal Htt fragment processed by caspase-3 and blocked alterations in lipid metabolism during serum withdrawal. To identify the diacylglycerol kinase mediating this effect, we knocked down all four DGK isoforms expressed in the brain (β, γ, ε, and ζ) using siRNA. Only the knockdown of the family member, DGKε, blocked striatal Hdh(111Q/111Q)-mediated toxicity. We also investigated the significance of these findings in vivo. First, we found that reduced function of the Drosophila DGKε homolog significantly improves Htt-induced motor dysfunction in a fly model of HD. In addition, we find that the levels of DGKε are increased in the striatum of R6/2 HD transgenic mice when compared with littermate controls. Together, these findings indicate that increased levels of kinase DGKε contribute to HD pathogenesis and suggest that reducing its levels or activity is a potential therapy for HD.

Vibrio fischeri exists in a symbiotic relationship with the Hawaiian bobtail squid, Euprymna scolopes, where the squid provides a home for the bacteria, and the bacteria in turn provide camouflage that helps protect the squid from night-time predators. Like other gram-negative organisms, V. fischeri expresses lipopolysaccharide (LPS) on its cell surface. The structure of the O-antigen and the core components of the LPS and their possible role in colonization of the squid have not previously been determined. In these studies, an O-antigen ligase mutant, waaL, was utilized to determine the structures of these LPS components and their roles in colonization of the squid. WaaL ligates the O-antigen to the core of the LPS; thus, LPS from waaL mutants lacks O-antigen. Our results show that the V. fischeri waaL mutant has a motility defect, is significantly delayed in colonization, and is unable to compete with the wild-type strain in co-colonization assays. Comparative analyses of the LPS from the wild-type and waaL strains showed that the V. fischeri LPS has a single O-antigen repeat composed of yersiniose, 8-epi-legionaminic acid, and N-acetylfucosamine. In addition, the LPS from the waaL strain showed that the core structure consists of L-glycero-D-manno-heptose, D-glycero-D-manno-heptose, glucose, 3-deoxy-D-manno-octulosonic acid, N-acetylgalactosamine, 8-epi-legionaminic acid, phosphate, and phosphoethanolamine. These studies indicate that the unusual V. fischeri O-antigen sugars play a role in the early phases of bacterial colonization of the squid.

Superoxide production from antimycin-inhibited complex III in isolated mitochondria first increased to a maximum then decreased as substrate supply was modulated in three different ways. In each case, superoxide production had a similar bell-shaped relationship to the reduction state of cytochrome b(566), suggesting that superoxide production peaks at intermediate Q-reduction state because it comes from a semiquinone in the outer quinone-binding site in complex III (Q(o)). Imposition of a membrane potential changed the relationships between superoxide production and b(566) reduction and between b(562) and b(566) redox states, suggesting that b(562) reduction also affects semiquinone concentration and superoxide production. To assess whether this behavior was consistent with the Q-cycle mechanism of complex III, we generated a kinetic model of the antimycin-inhibited Q(o) site. Using published rate constants (determined without antimycin), with unknown rate constants allowed to vary, the model failed to fit the data. However, when we allowed the rate constant for quinol oxidation to decrease 1000-fold and the rate constant for semiquinone oxidation by b(566) to depend on the b(562) redox state, the model fit the energized and de-energized data well. In such fits, quinol oxidation was much slower than literature values and slowed further when b(566) was reduced, and reduction of b(562) stabilized the semiquinone when b(566) was oxidized. Thus, superoxide production at Q(o) depends on the reduction states of b(566) and b(562) and fits the Q-cycle only if particular rate constants are altered when b oxidation is prevented by antimycin. These mechanisms limit superoxide production and short circuiting of the Q-cycle when electron transfer slows.

Complex I (NADH-ubiquinone oxidoreductase) can form superoxide during forward electron flow (NADH-oxidizing) or, at sufficiently high protonmotive force, during reverse electron transport from the ubiquinone (Q) pool (NAD(+)-reducing). We designed an assay system to allow titration of the redox state of the superoxide-generating site during reverse electron transport in rat skeletal muscle mitochondria: a protonmotive force generated by ATP hydrolysis, succinate:malonate to alter electron supply and modulate the redox state of the Q pool, and inhibition of complex III to prevent QH(2) oxidation via the Q cycle. Stepwise oxidation of the QH(2)/Q pool by increasing malonate concentration slowed the rates of both reverse electron transport and rotenone-sensitive superoxide production by complex I. However, the superoxide production rate was not uniquely related to the resultant potential of the NADH/NAD(+) redox couple. Thus, there is a superoxide producer during reverse electron transport at complex I that responds to Q pool redox state and is not in equilibrium with the NAD reduction state. In contrast, superoxide production during forward electron transport in the presence of rotenone was uniquely related to NAD redox state. These results support a two-site model of complex I superoxide production; one site in equilibrium with the NAD pool, presumably the flavin of the FMN moiety (site I(F)) and the other dependent not only on NAD redox state, but also on protonmotive force and the reduction state of the Q pool, presumably a semiquinone in the Q-binding site (site I(Q)).

Despite decades of speculation, the proton pumping mechanism of complex I (NADH-ubiquinone oxidoreductase) is unknown and continues to be controversial. Recent descriptions of the architecture of the hydrophobic region of complex I have resolved one vital issue: this region appears to have multiple proton transporters that are mechanically interlinked. Thus, transduction of conformational changes to drive the transmembrane transporters linked by a "connecting rod" during the reduction of ubiquinone (Q) can account for two or three of the four protons pumped per NADH oxidized. The remaining proton(s) must be pumped by direct coupling at the Q-binding site. Here, we present a mixed model based on a crucial constraint: the strong dependence on the pH gradient across the membrane (ΔpH) of superoxide generation at the Q-binding site of complex I. This model combines direct and indirect coupling mechanisms to account for the pumping of the four protons. It explains the observed properties of the semiquinone in the Q-binding site, the rapid superoxide production from this site during reverse electron transport, its low superoxide production during forward electron transport except in the presence of inhibitory Q-analogs and high protonmotive force, and the strong dependence of both modes of superoxide production on ΔpH., (© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.)